Electrochemical Biosensors Based on Polyaniline

Download Electrochemical Biosensors Based on Polyaniline

Post on 30-Nov-2015




1 download

Embed Size (px)




<ul><li><p>Chem. Anal. (Warsaw), 51, 839 (2006)</p><p>Keywords: Polyaniline; Biosensors</p><p>Electrochemical Biosensors Based on Polyaniline</p><p>by Di Wei1,2 and Ari Ivaska1*</p><p>1 Process Chemistry Center, c/o Laboratory of Analytical Chemistry, Abo Akademi University,Abo-Turku, FIN-20500, Finland</p><p>2 Graduate School of Materials Research (GSMR), Abo Akademi University,Abo-Turku, FIN-20500, Finland</p><p>Electrochemical biosensors based on conducting polymers offer many advantages and newpossibilities to detect biologically significant compounds. Various biosensors, such asenzyme sensors, DNA sensors and immunosensors based on polyaniline are reviewed inthis paper. Besides electrochemical detection methods, some new trends with the combina-tion of other techniques are also shortly discussed.</p><p>Biosensory elektrochemiczne z zastosowaniem polimerw przewodzcych oferuj licznezalety i wiele nowych moliwoci w oznaczaniu zwizkw o duym znaczeniu biologicznym.W niniejszym artykule przedstawiono przegld sensorw enzymatycznych, DNA orazimmunosensorw, stworzonych z uyciem polianiliny. Poza elektrochemicznymi metodamianalizy, przedyskutowano rwnie nowe kierunki wykorzystania innych technik anali-tycznych.</p><p>REVIEW</p><p>* Corresponding author. E-mail: ari.ivaska@abo.fi</p></li><li><p>840 D. Wei and A. Ivaska</p><p>A biosensor is an analytical device that converts the concentration of the analyteinto a signal (e.g. electrical signal) by integrating biological sensing element intoa transducer [1]. Biosensors are portable, simple-to-use, and high specificity analyti-cal tools, which are compatible with data-processing technologies. Therefore, biosen-sors will have promising applications in various fields, such as pharmacy, healthcare, pollution monitoring, food and agricultural product processing etc.</p><p>D%LRUHFHSWRU</p><p>(Q]\PH1XFOHLFDFLGV,PPXQRFKHPLFDOV2UJDQHOOH7LVVXHVHWF</p><p>(OHFWURFKHPLFDOWUDQVGXFHU&amp;RQGXFWLQJSRO\PHUV2SWLFDOWUDQVGXFHU3LH]RHOHFWULFFU\VWDOVHWF</p><p>E7UDQVGXFHU</p><p>F$PSOLILHU</p><p>3URFHVVLQJ</p><p>G0LFURHOHFWURQLFV</p><p>DWDLVSOD\</p><p>Figure 1. Basic structure of biosensors: (a) bioreceptor, (b) transducer converts biological recognitionto an electrical signal, (c) amplifier enhances the output from the transducer, and (d) microelec-tronics for data display</p><p>Figure 1 shows the main components of a biosensor. Enzymes, nucleic acids,antibodies, tissue slice, binding protein, whole cells or other bioreceptors have beenused as sensing probes in biosensor fabrications [2]. They have an intimate contact tothe transducer. The transducer can be an electrochemical, optical or piezoelectricaldevice. Bioreceptor (a) and transducer (b) together are sometimes referred to as bio-sensor membrane. A very important advancement in producing biosensor membranesis the use of electrically conducting membranes that contain the enzyme, cofactor,and mediator. There are many different kinds of conducting polymers that can bechosen. The conducting polymer can act as the electrochemical transducer to convertthe biological information into an electrical signal. It has been used in analyticalapplications already for a while [3]. There are some comprehensive reviews on theuse of conducting polymers in electrochemical sensors [49].</p><p>PANI is one of the most intensively investigated conducting polymers, particu-larly due to its excellent stability in different solutions, good electronic properties,and strong biomolecular interactions [10]. The emeraldine salt (ES) form, which canbe obtained in acidic conditions (pH = 2.5 ~ 3.0), is the only conducting state amongthe four basic states of PANI as shown in Scheme 1. Although PANI has been suc-cessfully applied in gas sensors [1113], the pH sensitivity seems unfavorable for itsapplication in biosensors, because most bioassays must be performed in neutral orslightly acidic conditions.</p></li><li><p>841Electrochemical biosensors based on polianiline</p><p>Scheme 1. Transitions between the four basic states of polyaniline: LEB, leucoemeraldine base (fullyreduced form); ES, emeraldine salt (the only conducting form); PNB, pernigraniline base (fullyoxidized form). Transitions between them are controlled by oxidation/reduction reactions. EB,emeraldine base (deprotonated form). Transition between ES and EB is controlled by pH</p><p>Recent research reveals that the N-substituted anilines do not have pH sensitivity[14]. This is due to that the alkyl chain, which is covalently bounded to the nitrogenatom, prevents formation of the EB form. Another derivative of PANI, self-dopedPANI, which is usually referred to sulfonated PANI, shows redox activity even insolutions with neutral pH [15]. Sulfonated PANI was used in amperometric enzymesensors with a sensitivity of 24.91 uA cm2 to detect H</p><p>2O</p><p>2 in pH 6.4 buffer solutions</p><p>[16]. It has also been demonstrated that the PANI blends that included negativelycharged co-components such as sulfonic acid or polyacrylic acids exhibit redox acti-vity in neutral aqueous solutions [1719]. Shi et al. reported poly(aniline-aniline boro-nic acid) wires generated on double-strand (ds)DNA templates [20]. These wires aredeposited on an electrode by means of electrostatic interactions, in which PANI exhi-bits redox activity also in solutions with neutral pH. The redox activity behavior atneutral pH values was also observed by Granot et al. [21]. They have developedPANI/single walled carbon nanotube hybrid systems, and these composites as matri-ces give enhanced bioelectrocatalytic activation of enzymes. These modifications arepotentially useful in electroanalytical applications of PANI in biosensors. However,PANI has already been used to detect different biological compounds by using itsproperties such as pH sensitivity [22], electrochromism [23] and conductivity [24].A nanobiodetector has been developed based on the changes of conductivity of PANImicro- and nano-fibrils in the presence of micro-organisms [25]. It was found that theelectrical response depends on the number of cells deposited on the PANI nano-network, and is specific to different kinds of micro-organisms. The minimum num-ber of cells to be detected by this sensor is below 100.</p><p>- 4 H+</p><p>Q</p><p>Q</p><p>1</p><p>1</p><p>1</p><p>1</p><p>Q</p><p>1</p><p>+</p><p>1</p><p>1</p><p>+</p><p>1</p><p>Q</p><p>(IIHFWRIS+</p><p>H</p><p>$</p><p>H</p><p>$</p><p>/(%</p><p>(6</p><p>(%</p><p>+</p><p>$</p><p>+</p><p>$</p><p>H</p><p>$</p><p>H</p><p>$</p><p>31%</p><p>$</p><p>$</p><p>1+</p><p>1+</p><p>1+</p><p>1+</p><p>1+</p><p>1+</p><p>1+</p><p>1+</p><p>+ 4 H+</p><p>+ +</p></li><li><p>842 D. Wei and A. Ivaska</p><p>ENZYME SENSORS</p><p>Potentiometric technique relates the potential to the analyte concentration (acti-vity) when the electrode reaction is at equilibrium and no current flows through theelectrodes. Whereas in amperometric biosensors the current is based on heteroge-neous electron transfer reaction i.e. the oxidation or reduction of the electroactivesubstances, and is proportional to their concentration.</p><p>The enzyme electrode is a combination of any electrochemical probe (ampero-metric, potentiometric or conductimetric) with a thin layer (10200 mm) of immobi-lized enzyme. There are many methods to immobilize the enzymes, such as adsorp-tion, physical entrapment in gels or membranes, crosslinking, covalent binding, anddoping etc. [26]. PANI has been used not only for the physical entrapment of theenzymes during electrodeposition, but also as a matrix for covalent enzyme immobi-lization [27].</p><p>Glucose biosensor</p><p>For determination of glucose, the reaction with glucose oxidase (GOD) enzymeelectrode is shown with the following equations:</p><p>b-glucose + O2 fi b-gluconic acid + H2O2</p><p>H2O2 fi O2 + 2H+ + 2e</p><p>The amount of hydrogen peroxide produced in equation (1) is usually determinedby amperometric method by oxidation at the working electrode according to equation(2). This is referred as an amperometric biosensor.</p><p>Equation (2) also shows that protons are produced in the follow-up oxidationreaction. If pH is measured by potentiometric method, and then related to the amountof glucose, the biosensor is referred to as a potentiometric biosensor. There are pHtransducers that have been developed for potentiometric biosensors [22, 28]. Althoughthe response of these types of biosensors is interfered by the buffer capacity of thesample, they, however, allow the direct, reagentless detection of the analyte, whilethe amperometric method requires several consecutive reactions.</p><p>Potentiometric glucose biosensor. The potentiometric detection of enzyme acti-vity is based on the measurement of the change in pH in the enzymatic layer on thesensor surface. The pH change (e.g. release of acid) is caused by oxidation of H</p><p>2O</p><p>2</p><p>produced by the enzymatic reaction as indicated in equation (2). The enzyme cata-lyzes the conversion of the target analyte in reactions consuming or releasing pro-tons. The sensitivity limit of a potentiometric enzyme electrode is the sensitivity of</p><p>(1)</p><p>(2)</p><p>GOD</p></li><li><p>843Electrochemical biosensors based on polianiline</p><p>the pH transducer. Conventional pH transducers, e.g. glass electrode or pH sensitivefield effect transistors, can be used for this purpose. The advantages of PANI asan advanced potentiometric pH transducer that can be used in biosensors, have beenreported by Karyakin et al. [28]. The chemically synthesized PANI (doped withcamphorsulfonic acid) showed reversible potentiometric response of approximately90 mV per pH unit over the pH range from 3 to 9. Some other processible PANI andself-dope PANI have also been used for this purpose. The pioneer potentiometricglucose biosensor based on PANI is shown in Figure 2 [22].</p><p>Figure 2. Prototype structure of potentiometric PANI-glucose oxidase (GOD) sensor [11]. Reproducedby the kind permission from American Chemical Society</p><p>The same strategy as with glucose biosensors has also been used in urea sensors,which reach a detection limit of 105 mol L1 [28]. The urea detection can also be donevia pH sensors made by PANI doped with tetraphenylborate [29]. On the surface ofthis PANI layer, urease was immobilized within either a poly(vinyl alcohol) matrix ormodified sol-gel glass. It was shown that the sensor response from the latter is betterand more stable.</p><p>Amperometric glucose biosensor. In amperometric glucose biosensors, theamount of hydrogen peroxide is usually detected by measuring the current during theoxidation reaction in equation (2). The GOD, which carries a negative charge, isnormally immobilized (doped) in PANI during the polymerization reaction when thePANI membrane is formed. The fibril of a PANI film is about 2000 [30], which isenough for immobilization of GOD into the film. The enzyme can also be adsorbedon the PANI film, but the doped film has better stability [31]. It has been shown thatthe glucose biosensor based on PANIpolyisoprene composite film has a highpermselectivity for hydrogen peroxide compared to ascorbic acid, therefore dimini-</p></li><li><p>844 D. Wei and A. Ivaska</p><p>shing the interference of ascorbic acid in detection [32]. An amperometric glucosebiosensor has also been made by depositing the horseradish peroxidase (HRP) ona PANImodified Pt electrode. HRP was electrostatically immobilized on the surfaceof the PANI film, and the electrocatalytic reduction of hydrogen peroxide under dif-fusion controlled conditions was monitored by a voltammetric method [33]. Ampero-metric glucose biosensors based on Prussian Blue and PANIGOD modified elec-trodes have also been developed [34].</p><p>The electron transfer pathways between immobilized enzyme molecules whichwere integrated with conducting polymers and the modified electrode have beenreviewed [35]. The electrons can transfer via redox mediators as well as the conduc-ting polymer wires directly. The understanding of these pathways is very importantto construct the conducting polymer based amperometric enzyme sensors. Raitmanet al. [36] have covalently attached N-6-(2-amiboethyl)flavin adenine dinucleotideas a linker between glucose oxidase and PANIpoly(acrylic acid) composite. Thisdirect electrical contact yielded very high electron transfer rates. It was shown thatthe glucose concentration controls the steady-state concentration ratio of oxidizedand reduced form of PANI. Yu et al. [37] have constructed an efficient electricalwiring of enzymes for biosensors with improved sensitivity. A film containing poly(sty-renesulfonate) and enzymes (HRP) covered a thin layer (4 nm) of sulfonated PANI,underneath which is a polycationic layer. It was shown that at least 90% of proteinswere electrically coupled to this kind of self-assembly electrode, and it has a detec-tion limit of 3 nmol L1 for hydrogen peroxide. Compared to classic PANI-basedbiosensors, an increase in linear response range with shorter response time was ob-served when entrapping enzymes into poly(styrenesulfonate)PANI composites [38].The composites were synthesized within the pores of track-etched polycarbonatemembranes, which resulted in immobilizing the enzymes during polymerization.Pan et al. [39] reported a method to prepare PANI glucose oxidase sensor via tem-plate process. The process contains three procedures. First, enzymes (glucose oxi-dases) were entrapped into polymer film directly during electropolymerization ofaniline. Second, the electrode is hydrolyzed in hydrochloric acid solution to removethe trapped enzymes from the PANI film. Finally, fresh glucose oxidase is immobi-lized again to obtain an active PANI glucose oxidase sensor. The stability and res-ponse was much enhanced in the sensor made by this process, since the denaturedand inactive glucose oxidase affected by aniline monomer during electropolymerizationhas been removed during hydrolysis. Amperometric enzyme biosensors based onPANI nanoparticles have also been reported [40]. HRP immobilized on the electrode-posited PANI nanoparticle film showed improved signal to noise ratio compared toHRP deposited directly in a PANI film.</p></li><li><p>845Electrochemical biosensors based on polianiline</p><p>Amino acid biosensor</p><p>Amino acids are basic components in living organism. The amino acid biosensoris widely used in many areas. Choline is a very important amino alcohol in the func-tions of human organism. Detection of choline is frequently needed in food and clini-cal analysis. Langer et al. have developed a choline sensor by immobilization ofcholine oxidase (ChO) to nanoporous PANI layers [41]. In this construction, PANIbehaves as a transducer. The oxidize nanoporous PANI layers trap and bound enzymeunits due to coulombic interactions between positively charged PANI and negativelycharged polar groups in the enzyme molecules. It was found that the electrical res-ponse of this sensor was substantially increased when PANI was charged and dis-charged several times in the presence of enzyme molecules by applying an oscillatingpotential. The structure of this PANIChO biosensor is shown in Figure 3. Its sensi-tivity is 5 A mmol L1 in the amperometric mode and 10 mV mmol L1 in the poten-tiometric mode of measurements. At the constant potential 0.4 V (vs Ag/AgCl), themeasured current response is linear vs. the choline concentration. It can detect cho-line with concentrations below 20 mmol L1 in the potentiometric mode.</p><p>Figure 3. The processes leading to generate an electrical response of Langers sensor owing toenzymemediated oxidation of choline. ChO: choline ox...</p></li></ul>