This article was downloaded by: [University Library Utrecht] On: 04 June 2014, At: 06:11 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 Enzyme Inhibition Based Biosensors: A Review Lata Sheo Bachan Upadhyay a & Nishant Verma a a Department of Biotechnology , National Institute of Technology , Raipur , India Accepted author version posted online: 03 Aug 2012.Published online: 02 Jan 2013. To cite this article: Lata Sheo Bachan Upadhyay & Nishant Verma (2013) Enzyme Inhibition Based Biosensors: A Review, Analytical Letters, 46:2, 225-241, DOI: 10.1080/00032719.2012.713069 To link to this article: http://dx.doi.org/10.1080/00032719.2012.713069 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 & Conditions of access and use can be found at http://www.tandfonline.com/page/terms- and-conditions
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This article was downloaded by: [University Library Utrecht]On: 04 June 2014, At: 06:11Publisher: 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
Enzyme Inhibition Based Biosensors: AReviewLata Sheo Bachan Upadhyay a & Nishant Verma aa Department of Biotechnology , National Institute of Technology ,Raipur , IndiaAccepted author version posted online: 03 Aug 2012.Publishedonline: 02 Jan 2013.
To cite this article: Lata Sheo Bachan Upadhyay & Nishant Verma (2013) Enzyme Inhibition BasedBiosensors: A Review, Analytical Letters, 46:2, 225-241, DOI: 10.1080/00032719.2012.713069
To link to this article: http://dx.doi.org/10.1080/00032719.2012.713069
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
Lata Sheo Bachan Upadhyay and Nishant VermaDepartment of Biotechnology, National Institute of Technology,Raipur, India
This article reviews the recent advances in the development of biosensors based on enzyme
inhibition. They are an important alternative as compared to conventional analytical techni-
ques due to their high selectivity and sensitivity. Since the most potent inhibitors of enzymes
are heavy metals and pesticides, these enzyme inhibition based biosensors have a wide appli-
cation in the field of environmental safety, food safety, and clinical analysis. Biosensors
based on the inhibition of enzymes such as glucose oxidase, urease, tyrosinase, cholinester-
ase, and some other enzymes are intended to be discussed in this review, concerning the
immobilization techniques involved and the sensitivity toward different inhibitors.
Keywords: Biosensors; Heavy metals; Inhibitors; Organophosphorus compounds; Pesticides
INTRODUCTION
Biosensors are the exceptional analytical system characterized by their highspecificity and sensitivity toward substance. They are the reagentless analyticaldevices, which are entirely different from other chemical sensors in terms of norequirement of sample processing, before or after analysis. Consequently, biosensorshave become an active area of research to detect various chemicals and biologicalcomponent for clinical, food and environmental monitoring (Amine et al. 2006). Con-ventional analytical techniques such as gas chromatography, liquid chromatography,thin film chromatography, and other techniques may fulfill these requirements, butrequire expensive instruments, unsuitable for in-fields analysis, are tedious and timeconsuming (Prieto-Simon et al. 2006). In contrast to direct monitoring of compo-nents, which is the major application of biosensors, inhibition based biosensor is gain-ing importance owing to high sensitivity and is, therefore, rapidly expanding its fieldof application. The significant applications of enzyme inhibition biosensors includesthe determination of pesticides (mainly organophosphorus compounds and carba-mates) followed by heavy-metal detection. Some papers are also reported in literature
Received 25 April 2012; accepted 1 July 2012.
Address correspondence to Dr. Lata Sheo Bachan Upadhyay, Assistant Professor, Department of
Biotechnology, National Institute of Technology, Raipur 492010 (C.G.), India. E-mail: contactlataupadhyay
@gmail.com
Analytical Letters, 46: 225–241, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032719.2012.713069
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reviewing the application of enzyme inhibition based biosensors in different fields(Turdean 2011; Amine et al. 2006; Arduini et al. 2009; Luque de Castro and Herrera2003; Trojanowicz 2002; Sole, Merkoci, and Alegret 2003; Patel 2002; Arduini et al.2010). In this review, we focused on different enzymes, engaged to develop biosensorbased on its activity inhibition, and provided an overview of the activity carried outsince 2006. Enzymes such as glucose oxidase, urease, tyrosinase, and cholinesterasehave been extensively used for biosensor development and thus described in detailin this review while others have been described in brief.
ENZYME BASED BIOSENSORS
It is the most common recognition system in which an enzyme, either mono ormulti enzyme, is immobilized in a thin layer at the transducer surface by differentimmobilization techniques. This immobilized enzyme consumed substrate or analytealong with a co-substrate (if any) and yield product(s). The biosensor response is thenachieved by either measuring the co-substrate consumption or product yield. This iscalled Direct monitoring of analytes as reported in determination of glucose (G. Liuand Lin 2006a; Z. Wang et al. 2009; Topiu Sulak et al. 2006; Deng et al. 2009),-Cholesterol (Tsai, Chen, and Lee 2008; Umar, Rahman, Al-Hajry, et al. 2009; Umar,Rahman, Vaseem, et al. 2009; Abdelwahab, Won, and Shim 2010), Urea (Premanodeand Toumazou 2007; Z. Yang et al. 2007; Barhoumi et al. 2006; Alqasaimeh, Heng,and Ahmad 2007), and organophosphate pesticides (Zourob, Simonian, et al. 2007;Viveros et al. 2006; Zourob, Ong, et al. 2007). Despite having advantages of beingsimple, portable, and a continuously operational configuration, these biosensors havesome limitations, when applied to the environmental monitoring. The major draw-backs comprise the limited number of environmental pollutants that can act as thesubstrate for the enzyme and the high detection limits.
Alternatively, indirect monitoring refers to the assessing of substance or inhibi-tors that specifically interact with immobilized enzyme and inhibits its biocatalyticproperties. Such inhibitors bind either to the enzyme or enzyme-substrate complexand further interfere with the enzymatic reactions. The beneficial aspect of indirectmonitoring is that most of the enzymes are susceptible to a very low concentrationof inhibitors, thus increase the sensitivity of biosensor. However, presence of someother types of inhibitors in the assay sample may inhibit the enzyme activity andtherefore produces unexpected results. Moreover, these types of biosensors alsorequire some substrate in addition to the analyte to be assay and, therefore, compli-cate the overall design of biosensor. The inhibition of enzyme can either be reversible,in which the binding of an inhibitor can be reversed by decreasing inhibitor concen-tration, or irreversible, in which binding of the inhibitor results in perpetual inhibitionof enzyme activity.
Enzyme Inhibition: Reversible Inhibition
This inhibition is characterized by a high rate of association and dissociation ofinhibitors with the enzyme. Consequently, the biosensor based on reversible inhi-bition can be repetitively regenerated and thus termed as ‘‘Multiple use biosensor.’’However, the biosensor response may vary considerably with each assay because
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some of the enzyme activity lost after every inhibition-regeneration step. Recentlydeveloped reversible enzyme inhibition biosensors have been reported for the deter-mination of heavy metals (Guascito et al. 2008; Ghica and Brett 2008), food preser-vatives like benzoic acid (Shan et al. 2008; Shan et al. 2007), carbamate insecticides(Bucur et al. 2006), organophosphorus insecticides (Vidal et al. 2006), mercury(J. X. Liu et al. 2009), nicotine (Sanchez-Paniagua Lopez et al. 2007), and other toxiccompounds (Dzyadevych et al. 2006). Based on the mechanism to inhibit the enzymeactivity, the inhibitors may further be classified into Competitive, Noncompetitive,and Uncompetitive inhibitors. Table 1 shows the characteristic behavior of each typeof inhibitor. In each case, the reaction rate slows down, which is the maximum in caseof uncompetitive inhibitors as compared to competitive and noncompetitive inhibi-tors. For an enzyme-catalyzed reaction, the effect of different types of the inhibitoron the rate vs. substrate concentration curve is shown in Fig 1.
Irreversible Inhibition
It is characterized by binding of an inhibitor to enzyme active site by covalentbonding and permanent inhibition of enzyme activity. It directed the formation of ahighly reactive inhibitor product that binds to the enzyme irreversibly and thusinhibits its activity (Turdean 2011). Since the enzyme is inactivated permanently,the biosensor based on irreversible inhibition may be termed as ‘‘Single use biosen-sor.’’ However, irreversible inhibition is entirely different from irreversible enzymeinactivation where some sort of nonspecific treatments like extreme of pH and tem-perature destroy the whole protein structure. Biosensors based on irreversibleenzyme have been reported for the detection of mercury ions (Frasco et al. 2007;Prakash et al. 2008) and organophosphorus pesticides (Periasamy, Umasankar,and Chen 2009; Prieto-Simon et al. 2006).
Table 1. Comparison of different binding mechanism of inhibitors and their effect on enzyme kinetics
Type of Inhibitor Binding Site Kinetic effects
Competitive
Inhibitor
. Inhibitor competitively binds to substrate binding site of
enzyme i.e., active site, due to its close resemblance to
substrate structure.
. Inhibition can be reversed by increasing the concentration
of substrate to a level where it out-competes inhibitor.
No change in
Vmax
but Km
increased
Non-Competitive
Inhibitor
. Inhibitor binds to a site on enzyme which is totally
different from active site.
. This inhibition cannot be reversed by increasing the
substrate concentration, as the binding sites are different
for both, inhibitor and substrate.
No change in Km
But Vmax
decreased
Un-Competitive
Inhibitor
. Inhibitor binds only to the Enzyme- Substrate (ES)
complexes.
. This enzyme-inhibitor-substrate complex (ESI) cannot
form product due to conformational changes in enzyme.
Both Vmax and
Km decreased
Note: Vmax is the maximum velocity of enzyme catalyzed reaction; Km is the Michaelis Menten constant
equivalent to the substrate concentration at which reaction attains half its maximal velocity.
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Biosensor Response
Biosensor response is generally determined by assessing the deviation inenzyme activity, after it is exposed to the inhibitor as shown in Fig. 2. The inhibitorsconcentration is usually quantified by measuring the percentage of inhibition, beforeand after exposure to the inhibitors. The percentage inhibition is calculated by theequation:
Ið%Þ ¼ A0�Ai
A0� 100
where I (%)¼Percentage of inhibition; Ao¼ the immobilized enzyme activity beforethe exposure to inhibitors; and Ai¼ the immobilized enzyme activity after theexposure to inhibitors.
With the help of enzyme inhibition based biosensors, not only the concentrationof inhibitors can be determined, but one can also study the kinetic characteristic ofinhibition processes. Here we can obtain information regarding catalytic mechanismof enzymes and their active sites. Two parameters reported to have a significant effecton the degree of inhibition includes: Incubation time and Inhibitor concentration(Turdean 2011). Both the factors must be considered before concluding the biosensorresponse. As the biosensor response is a function of substrate concentration, enzymeactivity, time and so on, the coupling of biosensor with flow injection analysis system(FIA) offers the possibility of controlling all these parameters (Prieto-Simon et al.2006) and, thus, improving the selectivity of biosensors. Such an approach is desirablefor the real time analysis and repetitive measurements of inhibitors. The FIA biosen-sors have been extensively used to determine the environmental pollutants likeorganophosphorus pesticides (Wei et al. 2009; Li et al. 2007; Shi et al. 2006; G. Liu
Figure 1. Reaction rate vs. substrate concentration graph showing the relative rate in the absence and
presence of different types of inhibitors.
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and Lin 2006b; Zheng et al. 2006). However, in the case of disposable biosensors orstrips, such as glucose biosensor and urea biosensor, incorporation of FIA is of no use.
Immobilization Effect on Biosensor Response
Several immobilization techniques such as covalent binding, physical entrap-ment, adsorption, cross-linking, and encapsulation have been reported in literaturebut not a single technique can be considered as a universal method of immobilizationto achieve the better biosensor response. This is the most important factor to beconsidered while designing a biosensor, to attain higher sensitivity, and functionalstability. The so-called immobilization effect alters the enzyme in the following ways(Kobayashi and Laidler 1974):
. Substrate affinity of an enzyme changes due to conformational change in enzymestructure, caused by immobilization.
. Immobilization may also result in variation in the dissociation equilibrium ofcharged groups of the active center.
. Apparent kinetic constants tend to vary by a non-uniform distribution ofsubstrate and=or product between enzyme matrix and the surrounding solution.
. Immobilization in a multi-layer system affects the reaction rate by creating adiffusional barrier between substrate and immobilized enzyme.
Figure 2. Diagrammatic representation of the working principle of enzyme inhibition based biosensors.
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Several different approaches of enzyme immobilization have been reported in litera-ture for biosensor development, each vary for its sensitivity toward the inhibitors. Acomparative study of biosensor response for anticholinesterase pesticides was done,by immobilizing acetylcholinesterase with glutaraldehyde in presence of BSA (bovineserum albumin) and by photopolymerization with poly(vinyl alcohol) bearing styryl-pyridinium groups (PVA-SBQ) (Silva Nunes, Jeanty, andMarty 2004). They reportedthat immobilization with glutaraldehyde produces robust and reproducible biosen-sors, but need higher enzyme content to achieve current values as compared toPVA-SBQ immobilized AChE. For the determination of mercury ions, urease wasimmobilized in poly(vinylferrocenium) film (Kuralay, Ozyoruk, and Yıldız 2007)and nano-structured polyaniline-nafion composite film (Do, Lin, and Ohara 2011).The detection limit in the latter case was found to be lower than poly(vinylferroce-nium) film immobilized urease. For the detection of organophosphate pesticides, acomparative analysis was performed by employing different techniques like entrap-ment, covalent attachment, cross-linking, and dispersion, for tyrosinase immobiliza-tion (Vidal et al. 2006). They reported that entrapment of tyrosinase withinelectropolymerized poly(o-phenylenediamine) polymer provided higher sensitivityto the biosensor.
Enzyme Inhibition Based Biosensors
Glucose oxidase inhibition. Glucose oxidase (EC 1.1.3.4) is an oxido-reductase that catalyzes the oxidation of glucose to hydrogen peroxide and glucono-lactone. The first biosensor developed by Clark for the determination of glucose wasbased on enzyme glucose oxidase (Clark and Lyons 2006). However, it was not basedon the inhibition of enzyme but on the consumption of oxygen (Belluzo, Ribone, andLagier 2008). In last five years, some papers were published on biosensors regardingglucose oxidase inhibition and most of them were for the determination of heavymetals (Ghica and Brett 2008; Guascito et al. 2008; J. X. Liu et al. 2009; Samphaoet al. 2012). An electrochemical biosensor was developed for mercury determinationin compost extract by using ferrocene as an electron transfer mediator (J. X. Liu et al.2009). The enzyme was immobilized on an aniline membrane supported on a plati-num electrode using glutaraldehyde as the cross-linking agent. Its detection limitwas lower as compared to the biosensor, developed by immobilizing glucose oxidaseon a carbon paste electrode modified with manganese dioxide (Samphao et al. 2012).Additionally, the latter biosensor also reported to have the sensitivity toward otherinterfering ions such as zinc and chromium. In another method, glucose oxidasewas immobilized on a carbon film electrode with poly (neutral red) as a mediator(Ghica and Brett 2008). The biosensor showed good sensitivity toward copper, cad-mium, lead, and zinc with the detection limits in microgram per mL. For herbicidesdetection in water samples, an amperometric biosensor was developed byself-assembling of the enzyme on a copper electrode surface (Q. Yang et al. 2010).The biosensor reported to have the sensitivity in nanomoles toward atrazine.Table 2 summarizes some of the biosensor based on glucose oxidase inhibition.
Tyrosinase inhibition. Tyrosinase (EC 1.14.18.1), also known as monophe-nol monoxygenase, is a copper containing enzyme that catalyzes the oxidation of
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phenols, such as tyrosine. In recent years, many biosensors have been developedbased on the inhibition of tyrosinase enzyme activity by different inhibitors. Theywere mostly used for the determination of pesticides (Campanella et al. 2006;Campanella et al. 2007; T. Liu et al. 2011; Tanimoto de Albuquerque and Ferreira2007; Vidal et al. 2006; X. Wang et al. 2006), food preservatives (Shan et al. 2008),reduced thiols (L. Liu et al. 2009), and fluorides (Asav, Yorganci, and Akyilmaz2009). All of them were amperometric biosensor except one, which was developedby X. Wang et al. (2006), for the determination of pesticides. However, for the deter-mination of same pesticides such as diazinon and carbaryl, the sensitivity of the con-ductometric biosensor was found to be low compared to the amperometric biosensordeveloped by Tanimoto de Albuquerque and Ferreira (2007). Benzoic acid which isgenerally used as food preservatives and an active ingredient of cosmetics againsthyperpigmentation, was determined by its inhibition effect on tyrosinase immobi-lized by calcium carbonate nano materials (Shan et al. 2008) and glutaraldehydeactivated streptavidine magnetic particles (Sima et al. 2011). T. Liu et al. (2011)immobilized tyrosinase on platinum electrodes via electrostatic interaction for thedetection of organophosphorus pesticides in ppb range. Different immobilizationtechniques were worked out by Vidal et al. (2006), to increase the sensitivity oftyrosinase toward dichlorovos. They reported that entrapment of enzyme withinelectropolymerized poly(o- phenylenediamine) polymer gave higher performance.As the organophosphorus and carbamates pesticides are least soluble in aqueousphase, a different approach was carried out using enzyme inhibition OPEEEs(enzyme electrode working in organic phase), operated in water saturated chloro-form medium (Campanella et al. 2006; Campanella et al. 2007). Table 3 lists someof the biosensors based on tyrosinase inhibition.
Urease inhibition. Urease (EC: 3.5.1.5) is a bi-metallic enzyme, which cata-lyzes the hydrolysis of urea into ammonia and carbon dioxide. It was the first enzymeto be crystallized out from jack bean (Balasubramanian and Ponnuraj 2010). Severalbiosensors with different transducer types have been reported based on the inhibitionof activity of urease enzyme. These biosensors were mostly used for the determinationof heavy metals like copper, cadmium, chromium, lead, and mercury. A renewablepotentiometric biosensor for the determination of mercury ions was developed byimmobilizing urease on self-assembled gold nanoparticles (Y. Yang et al. 2006). Thisbiosensor was very effective in terms of faster response, low detection limit, and can
Table 2. Glucose oxidase inhibition based biosensors with comparative analysis of
their detection limit toward different inhibitors
Transducer Type Inhibitors Detection Limit References
Amperometric Hg(II)
Cd(II)
Cu(II)
Pb(II)
Zn(II)
Atrazine
0.49mg=L0.5mg=L
1mg=L6mg=L3mg=L9mg=L
39 nmole=L
(J. X. Liu et al. 2009)
(Samphao et al. 2012)
(Ghica and Brett 2008)
(Ghica and Brett 2008)
(Ghica and Brett 2008)
(Ghica and Brett 2008)
(Q. Yang et al. 2010)
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be regenerated easily. An amperometric biosensor was also developed by immobiliz-ing urease on poly (vinylferrocenium) film for the detection of mercury ions (Kuralayet al. 2007). This biosensor was also used to study the interference effect of metal ionssuch as copper, cadmium, chromium, iron, zinc, and lead ions. From the clinical pointof analysis, an amperometric biosensor was developed for the detection of mercuryions by immobilizing urease on gold modified screen printed carbon electrode andscreen printed carbon electrode alone (Domınguez-Renedo et al. 2009). Theyreported higher sensitivity of gold modified electrode than unmodified one. Anotheramperometric biosensor for mercury determination was developed by immobilizingurease on nano-structured polyaniline-nafion film=Au=Al2O3 sensing electrode,which had the detection limit in parts per million (Do et al. 2011). Recently, Sol-gelapproach has been employed to develop optical fiber based biosensor for the detec-tion of heavy metals (Gani, Ashari, and Kuswandi 2010) and cadmium (Verma,Kumar, and Kaur 2010), in aqueous sample and milk respectively. Same approachwas also reported for development of an amperometric biosensor (Nepomuscene,Daniel, and Krastanov 2007) and conductometric biosensor (Ilangovan et al.2006). In the latter case, sensitivity of urease for different metals ions was found tobe Cu2þ>Cd2þ>Pb2. Table 4 provides an outline of the biosensors based on ureaseinhibition.
Cholinesterase inhibition. Cholinesterase is a family of enzymes that cata-lyze the hydrolysis of the neurotransmitter acetylcholine, resulting in the production
Table 3. Tyrosinase inhibition based biosensors with comparative analysis of their detection limit toward
inhibitors
Transducer Type Inhibitors Detection Limit References
Amperometric Methyl parathion
Diazinon
Carbofuran
Carbaryl
Benzoic acid
Kojic acid
Ascorbic acid
Azelaic acid
Chlorpyrifos
Profenofos
Malathion
Dichlorovos
Glutathione
Cysteine
Fluoride
Paraoxon
Malathion
Parathion-ethyl
Triazine pesticides
6–100mg=L19–50mg=L5–90mg=L10–50mg=L
5.6� 10�7 to 9.2� 10�5mole=L
IC50¼ 7.2� 10�5mole=L
IC50¼ 3.7� 10�6mole=L
IC50¼ 1.2� 10�5mole=L
IC50¼ 1.3� 10�4mole=L
0.2 mg=L0.8 mg=L3mg=L
0.06mmole=L
0.09mmole=L
0.06mmole=L
1.0–20mmole=L
0.01� 10�6–0.1� 10�6mole=L
0.01� 10�6–0.1� 10�6mole=L
0.01� 10�6–0.1� 10�6mole=L
0.5� 10�9mole=L
(Tanimoto de Albuquerque
and Ferreira 2007)
(Shan et al. 2008)
(Sima et al. 2011)
(Sima et al. 2011)
(T. Liu et al. 2011)
(Vidal et al. 2006)
(L. Liu et al. 2009)
(Asav et al. 2009)
(Campanella et al. 2007)
(Campanella et al. 2006)
Conductometric Diazinon
Alachlor
Carbaryl
5.0� 10�8mole=L
1.5� 10�7mole=L
2.0� 10�7mole=L
(X. Wang et al. 2006)
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of choline and acetic acid. This reaction is essential for the cholinergic neuron to cometo a resting state after activation. Based on the type of substrate utilized, it can furtherbe divided into two types: Acteylcholinesterase (AChE; EC 3.1.1.7) and Butyrylcho-linesterase (BChE; EC 3.1.1.8). The former hydrolyses acetylcholine while the latterhydrolyses butyrylcholine. Cholinesterase-based biosensors are widely used as ananalytical device for detection of organophosphorus compound and carbamate pes-ticides (Amine et al. 2006). The organophosphorus compounds partly inhibit the bio-logical activity of acetylcholinesterase through phosphorylation of the serine groupand the biosensor principle lies on the detection of thiocholine, hydrogen peroxide,and pH change due to hydrolysis reaction (Jaffrezic-Renault 2001). They are veryeffective tools for the assay of anticholinergic compounds (Pohanka 2009). ASPR-sensor system was developed with the help of alkanethiol self-assembled mono-layer for the detection of organophosphate pesticide and carbamates insecticide(Mauriz et al. 2006). Its sensitivity toward chloropyrifos, an organophosphate pesti-cide, was found to be very high as compared to the optical fiber biosensor developedby sol-gel film immobilized acetylcholinesterase (Kuswandi, Fikriyah, and Gani2008). To prevent leaching problems, an optical biosensors was developed, consistingof lipophilic chromoionophore (ETH5294) doped sol gel film interfaced with anothersol-gel film immobilized acetylcholinesterase (Wong et al. 2006). During the last fiveyears, amperometric mode of detection has been widely used for biosensor design.Inhibitors detected by this mode of transduction included paraoxon (Arduini et al.2006; G. Liu and Lin 2006b; Pohanka, Jun, and Kuca 2008; Pohanka, Kuca, andJun 2008; Sinha et al. 2010; Waibel et al. 2006; Zhao et al. 2009), methyl paraoxon(Valdes-Ramırez et al. 2008), parathion (Waibel et al. 2006; Zhao et al. 2009), methylparathion (Gong, Wang, and Zhang 2009), carbaryl (Arduini et al. 2006; Bucur et al.2006; Ion et al. 2010; Valdes-Ramırez et al. 2008), aldicarb (Arduini et al. 2006), car-bofuran (Bucur et al. 2006; Cortina, del Valle, and Marty 2008; Laschi et al. 2007;Valdes-Ramırez et al. 2008), pirimicarb (Bucur et al. 2006), dichlorovos (Cortinaet al. 2008; Shi et al. 2006; Valdes-Ramırez et al. 2008), chloropyrifos oxon-methyl(Arduini et al. 2006), nerve agents (Arduini et al. 2007; Pohanka et al. 2009), diazinon(Somerset et al. 2007), fenthion (Somerset et al. 2007), trichlorfon (Li et al. 2007), andtriazophos (Du, Cai, et al. 2007; Du, Huang, et al. 2007). Among these, few are
Table 4. Urease inhibition based biosensors with comparative analysis of their detection limit toward
different inhibitors
Transducer Type Inhibitors Detection Limit References
Potentiometric Hg(II) 0.05mmole=L (Y. Yang et al. 2006)
reported to be based on enzyme butyrylcholinesterase (Arduini et al. 2007; Arduiniet al. 2006). A comparative study was done by using co-phthalocyanine and Prussianblue modified screen printed electrode for the detection of different pesticides byemploying both enzymes (Arduini et al. 2006). AChE based biosensor reported tohave higher sensitivity for aldicarb and carbaryl while BChE based biosensor hadhigher affinity toward paraoxon and chloropyrifos methyl oxon. Sol-gel approachwas used to develop a bi-enzymatic biosensor by immobilizing nippostrongylusbrasi-liensis AChE and cytochrome P450 BM-3 (CYP102-A1) mutant, through sol-gelprocess (Waibel et al. 2006). The biosensor was able to detect organophosphate deri-vatives, such as paraoxon and parathion in food materials. Table 5 summarizes thedifferent types of biosensors based on cholinesterase inhibition.
Other enzymes inhibition based biosensors. Apart from the enzymes dis-cussed previously, some other enzymes are also reported in literature, which are nota part of extensive research for the development of inhibition-based biosensors(Table 6). It includes choline oxidase, polyphenol oxidase, nitrate reductase, inver-tase, alkaline phosphatase, and horseradish peroxidase=catalase. For the detectionof organophosphorus compounds, a monoenzymatic amperometric biosensor wasdeveloped by immobilizing choline oxidase on a graphite electrode (Tavakolia andGhourchianb 2010). This biosensor was able to detect inhibitors with the detectionlimit of 0.4 mM. Polyphenol oxidase was employed to develop an amperometricbiosensor for the detection of benzoic acid in samples such as milk, yogurt, andcold drinks (Shan et al. 2007). Heavy metal analysis was also performed by employ-ing enzymes such as nitrate reductase (X. J. Wang et al. 2009) and invertase(Bagal-Kestwal et al. 2008). For the detection of herbicide, 2,4-dichlorophenoxyace-tic acid, an amperometric biosensor was developed by immobilizing alkaline phos-phatase in sol-gel=chitosan membrane alongwith Fe3O4 nanoparticles (Loh et al.2008). The use of nanoparticles reported to increase the sensitivity of biosensor bytwo fold. Contrary to these mono-enzymatic biosensors, a novel bi-enzymatic bio-sensor was developed by entrapping horseradish peroxidase and catalase separatelyinto layered double hydroxides (an anionic clay) (Chen et al. 2008). On exposing tonitrite, an inhibitor, the latter biosensor showed increment in signals, with a detec-tion limit of 4 mM.
Table 6. List of other enzymes reported to be used in biosensors for inhibitor detection
HRP=Catalase Amperometric Nitrite 4 mmole=L (Chen et al. 2008)
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CONCLUSION
Despite the ever-increasing number in enzyme inhibition based biosensors in thelast decades, some issues still raise a barricade in their broader area of application.Sensitivity of the enzyme inhibition biosensors for the compound other than the ana-lyte to be assayed is the main problem. These biosensors usually act as an ideal bio-sensor in lab analysis but become unpredictable when apply for on-site analysis. Thismay be due to the multi-analyte nature of the sample, which hinders with the selec-tivity of biosensors. However, it can sort out by employing a protective layer, allow-ing only the desired analytes to pass through but this may be a rate-limiting step inbiosensor response. Better understanding of the interference mechanism of heavymetals and pesticides, may help in increasing the selectivity of biosensor, and thisapproach needs to be worked out to design more precise and reliable devices.Additionally, the biosensor should always be developed by considering the para-meters as specified by various regulating agencies. It will be more helpful in their com-mercialization and is eminent in the replacement of conventional methods.
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