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Research ArticleConductive Composite Biosensor System forElectrochemical Indinavir Drug Detection
Natasha Ross Nicolette Hendricks-Leukes Rachel Fanelwa AjayiPriscilla Baker and Emmanuel I Iwuoha
SensorLab Department of Chemistry University of Western Cape Private Bag X17 Bellville 7535 South Africa
Correspondence should be addressed to Natasha Ross nrossuwcacza
Received 3 July 2015 Accepted 26 August 2015
Academic Editor Josefina Pons
Copyright copy 2015 Natasha Ross 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
Indinavir is a protease inhibitor antiretroviral (ARV) drug which forms part of the highly active antiretroviral therapy during thetreatment of HIVAIDS Indinavir undergoes first-pass metabolism through the cytochrome P450 (CYP) enzymes in the humanliver ofwhichCYP3A4 is themost influential isoenzymeMultidrug combination therapy and as such therapeutic drugmonitoring(TDM) during HIVAIDS treatment are therefore critical to prevent adverse interactions The conventional sensitive and specificassays available for quantifying ARV drugs however suffer from distinct disadvantages In this regard biosensors can be used toprovide real time information on themetabolic profile of the drug In this study a biosensor with cobalt(III) sepulchrate trichlorideCo(Sep)3+ as diffusional mediator was constructed The biosensor platform consisted of CYP3A4 immobilized onto a goldnanoparticle (GNP) overoxidized polypyrrole (OvOxPpy) carrier matrixThe biosensor exhibited reversible electrochemistry withformal potential determined as minus624 plusmn 5mV from voltammetric analysis with overall electron transfer being diffusion controlledThe biosensor showed typical electrocatalytic response to dioxygen (O
2) exemplified by the distinct increase in the cathodic peak
current (119868119901119888) A concentration-dependent increase in 119868
119901119888was observed in response to consecutive additions of Indinavir
1 Introduction
Antiretroviral (ARV) drugs are ubiquitously associated withtreatment ofHIVAIDS [1]These drugs work bymaintaininga plasma level concentration at the virus (HIV) replicationsite thereby inhibiting viral mutation and viral resistanceand suppressing viral load of patients [2] Treatment isimplemented through highly active antiretroviral therapy(HAART) which is a multidrug combination regimen con-sisting of ARV drugs from three basic groups includingthe nucleoside reverse transcriptase inhibitors (NRTIs) non-nucleoside reverse transcriptase inhibitors (NNRTIs) andprotease inhibitors (PIs) The PIs as well as the NNR-TIs undergo extensive phase I metabolism through thecytochrome P450 (CYP) enzymes present in the liver ofwhich the most influential isoenzyme involved is CYP3AOne of the major aspects associated with HAART treatmentis adverse drug interactions and as such [3] the need fortherapeutic drug monitoring during HIVAIDS treatmentis pivotal [4] Sensitive and specific assays available for
quantifying ARV drugs are based on high performanceliquid-chromatography andor gas chromatography (HPLCandGC) with ultraviolet detection or HPLC andor GC-massspectrometry These methods however are very complexrequiring extensive sample preparation and skilled operatorsare expensive and are not designed to give rapid analysis andreal time measurements In this regard biosensors can beused to provide real time information on themetabolic profileof the drug necessary for modulating therapy to the patientrsquosbenefit During the reduction of P450s according to thescheme Fe3+ + eminus rarr Fe2+ NADPH or NADH is exhausted[5] Therefore P450s and enzyme electrodes based on P450sare promising for the application of biosensors Howeverachieving effective electrical communication between theelectrode and the enzyme in the case of heme-enzymessuch as the CYP3A4 enzyme can be problematic [6] Thisis because the electrochemically active heme-centre is buriedin the protein structure to gain hydrophobic environmentfor catalysis thus a longer direct electron transfer distancebetween the prosthetic group and the electrode surface
Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 630408 7 pageshttpdxdoiorg1011552015630408
2 Journal of Chemistry
Therefore reduction in the size of enzyme carrier materialscan greatly improve the efficiency of immobilized enzymes[7] Numerous publications indicate organic conductingpolymers as a convenient biosensor component forming anappropriate environment for the immobilization of enzymeat the electrode surface [8] Amongst the studied polymerspolypyrrole (Ppy) has attracted considerable attention Thispolymer can be easily prepared by electrochemical oxidationof the monomer in the presence of suitable dopant ionsConcomitantly overoxidation is an irreversible processesassociated with loss of conductivity However overoxidized-polypyrrole (OvOxPpy) is an excellent matrix materialused for deposition of metal nanoclusters Au nanoparticles(GNP) in particular due to their unique properties such asgood conductivity useful electrocatalytic and biocompati-bility were considered excellent integration material in theelectrochemical biosensors [9] The presence of GNPs givesimmobilized protein molecules more freedom of orientationand as such permits proteins to orient in conformationswhich aremore favorable for direct electron transfer [10]Thegold particles form the conducting electrode and thus thesite of electron transfer when anchored to the substrate [11]The usual methods for enzyme immobilization include directphysical adsorption [12 13] encapsulation into hydrogel [14]cross-linking [15] and covalent binding [16] However akey requirement of enzyme immobilization is attachmentwithout the bioactivity being sacrificed [17 18] Here theGNP|OvOxPpymodified film provides an attractive templatefor enzyme immobilization due to the fast inert low temper-ature process
In this work OvOxPpy-film [19] infused with conduc-tive Au nanoparticles was constructed on a glassy carbonelectrode and used as platform for CYP3A4 enzyme immo-bilization to fabricate a biosensor for selective and sensitivedetection of Indinavir (ARV drugs) in the large presence ofPBS at pH 741 The goal was to obtain results with precisionand accuracy and at increasingly lower concentration levelsof the substance being determined
Electroanalytical Methods Applied for the Determination ofAnti-HIV Drugs Indinavir [1(1S2R)5(S)]-235-trideoxy-N-(23-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(11-dimeth-ylethyl)-amino]carbonyl]-4-(3-pyridinylmethyl)-1-piperaz-inyl]-2-phenylmethyl)-D-erythro-pentonamide sulphate isHIV protease inhibitor metabolized by the cytochromeP450 system It has been probed by various capillaryelectrophoretic spectrophotometric and liquid chromato-graphic methods however all the reported methods arearduous and time-consuming and require highly sophisti-cated equipment [20] Up to date a great deal of biosensorswere developed for determination of Indinavir Howeveronly one paper reported anodic voltammetry using glassycarbon electrode Ignaszak et al [21] developed a noveltherapeutic biosensor for the determination of IND In thisstudy pH 60 Britton-Robinson buffer solution was selectedfor analytical medium in which IND exhibited diffusioncontrolled oxidative peak at +089V Also the peak currentvaried linearly with drug concentration in the range between28 times 10minus7 and 50 times 10minus5M The constructed biosensor also
showed quasi-reversible electrochemistry of the Fe3+Fe2+redox species of the heme thiolate CYP3A4 enzyme underaerobic and anaerobic conditions with a detection limit andresponse time of 6158 times 10minus2mgLminus1 and 40 s respectively
2 Experimental
21 Reagents and Instruments The gold nanoparticles wereproduced by the reduction of Au(III) involving the use ofHAuCl
4in the presence of KCl Cytochrome P450 3A4
(CYP3A4) (Mw 52 000) was prepared by genetic engineer-ing with subsequent purification from a human CYP3A4cDNA clonemdashoverexpressed in Escherichia coli (E coli) cellsconsisting of only the terminal oxidase (heme domain)mdashprepared as 817 120583Msolution Cobalt(III) sepulchrate trichlo-ride Co(Sep)3+ and serum from bovine albuminwere prod-ucts of Sigma-Aldrich Dipotassium hydrogen phosphate(K2HPO4) and potassium dihydrogen phosphate (KH
2PO4)
monohydrate salts and KCl were purchased from Sigma-Aldrich Electrolyte solutions were 50mM of PBS (pH74) containing 100mM KCl as the supporting electrolyteWorking solution of Indinavir was prepared from Indinavirsulphate obtained through extraction from the capsules(Crixivan)mdashpurchased from Merck amp Co Inc Ultrapurewater with resistivity 182MΩ cm was prepared using Mil-lipore Synergy water purification system All reagents wereof analytical grade Electrochemical experiments were per-formed with BAS 100W electrochemical workstation andelectrodes fromBioAnalytical Systems BAS USA Cyclic andsquare wave voltammograms were recorded with a computerinterfaced to the BAS 100Wworkstation A glassy carbon diskelectrode (GCE) of area 0071 cm2 (diameter 3mm) was usedas the working electrode A platinum wire and AgAgCl (3MNaCl) electrodes were used as auxiliary and reference elec-trodes respectively SEM analysis was performed on JEOLJSM-7500F Scanning ElectronMicroscope All voltammetricresults are reported with respect to AgAgCl
22 Preparation of Biosensor Platform GNP|OvOxPpy||GCEPpy was electrochemically prepared from an aqueous solu-tion of 015M monomer by potentiostatic method in 01MLiClO
4supporting electrolyte Polymerization was done
at a constant potential of +800mV (versus AgAgCl) for120 s under argon atmosphere The Ppy-modified GCE wasthen carefully rinsed with milli-Q water and subsequentlyplaced into an electrolytic cell containing 01M NaOH andoveroxidation was done at +1000mV The process usuallytook approximately 450 s Gold nanoparticles were thenelectrodeposited from an argon degassed solution of 100mMKCl containing 05mMHAuCl
4 Electrodepositionwas done
potentiodynamically by cycling the potential from 200 tominus1000mV at a scan rate of 50mVs for 15 cycles
23 Preparation of CYP3A4-Based Biosensor CYP3A4 wasimmobilized on the GNP|OvOxPpy||GCE by drop-coating5 120583L of 163 120583M enzyme solution containing 20mg BSAwas carefully drop-coated onto the GNP-modified GCE Theenzyme-modified electrode was then covered with a tight
Journal of Chemistry 3
C
Cu
Cu
Cu
CuCu Au
Au
Au
AuAu
AuAuAu
Au
AuAu
AuOK
K
EDX acquiring HAADF area 1
(a) (b) (c)
(d)
20
40
60
10 15 205Energy (keV)
Figure 1 SEM of Ppy||GCE (a) OvOxPpy||GCE (b) and GNP|OxPpy||GCE (c) respectively at 30 000Mag (scale = 1 120583m) with the EDXspectrum (d)
fitting lid for the first 10min to form a uniform layer afterwhich the lid was removed and the enzyme layer was slightlydried for another 40min but still retaining wet camera Thiswas then followed by the deposition of 4 120583L agarose (1wv) after which the prepared biosensor was immediatelyplaced at 4∘C for at least 1 h The biosensor was denoted byCYP3A4|GNP|OvOxPpy||GCE
24 Electrochemical Procedures All voltammetric analysiswas done at 25∘C in the presence of 1mM Co(Sep)3+ diffu-sional electron transfermediator dissolved in either degassedor undegassed PBS (pH 74) Cyclic voltammograms wererecorded in the presence and absence of oxygen andorIndinavir at a potential scan rate (]) of 10mVs versusAgAgCl from an initial potential 119864
119894= minus200mV to a switch
potential 119864120582= minus1000mV Oysteryoung-type square wave
voltammetry (SWV) was done at an amplitude of 75mV andfrequency of 5Hz The analysis of the CVs gave the formalpotentialsmeasured as themidpoint potential (119864∘1015840)The peakseparation Δ119864
119901(the difference between 119864
119901119888and 119864
119901119886) and
the anodic to cathodic peak current ratio (119868119901119886119868119901119888) of the
biosensor responses were also determined
3 Results and Discussion
31 Structure of Composite Polypyrrole Materials Morpho-logical characterization of the electrode platform during elec-trode assembly process was conducted by SEM analysis asshown in Figure 1The SEM images of the Ppy-modifiedGCE
(a) OvOxPpy-film (b) and GNP|OvOxPpy||GCE (c) com-posite film were recorded at a magnification of 30 000 with ascale of 1120583mTheEDX spectrum (d) shows elemental analysisof a selected area on Figure 1(c) Figure 1(b) clearly showsthat overoxidation affects themorphological properties of thepolypyrrole film and as such the OvOxPpy-film exhibited astructural morphology that was significantly different fromthat of the conductive Ppy-film [22] The OvOxPpy-filmhad a typical ldquocauliflower-likerdquo structure constituted byspherical micrograins Prior research has established thatoveroxidation is initiated by hydroxyl radicals that form dueto the oxidation of water of which the overall process may bedescribed by [23]
Ppy + 119909HOminus 997888rarr OvOxPpy + 119899eminus
+ 119898H+ + sdot sdot sdot(1)
Figure 1(c) shows the porous OvOxPpy-film to be anexcellent matrix for the electrodeposition of gold nanoparti-cles which had good surface coverage across the film withuniform size and diameters of approximately 100 nm TheEDX spectrum confirms the presence of gold within theOvOxPpy matrix
During overoxidation a large current is passed whichdecreased as oxidation progressed eventually levelling offand fluctuating slightly around one value which indicatesthe completion of the overoxidation process as shown inFigure 2 During overoxidation nucleophilic attack of theradical cationic pyrrole units in the polymer by hydroxide
4 Journal of Chemistry
3002001000 400Time (sec)
minus300
minus250
minus200
minus150
minus100
minus50
I(120583
A)
Figure 2 Current time curve for polypyrrole overoxidation(OvOxPpy||GCE)
(a)
(b) (c)
minus150
minus100
minus50
0
50
100
150
I(120583
A)
minus1200 minus1000 minus800 minus600 minus400 minus200 0 200minus1400E (mV) versus AgAgCl (3M NaCl)
(a) Bare GCE in PBS
minus35
0
minus10
50minus
700
350
700
10500
(b) OvOxPpyGCE in PBS(c) GNP|OvOxPpyGCE in PBS
120583A OvOxPPyGCEEpa
GNP|OvOxPPyGCEEpc
GNP|OvOxPPyGCEEpa
OvOxPPyGCEEpc
Figure 3 Cyclic voltammogram of GCE (a) OvOxPpy||GCE(b) and GNP|OvOxPpy||GCE (c) in PBS (pH 74) at 70mVsversus AgAgCl (inset) SWV of OvOxPpy||GCE and theGNP|OvOxPpy||GCE composite film
ions takes place resulting in loss of the conjugated structureand electrical conductivity Expulsion of the ClO
4
minus dopantions results in the uniformly sized nanopores within thepolymer film
32 Electron Transfer Systems The modification of theglassy carbon electrode during the film assembly waselectrochemically characterized by cyclic voltammetry andsquare wave voltammetry From Figure 3 it is evidentthat the bare glassy carbon electrode shows no observableelectrochemistry whereas the background current of theGNP|OvOxPpy||GCE composite is two orders of magnitudehigher as compared to that of the overoxidized-polypyrrole
film Moreover the forward and reverse square waves in theinset exhibit a similar trend with regard to magnitude ofthe background current of GNP|OvOxPpy||GCE compositefilm as compared to that of the OvOxPpy||GCE film and assuch corroborate the findings shown in cyclic voltammetricresults
33 Effect of Cobalt(III) Sepulchrate Trichloride As statedin previous sections all of the electrochemical studies per-taining to the fabricated CYP3A4-based biosensor wereconducted in the presence of diffusional electron transfermediator Co(Sep)3+ The main requirement for a goodmediator is that it exhibits fast reversible electrochemistrywhile also showing a redox response sufficiently negativein order to in turn reduce the enzyme heme group Priorinvestigations have established Co(Sep)3+ as superior choiceto facilitate electrical communication between redox centresof P450 enzymes and the electrode [24] Figure 4(a) showsthe CV taken at 100mVs and SWV recorded at a frequencyof 5Hz This revealed that as the potential was scanned fromminus200 to minus800mV the reduction current steadily increased toa maximum value after which the current decreased untilthe switching potentialmdashcathodic and anodic peak currents(119868119901119888
and 119868119901119886) shown to be fairly similar in magnitude The
CV exhibited well defined corresponding cathodic (119864119901119888)
and anodic peaks (119864119901119886) for which peak potentials were
determined as minus650- (plusmn5) and minus589 (plusmn5)mV respectivelywith corresponding 119864∘1015840 determined as 620 plusmn 5mV Thepeak potential difference (Δ119864) determined from CV resultswas approximately 61 plusmn 5mV Square wave voltammetrywas used to corroborate CV results The SW in Figure 4(b)shows well defined forward (cathodic) and reverse (anodic)square waves with 119868
119901119888shown to be slightly higher than
119868119901119886 by approximately half an order of magnitude The
peak potentials corresponded to values of minus631 and minus620 plusmn5mV for cathodic and anodic square waves respectively Incomparing CV and SW results it is well known that squarewave voltammetry has a higher signal-to-noise ratio andas such differences are considered well within experimentalvariability
34 Monooxygenation of CYP3A4 Figure 5 shows the cyclicvoltammograms of the biosensor containing the immobilizedredox species at different scan rates varying from 10 to100mVs The inset shows the linear variation of anodic andcathodic peak currents (119868
119901119886and 119868119901119888) with square root of scan
rate (]12) On increasing the scan rates the biosensor CVpeak currents increased gradually and peak potentials stayedfairly stable concomitantly resulting in little change in peak-to-peak separation with each consecutive scan rate change119868119901119888
and 119868119901119886
also increase linearly with ]12 Based on theseresults it may be deduced that the CYP3A4-film was stableand exhibited reversible electrochemistry constituted by adiffusion-controlled electron transfer process The diffusioncoefficient 119863 calculated using the Randle Sevcik equation is25 times 10minus8 cm2 sminus1 where 119879 = 25∘C 119877 = 8314 Jmolminus1 Kminus1 119865 =
Journal of Chemistry 5
minus75
minus50
minus25
00
25
50
75
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus585 plusmn 5mVEpa
(plusmn) minus650 plusmn 5mVEpc
(a)
minus12
minus10
minus8
minus6
minus4
minus2
0
2
4
6
8
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus631mVEpa
(plusmn) minus620mVEpc
(b)
Figure 4 Cyclic (a) and square wave (SW) (b) voltammograms of CYP3A4-based biosensor in argon-saturated PBS (pH 74 100mM KCl)containing 1mM Co(Sep)3+ in solution The biosensor contained 163120583MCYP3A4
Anodic Ipa
Cathodic Ipc
10mV sminus1
100mV sminus1
minus800 minus600 minus400 minus200minus1000
minus8
minus6
minus4
minus2
0
2
4
6
I(120583
A) 4 6 8 102
12 (mV)12
minus8minus6minus4minus2
0246
I(120583
A)
E (mV) versus AgAgCl (3M NaCl)
Figure 5 Cyclic voltammograms of biosensor (CYP3A4|GNPs|OvOxPpy||GCE) in argon-saturated PBS (50mM 100mMKCl) containing 1mM CoSep3+
96486Cmolminus1 119862 = 1 times 10minus5mol cmminus3 119899 = 1 119860 = 0071 cm2and slope = 467 times 10minus3
119868119901
= 04463119899119865119860[119899119865
119877119879]
12
11986312]12119862analyte
(2)
Concomitantly the surface concentration Γlowast(mol cmminus2) of the adsorbed electroactive species maybe estimated accordingly
119868119901119886
]=11989921198652119860Γlowast
4119877119879 (3)
where 119877 119865 119879 and 119899 are the gas constant Faraday constanttemperature and number of electrons transferred respec-tivelyThe ion exchange behaviour of conducting polypyrroleis associated by a one-electron transfer process in which themonomers are oxidized by oxidizing agents or catalysts toproduce the radical polymer cationThus for the one electrontransfer process at 25∘C Γlowast value evaluated from the slope ofthe anodic peak is 699 times 10minus7mol cmminus2
35 Mechanism of Biosensor Response to Indinavir Voltam-metric responses of the biosensor were recorded at a scanrate of 10mVs in the absence and presence of oxygen(O2) andor Indinavir in order to observe enzyme-oxygen-
substrate interactions and are shown in Figures 6(a) 6(b)and 6(c) respectively In the presence of O
2 there was a
significant increase in 119868119901119888 This is a common phenomenon
which has been recorded in literature reports with regardto biosensor studies with P450 [25] O
2is the natural
cosubstrate of CYP3A4 and P450 enzymes in general andbinds to the ferrous ion of the enzyme heme group at avery fast rate therefore the distinctive 119868
119901119888increase can be
regarded as a chemical reaction which immediately followsFe3+Fe2+ electron transfer process a phenomenon which inthis case may be described as the electrocatalytic responseof the enzyme to O
2 The O
2-CYP3A4 interaction is greatly
enhanced in the presence of substrate and as such in thepresence of Indinavir (25 120583M) there is a strong amplificationin 119868119901119888
6 Journal of Chemistry
Biosensor in oxygen-saturated
Biosensor in oxygen-saturated
c
b
a Biosensor in argon-saturated
minus8
minus7
minus6
minus5
minus4
minus3
minus2
minus1
0
1
I(120583
A)
minus800 minus600 minus400 minus200minus1000
PBS (0120583M Indinavir)
PBS (0120583M Indinavir)
PBS (25 120583M Indinavir)
E (mV) versus AgAgCl (3M NaCl)
Figure 6 Biosensor in argon-saturated PBS (0 120583M Indinavir) (a)oxygen-saturated PBS (0 120583M Indinavir) (b) and oxygen-saturatedPBS containing 25120583M Indinavir (c)
Indinavir
minus10
minus8
minus6
minus4
minus2
0
I(120583
A)
minus800 minus600 minus400 minus200minus1000
02 120583M
10120583M minus10minus9minus8minus7minus6minus5minus4minus3minus2
I(120583
A)
04 06 08 1002Indinavir (120583M)
E (mV) versus AgAgCl (3M NaCl)
Figure 7 CYP3A4-based biosensor in the presence of increasingIndinavir concentrations (02 to 10 120583M) Inset cathodic peak currentversus Indinavir concentration
Figure 7 shows the voltammetric response of the biosen-sor in oxygen-saturated PBS (pH 74 100mM KCl) con-taining 1mM Co(Sep)3+ diffusional mediator at a scan rateof 10mVs The CVs reveal that the biosensor exhibits aconcentration-dependent increase in 119868
119901119888with each addition
of Indinavir up to the final concentration of 10 120583M Thesefindings directly correspond with previous investigations[26 27] and thus it may be deduced that the substrateIndinavir is metabolised by the reduced CYP3A4 which inturn is mediated by the reduced Co(Sep)2+ The increasingrate of dioxygen to the heme in the presence of substrate isexemplified by the increase in the reduction current (119868
119901119888) as
shown in the cyclic voltammograms The response profile ofthe biosensor as shown by CV is typical of cytochrome P450enzyme in particular and heme-enzyme in general therebyadequately proving the strong catalytic effect andmetabolism
of Indinavir by CYP3A4 These favourable features of themodified electrode offer great promise for its sensing appli-cations The inset shows that the catalytic current is linearversus Indinavir concentration in the range of 02ndash10 120583Manda correlation coefficient of 09908Thedetection limit (signal-to-noise ratio is 3) is 0001120583mol Lminus1 The detection limit iswell below the plasma concentration of Indinavir (8 h afterintake) which ranges from 013 to 86mg Lminus1
4 Conclusions
An ideal nanocomposite support material consisting of GNPinfused OvOxPpy was successfully constructed based ona rapid electrochemical technique The obtained platformserved as an ideal host for enzyme immobilization for thedevelopment of a user-friendly and low-cost biosensor forIndinavir The results demonstrated that Au nanoparticlesenhanced the catalytic activity ofOvOxPpy by giving negativeshifts of CV peak potentials and increasing peak current ofthe enzyme Due to the synergy of OvOxPpy and the GNPsGNP|OvOxPpy||GCE had good stability sensitivity andselectivity to determine Indinavir in a large presence of PBS(pH 721) The biosensor response to Indinavir in oxygenatedPBS also showed significant increase GNP|OvOxPpy||GCEdisplayed strong catalytic activity towards the oxidation ofIndinavir and reveals the response of the active specieswith distinctive voltammetric peaks Low concentrationsrsquodetection of Indinavir demonstrates the ability of biosensorplatform to perform effective point-of-care diagnostics Thebiosensor constructionmethod developed in this study holdsgreat promise for ARV drugsrsquo (Indinavir) determination inreal samples and use for effective therapeutic drug monitor-ing in real time
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This research was financially supported by South AfricarsquosNational Research Foundation (NRF)
References
[1] N A Thompson ldquoLatin Postrdquo 2015 httpwwwlatinpostcomarticles47066
[2] H F Gunthard J A Aberg J J Eron et al ldquoAntiretroviraltreatment of adult HIV infection 2014 recommendations of theInternational Antiviral Society-USA panelrdquo The Journal of theAmericanMedical Association vol 312 no 4 pp 410ndash425 2014
[3] H Namme Luma M-S Doualla S-P Choukem et alldquoAdverse drug reactions ofHighlyActiveAntiretroviralTherapy(HAART) in HIV infected patients at the General HospitalDouala Cameroon a cross sectional studyrdquo The Pan AfricanMedical Journal vol 12 p 87 2012
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
2 Journal of Chemistry
Therefore reduction in the size of enzyme carrier materialscan greatly improve the efficiency of immobilized enzymes[7] Numerous publications indicate organic conductingpolymers as a convenient biosensor component forming anappropriate environment for the immobilization of enzymeat the electrode surface [8] Amongst the studied polymerspolypyrrole (Ppy) has attracted considerable attention Thispolymer can be easily prepared by electrochemical oxidationof the monomer in the presence of suitable dopant ionsConcomitantly overoxidation is an irreversible processesassociated with loss of conductivity However overoxidized-polypyrrole (OvOxPpy) is an excellent matrix materialused for deposition of metal nanoclusters Au nanoparticles(GNP) in particular due to their unique properties such asgood conductivity useful electrocatalytic and biocompati-bility were considered excellent integration material in theelectrochemical biosensors [9] The presence of GNPs givesimmobilized protein molecules more freedom of orientationand as such permits proteins to orient in conformationswhich aremore favorable for direct electron transfer [10]Thegold particles form the conducting electrode and thus thesite of electron transfer when anchored to the substrate [11]The usual methods for enzyme immobilization include directphysical adsorption [12 13] encapsulation into hydrogel [14]cross-linking [15] and covalent binding [16] However akey requirement of enzyme immobilization is attachmentwithout the bioactivity being sacrificed [17 18] Here theGNP|OvOxPpymodified film provides an attractive templatefor enzyme immobilization due to the fast inert low temper-ature process
In this work OvOxPpy-film [19] infused with conduc-tive Au nanoparticles was constructed on a glassy carbonelectrode and used as platform for CYP3A4 enzyme immo-bilization to fabricate a biosensor for selective and sensitivedetection of Indinavir (ARV drugs) in the large presence ofPBS at pH 741 The goal was to obtain results with precisionand accuracy and at increasingly lower concentration levelsof the substance being determined
Electroanalytical Methods Applied for the Determination ofAnti-HIV Drugs Indinavir [1(1S2R)5(S)]-235-trideoxy-N-(23-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(11-dimeth-ylethyl)-amino]carbonyl]-4-(3-pyridinylmethyl)-1-piperaz-inyl]-2-phenylmethyl)-D-erythro-pentonamide sulphate isHIV protease inhibitor metabolized by the cytochromeP450 system It has been probed by various capillaryelectrophoretic spectrophotometric and liquid chromato-graphic methods however all the reported methods arearduous and time-consuming and require highly sophisti-cated equipment [20] Up to date a great deal of biosensorswere developed for determination of Indinavir Howeveronly one paper reported anodic voltammetry using glassycarbon electrode Ignaszak et al [21] developed a noveltherapeutic biosensor for the determination of IND In thisstudy pH 60 Britton-Robinson buffer solution was selectedfor analytical medium in which IND exhibited diffusioncontrolled oxidative peak at +089V Also the peak currentvaried linearly with drug concentration in the range between28 times 10minus7 and 50 times 10minus5M The constructed biosensor also
showed quasi-reversible electrochemistry of the Fe3+Fe2+redox species of the heme thiolate CYP3A4 enzyme underaerobic and anaerobic conditions with a detection limit andresponse time of 6158 times 10minus2mgLminus1 and 40 s respectively
2 Experimental
21 Reagents and Instruments The gold nanoparticles wereproduced by the reduction of Au(III) involving the use ofHAuCl
4in the presence of KCl Cytochrome P450 3A4
(CYP3A4) (Mw 52 000) was prepared by genetic engineer-ing with subsequent purification from a human CYP3A4cDNA clonemdashoverexpressed in Escherichia coli (E coli) cellsconsisting of only the terminal oxidase (heme domain)mdashprepared as 817 120583Msolution Cobalt(III) sepulchrate trichlo-ride Co(Sep)3+ and serum from bovine albuminwere prod-ucts of Sigma-Aldrich Dipotassium hydrogen phosphate(K2HPO4) and potassium dihydrogen phosphate (KH
2PO4)
monohydrate salts and KCl were purchased from Sigma-Aldrich Electrolyte solutions were 50mM of PBS (pH74) containing 100mM KCl as the supporting electrolyteWorking solution of Indinavir was prepared from Indinavirsulphate obtained through extraction from the capsules(Crixivan)mdashpurchased from Merck amp Co Inc Ultrapurewater with resistivity 182MΩ cm was prepared using Mil-lipore Synergy water purification system All reagents wereof analytical grade Electrochemical experiments were per-formed with BAS 100W electrochemical workstation andelectrodes fromBioAnalytical Systems BAS USA Cyclic andsquare wave voltammograms were recorded with a computerinterfaced to the BAS 100Wworkstation A glassy carbon diskelectrode (GCE) of area 0071 cm2 (diameter 3mm) was usedas the working electrode A platinum wire and AgAgCl (3MNaCl) electrodes were used as auxiliary and reference elec-trodes respectively SEM analysis was performed on JEOLJSM-7500F Scanning ElectronMicroscope All voltammetricresults are reported with respect to AgAgCl
22 Preparation of Biosensor Platform GNP|OvOxPpy||GCEPpy was electrochemically prepared from an aqueous solu-tion of 015M monomer by potentiostatic method in 01MLiClO
4supporting electrolyte Polymerization was done
at a constant potential of +800mV (versus AgAgCl) for120 s under argon atmosphere The Ppy-modified GCE wasthen carefully rinsed with milli-Q water and subsequentlyplaced into an electrolytic cell containing 01M NaOH andoveroxidation was done at +1000mV The process usuallytook approximately 450 s Gold nanoparticles were thenelectrodeposited from an argon degassed solution of 100mMKCl containing 05mMHAuCl
4 Electrodepositionwas done
potentiodynamically by cycling the potential from 200 tominus1000mV at a scan rate of 50mVs for 15 cycles
23 Preparation of CYP3A4-Based Biosensor CYP3A4 wasimmobilized on the GNP|OvOxPpy||GCE by drop-coating5 120583L of 163 120583M enzyme solution containing 20mg BSAwas carefully drop-coated onto the GNP-modified GCE Theenzyme-modified electrode was then covered with a tight
Journal of Chemistry 3
C
Cu
Cu
Cu
CuCu Au
Au
Au
AuAu
AuAuAu
Au
AuAu
AuOK
K
EDX acquiring HAADF area 1
(a) (b) (c)
(d)
20
40
60
10 15 205Energy (keV)
Figure 1 SEM of Ppy||GCE (a) OvOxPpy||GCE (b) and GNP|OxPpy||GCE (c) respectively at 30 000Mag (scale = 1 120583m) with the EDXspectrum (d)
fitting lid for the first 10min to form a uniform layer afterwhich the lid was removed and the enzyme layer was slightlydried for another 40min but still retaining wet camera Thiswas then followed by the deposition of 4 120583L agarose (1wv) after which the prepared biosensor was immediatelyplaced at 4∘C for at least 1 h The biosensor was denoted byCYP3A4|GNP|OvOxPpy||GCE
24 Electrochemical Procedures All voltammetric analysiswas done at 25∘C in the presence of 1mM Co(Sep)3+ diffu-sional electron transfermediator dissolved in either degassedor undegassed PBS (pH 74) Cyclic voltammograms wererecorded in the presence and absence of oxygen andorIndinavir at a potential scan rate (]) of 10mVs versusAgAgCl from an initial potential 119864
119894= minus200mV to a switch
potential 119864120582= minus1000mV Oysteryoung-type square wave
voltammetry (SWV) was done at an amplitude of 75mV andfrequency of 5Hz The analysis of the CVs gave the formalpotentialsmeasured as themidpoint potential (119864∘1015840)The peakseparation Δ119864
119901(the difference between 119864
119901119888and 119864
119901119886) and
the anodic to cathodic peak current ratio (119868119901119886119868119901119888) of the
biosensor responses were also determined
3 Results and Discussion
31 Structure of Composite Polypyrrole Materials Morpho-logical characterization of the electrode platform during elec-trode assembly process was conducted by SEM analysis asshown in Figure 1The SEM images of the Ppy-modifiedGCE
(a) OvOxPpy-film (b) and GNP|OvOxPpy||GCE (c) com-posite film were recorded at a magnification of 30 000 with ascale of 1120583mTheEDX spectrum (d) shows elemental analysisof a selected area on Figure 1(c) Figure 1(b) clearly showsthat overoxidation affects themorphological properties of thepolypyrrole film and as such the OvOxPpy-film exhibited astructural morphology that was significantly different fromthat of the conductive Ppy-film [22] The OvOxPpy-filmhad a typical ldquocauliflower-likerdquo structure constituted byspherical micrograins Prior research has established thatoveroxidation is initiated by hydroxyl radicals that form dueto the oxidation of water of which the overall process may bedescribed by [23]
Ppy + 119909HOminus 997888rarr OvOxPpy + 119899eminus
+ 119898H+ + sdot sdot sdot(1)
Figure 1(c) shows the porous OvOxPpy-film to be anexcellent matrix for the electrodeposition of gold nanoparti-cles which had good surface coverage across the film withuniform size and diameters of approximately 100 nm TheEDX spectrum confirms the presence of gold within theOvOxPpy matrix
During overoxidation a large current is passed whichdecreased as oxidation progressed eventually levelling offand fluctuating slightly around one value which indicatesthe completion of the overoxidation process as shown inFigure 2 During overoxidation nucleophilic attack of theradical cationic pyrrole units in the polymer by hydroxide
4 Journal of Chemistry
3002001000 400Time (sec)
minus300
minus250
minus200
minus150
minus100
minus50
I(120583
A)
Figure 2 Current time curve for polypyrrole overoxidation(OvOxPpy||GCE)
(a)
(b) (c)
minus150
minus100
minus50
0
50
100
150
I(120583
A)
minus1200 minus1000 minus800 minus600 minus400 minus200 0 200minus1400E (mV) versus AgAgCl (3M NaCl)
(a) Bare GCE in PBS
minus35
0
minus10
50minus
700
350
700
10500
(b) OvOxPpyGCE in PBS(c) GNP|OvOxPpyGCE in PBS
120583A OvOxPPyGCEEpa
GNP|OvOxPPyGCEEpc
GNP|OvOxPPyGCEEpa
OvOxPPyGCEEpc
Figure 3 Cyclic voltammogram of GCE (a) OvOxPpy||GCE(b) and GNP|OvOxPpy||GCE (c) in PBS (pH 74) at 70mVsversus AgAgCl (inset) SWV of OvOxPpy||GCE and theGNP|OvOxPpy||GCE composite film
ions takes place resulting in loss of the conjugated structureand electrical conductivity Expulsion of the ClO
4
minus dopantions results in the uniformly sized nanopores within thepolymer film
32 Electron Transfer Systems The modification of theglassy carbon electrode during the film assembly waselectrochemically characterized by cyclic voltammetry andsquare wave voltammetry From Figure 3 it is evidentthat the bare glassy carbon electrode shows no observableelectrochemistry whereas the background current of theGNP|OvOxPpy||GCE composite is two orders of magnitudehigher as compared to that of the overoxidized-polypyrrole
film Moreover the forward and reverse square waves in theinset exhibit a similar trend with regard to magnitude ofthe background current of GNP|OvOxPpy||GCE compositefilm as compared to that of the OvOxPpy||GCE film and assuch corroborate the findings shown in cyclic voltammetricresults
33 Effect of Cobalt(III) Sepulchrate Trichloride As statedin previous sections all of the electrochemical studies per-taining to the fabricated CYP3A4-based biosensor wereconducted in the presence of diffusional electron transfermediator Co(Sep)3+ The main requirement for a goodmediator is that it exhibits fast reversible electrochemistrywhile also showing a redox response sufficiently negativein order to in turn reduce the enzyme heme group Priorinvestigations have established Co(Sep)3+ as superior choiceto facilitate electrical communication between redox centresof P450 enzymes and the electrode [24] Figure 4(a) showsthe CV taken at 100mVs and SWV recorded at a frequencyof 5Hz This revealed that as the potential was scanned fromminus200 to minus800mV the reduction current steadily increased toa maximum value after which the current decreased untilthe switching potentialmdashcathodic and anodic peak currents(119868119901119888
and 119868119901119886) shown to be fairly similar in magnitude The
CV exhibited well defined corresponding cathodic (119864119901119888)
and anodic peaks (119864119901119886) for which peak potentials were
determined as minus650- (plusmn5) and minus589 (plusmn5)mV respectivelywith corresponding 119864∘1015840 determined as 620 plusmn 5mV Thepeak potential difference (Δ119864) determined from CV resultswas approximately 61 plusmn 5mV Square wave voltammetrywas used to corroborate CV results The SW in Figure 4(b)shows well defined forward (cathodic) and reverse (anodic)square waves with 119868
119901119888shown to be slightly higher than
119868119901119886 by approximately half an order of magnitude The
peak potentials corresponded to values of minus631 and minus620 plusmn5mV for cathodic and anodic square waves respectively Incomparing CV and SW results it is well known that squarewave voltammetry has a higher signal-to-noise ratio andas such differences are considered well within experimentalvariability
34 Monooxygenation of CYP3A4 Figure 5 shows the cyclicvoltammograms of the biosensor containing the immobilizedredox species at different scan rates varying from 10 to100mVs The inset shows the linear variation of anodic andcathodic peak currents (119868
119901119886and 119868119901119888) with square root of scan
rate (]12) On increasing the scan rates the biosensor CVpeak currents increased gradually and peak potentials stayedfairly stable concomitantly resulting in little change in peak-to-peak separation with each consecutive scan rate change119868119901119888
and 119868119901119886
also increase linearly with ]12 Based on theseresults it may be deduced that the CYP3A4-film was stableand exhibited reversible electrochemistry constituted by adiffusion-controlled electron transfer process The diffusioncoefficient 119863 calculated using the Randle Sevcik equation is25 times 10minus8 cm2 sminus1 where 119879 = 25∘C 119877 = 8314 Jmolminus1 Kminus1 119865 =
Journal of Chemistry 5
minus75
minus50
minus25
00
25
50
75
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus585 plusmn 5mVEpa
(plusmn) minus650 plusmn 5mVEpc
(a)
minus12
minus10
minus8
minus6
minus4
minus2
0
2
4
6
8
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus631mVEpa
(plusmn) minus620mVEpc
(b)
Figure 4 Cyclic (a) and square wave (SW) (b) voltammograms of CYP3A4-based biosensor in argon-saturated PBS (pH 74 100mM KCl)containing 1mM Co(Sep)3+ in solution The biosensor contained 163120583MCYP3A4
Anodic Ipa
Cathodic Ipc
10mV sminus1
100mV sminus1
minus800 minus600 minus400 minus200minus1000
minus8
minus6
minus4
minus2
0
2
4
6
I(120583
A) 4 6 8 102
12 (mV)12
minus8minus6minus4minus2
0246
I(120583
A)
E (mV) versus AgAgCl (3M NaCl)
Figure 5 Cyclic voltammograms of biosensor (CYP3A4|GNPs|OvOxPpy||GCE) in argon-saturated PBS (50mM 100mMKCl) containing 1mM CoSep3+
96486Cmolminus1 119862 = 1 times 10minus5mol cmminus3 119899 = 1 119860 = 0071 cm2and slope = 467 times 10minus3
119868119901
= 04463119899119865119860[119899119865
119877119879]
12
11986312]12119862analyte
(2)
Concomitantly the surface concentration Γlowast(mol cmminus2) of the adsorbed electroactive species maybe estimated accordingly
119868119901119886
]=11989921198652119860Γlowast
4119877119879 (3)
where 119877 119865 119879 and 119899 are the gas constant Faraday constanttemperature and number of electrons transferred respec-tivelyThe ion exchange behaviour of conducting polypyrroleis associated by a one-electron transfer process in which themonomers are oxidized by oxidizing agents or catalysts toproduce the radical polymer cationThus for the one electrontransfer process at 25∘C Γlowast value evaluated from the slope ofthe anodic peak is 699 times 10minus7mol cmminus2
35 Mechanism of Biosensor Response to Indinavir Voltam-metric responses of the biosensor were recorded at a scanrate of 10mVs in the absence and presence of oxygen(O2) andor Indinavir in order to observe enzyme-oxygen-
substrate interactions and are shown in Figures 6(a) 6(b)and 6(c) respectively In the presence of O
2 there was a
significant increase in 119868119901119888 This is a common phenomenon
which has been recorded in literature reports with regardto biosensor studies with P450 [25] O
2is the natural
cosubstrate of CYP3A4 and P450 enzymes in general andbinds to the ferrous ion of the enzyme heme group at avery fast rate therefore the distinctive 119868
119901119888increase can be
regarded as a chemical reaction which immediately followsFe3+Fe2+ electron transfer process a phenomenon which inthis case may be described as the electrocatalytic responseof the enzyme to O
2 The O
2-CYP3A4 interaction is greatly
enhanced in the presence of substrate and as such in thepresence of Indinavir (25 120583M) there is a strong amplificationin 119868119901119888
6 Journal of Chemistry
Biosensor in oxygen-saturated
Biosensor in oxygen-saturated
c
b
a Biosensor in argon-saturated
minus8
minus7
minus6
minus5
minus4
minus3
minus2
minus1
0
1
I(120583
A)
minus800 minus600 minus400 minus200minus1000
PBS (0120583M Indinavir)
PBS (0120583M Indinavir)
PBS (25 120583M Indinavir)
E (mV) versus AgAgCl (3M NaCl)
Figure 6 Biosensor in argon-saturated PBS (0 120583M Indinavir) (a)oxygen-saturated PBS (0 120583M Indinavir) (b) and oxygen-saturatedPBS containing 25120583M Indinavir (c)
Indinavir
minus10
minus8
minus6
minus4
minus2
0
I(120583
A)
minus800 minus600 minus400 minus200minus1000
02 120583M
10120583M minus10minus9minus8minus7minus6minus5minus4minus3minus2
I(120583
A)
04 06 08 1002Indinavir (120583M)
E (mV) versus AgAgCl (3M NaCl)
Figure 7 CYP3A4-based biosensor in the presence of increasingIndinavir concentrations (02 to 10 120583M) Inset cathodic peak currentversus Indinavir concentration
Figure 7 shows the voltammetric response of the biosen-sor in oxygen-saturated PBS (pH 74 100mM KCl) con-taining 1mM Co(Sep)3+ diffusional mediator at a scan rateof 10mVs The CVs reveal that the biosensor exhibits aconcentration-dependent increase in 119868
119901119888with each addition
of Indinavir up to the final concentration of 10 120583M Thesefindings directly correspond with previous investigations[26 27] and thus it may be deduced that the substrateIndinavir is metabolised by the reduced CYP3A4 which inturn is mediated by the reduced Co(Sep)2+ The increasingrate of dioxygen to the heme in the presence of substrate isexemplified by the increase in the reduction current (119868
119901119888) as
shown in the cyclic voltammograms The response profile ofthe biosensor as shown by CV is typical of cytochrome P450enzyme in particular and heme-enzyme in general therebyadequately proving the strong catalytic effect andmetabolism
of Indinavir by CYP3A4 These favourable features of themodified electrode offer great promise for its sensing appli-cations The inset shows that the catalytic current is linearversus Indinavir concentration in the range of 02ndash10 120583Manda correlation coefficient of 09908Thedetection limit (signal-to-noise ratio is 3) is 0001120583mol Lminus1 The detection limit iswell below the plasma concentration of Indinavir (8 h afterintake) which ranges from 013 to 86mg Lminus1
4 Conclusions
An ideal nanocomposite support material consisting of GNPinfused OvOxPpy was successfully constructed based ona rapid electrochemical technique The obtained platformserved as an ideal host for enzyme immobilization for thedevelopment of a user-friendly and low-cost biosensor forIndinavir The results demonstrated that Au nanoparticlesenhanced the catalytic activity ofOvOxPpy by giving negativeshifts of CV peak potentials and increasing peak current ofthe enzyme Due to the synergy of OvOxPpy and the GNPsGNP|OvOxPpy||GCE had good stability sensitivity andselectivity to determine Indinavir in a large presence of PBS(pH 721) The biosensor response to Indinavir in oxygenatedPBS also showed significant increase GNP|OvOxPpy||GCEdisplayed strong catalytic activity towards the oxidation ofIndinavir and reveals the response of the active specieswith distinctive voltammetric peaks Low concentrationsrsquodetection of Indinavir demonstrates the ability of biosensorplatform to perform effective point-of-care diagnostics Thebiosensor constructionmethod developed in this study holdsgreat promise for ARV drugsrsquo (Indinavir) determination inreal samples and use for effective therapeutic drug monitor-ing in real time
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This research was financially supported by South AfricarsquosNational Research Foundation (NRF)
References
[1] N A Thompson ldquoLatin Postrdquo 2015 httpwwwlatinpostcomarticles47066
[2] H F Gunthard J A Aberg J J Eron et al ldquoAntiretroviraltreatment of adult HIV infection 2014 recommendations of theInternational Antiviral Society-USA panelrdquo The Journal of theAmericanMedical Association vol 312 no 4 pp 410ndash425 2014
[3] H Namme Luma M-S Doualla S-P Choukem et alldquoAdverse drug reactions ofHighlyActiveAntiretroviralTherapy(HAART) in HIV infected patients at the General HospitalDouala Cameroon a cross sectional studyrdquo The Pan AfricanMedical Journal vol 12 p 87 2012
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 3
C
Cu
Cu
Cu
CuCu Au
Au
Au
AuAu
AuAuAu
Au
AuAu
AuOK
K
EDX acquiring HAADF area 1
(a) (b) (c)
(d)
20
40
60
10 15 205Energy (keV)
Figure 1 SEM of Ppy||GCE (a) OvOxPpy||GCE (b) and GNP|OxPpy||GCE (c) respectively at 30 000Mag (scale = 1 120583m) with the EDXspectrum (d)
fitting lid for the first 10min to form a uniform layer afterwhich the lid was removed and the enzyme layer was slightlydried for another 40min but still retaining wet camera Thiswas then followed by the deposition of 4 120583L agarose (1wv) after which the prepared biosensor was immediatelyplaced at 4∘C for at least 1 h The biosensor was denoted byCYP3A4|GNP|OvOxPpy||GCE
24 Electrochemical Procedures All voltammetric analysiswas done at 25∘C in the presence of 1mM Co(Sep)3+ diffu-sional electron transfermediator dissolved in either degassedor undegassed PBS (pH 74) Cyclic voltammograms wererecorded in the presence and absence of oxygen andorIndinavir at a potential scan rate (]) of 10mVs versusAgAgCl from an initial potential 119864
119894= minus200mV to a switch
potential 119864120582= minus1000mV Oysteryoung-type square wave
voltammetry (SWV) was done at an amplitude of 75mV andfrequency of 5Hz The analysis of the CVs gave the formalpotentialsmeasured as themidpoint potential (119864∘1015840)The peakseparation Δ119864
119901(the difference between 119864
119901119888and 119864
119901119886) and
the anodic to cathodic peak current ratio (119868119901119886119868119901119888) of the
biosensor responses were also determined
3 Results and Discussion
31 Structure of Composite Polypyrrole Materials Morpho-logical characterization of the electrode platform during elec-trode assembly process was conducted by SEM analysis asshown in Figure 1The SEM images of the Ppy-modifiedGCE
(a) OvOxPpy-film (b) and GNP|OvOxPpy||GCE (c) com-posite film were recorded at a magnification of 30 000 with ascale of 1120583mTheEDX spectrum (d) shows elemental analysisof a selected area on Figure 1(c) Figure 1(b) clearly showsthat overoxidation affects themorphological properties of thepolypyrrole film and as such the OvOxPpy-film exhibited astructural morphology that was significantly different fromthat of the conductive Ppy-film [22] The OvOxPpy-filmhad a typical ldquocauliflower-likerdquo structure constituted byspherical micrograins Prior research has established thatoveroxidation is initiated by hydroxyl radicals that form dueto the oxidation of water of which the overall process may bedescribed by [23]
Ppy + 119909HOminus 997888rarr OvOxPpy + 119899eminus
+ 119898H+ + sdot sdot sdot(1)
Figure 1(c) shows the porous OvOxPpy-film to be anexcellent matrix for the electrodeposition of gold nanoparti-cles which had good surface coverage across the film withuniform size and diameters of approximately 100 nm TheEDX spectrum confirms the presence of gold within theOvOxPpy matrix
During overoxidation a large current is passed whichdecreased as oxidation progressed eventually levelling offand fluctuating slightly around one value which indicatesthe completion of the overoxidation process as shown inFigure 2 During overoxidation nucleophilic attack of theradical cationic pyrrole units in the polymer by hydroxide
4 Journal of Chemistry
3002001000 400Time (sec)
minus300
minus250
minus200
minus150
minus100
minus50
I(120583
A)
Figure 2 Current time curve for polypyrrole overoxidation(OvOxPpy||GCE)
(a)
(b) (c)
minus150
minus100
minus50
0
50
100
150
I(120583
A)
minus1200 minus1000 minus800 minus600 minus400 minus200 0 200minus1400E (mV) versus AgAgCl (3M NaCl)
(a) Bare GCE in PBS
minus35
0
minus10
50minus
700
350
700
10500
(b) OvOxPpyGCE in PBS(c) GNP|OvOxPpyGCE in PBS
120583A OvOxPPyGCEEpa
GNP|OvOxPPyGCEEpc
GNP|OvOxPPyGCEEpa
OvOxPPyGCEEpc
Figure 3 Cyclic voltammogram of GCE (a) OvOxPpy||GCE(b) and GNP|OvOxPpy||GCE (c) in PBS (pH 74) at 70mVsversus AgAgCl (inset) SWV of OvOxPpy||GCE and theGNP|OvOxPpy||GCE composite film
ions takes place resulting in loss of the conjugated structureand electrical conductivity Expulsion of the ClO
4
minus dopantions results in the uniformly sized nanopores within thepolymer film
32 Electron Transfer Systems The modification of theglassy carbon electrode during the film assembly waselectrochemically characterized by cyclic voltammetry andsquare wave voltammetry From Figure 3 it is evidentthat the bare glassy carbon electrode shows no observableelectrochemistry whereas the background current of theGNP|OvOxPpy||GCE composite is two orders of magnitudehigher as compared to that of the overoxidized-polypyrrole
film Moreover the forward and reverse square waves in theinset exhibit a similar trend with regard to magnitude ofthe background current of GNP|OvOxPpy||GCE compositefilm as compared to that of the OvOxPpy||GCE film and assuch corroborate the findings shown in cyclic voltammetricresults
33 Effect of Cobalt(III) Sepulchrate Trichloride As statedin previous sections all of the electrochemical studies per-taining to the fabricated CYP3A4-based biosensor wereconducted in the presence of diffusional electron transfermediator Co(Sep)3+ The main requirement for a goodmediator is that it exhibits fast reversible electrochemistrywhile also showing a redox response sufficiently negativein order to in turn reduce the enzyme heme group Priorinvestigations have established Co(Sep)3+ as superior choiceto facilitate electrical communication between redox centresof P450 enzymes and the electrode [24] Figure 4(a) showsthe CV taken at 100mVs and SWV recorded at a frequencyof 5Hz This revealed that as the potential was scanned fromminus200 to minus800mV the reduction current steadily increased toa maximum value after which the current decreased untilthe switching potentialmdashcathodic and anodic peak currents(119868119901119888
and 119868119901119886) shown to be fairly similar in magnitude The
CV exhibited well defined corresponding cathodic (119864119901119888)
and anodic peaks (119864119901119886) for which peak potentials were
determined as minus650- (plusmn5) and minus589 (plusmn5)mV respectivelywith corresponding 119864∘1015840 determined as 620 plusmn 5mV Thepeak potential difference (Δ119864) determined from CV resultswas approximately 61 plusmn 5mV Square wave voltammetrywas used to corroborate CV results The SW in Figure 4(b)shows well defined forward (cathodic) and reverse (anodic)square waves with 119868
119901119888shown to be slightly higher than
119868119901119886 by approximately half an order of magnitude The
peak potentials corresponded to values of minus631 and minus620 plusmn5mV for cathodic and anodic square waves respectively Incomparing CV and SW results it is well known that squarewave voltammetry has a higher signal-to-noise ratio andas such differences are considered well within experimentalvariability
34 Monooxygenation of CYP3A4 Figure 5 shows the cyclicvoltammograms of the biosensor containing the immobilizedredox species at different scan rates varying from 10 to100mVs The inset shows the linear variation of anodic andcathodic peak currents (119868
119901119886and 119868119901119888) with square root of scan
rate (]12) On increasing the scan rates the biosensor CVpeak currents increased gradually and peak potentials stayedfairly stable concomitantly resulting in little change in peak-to-peak separation with each consecutive scan rate change119868119901119888
and 119868119901119886
also increase linearly with ]12 Based on theseresults it may be deduced that the CYP3A4-film was stableand exhibited reversible electrochemistry constituted by adiffusion-controlled electron transfer process The diffusioncoefficient 119863 calculated using the Randle Sevcik equation is25 times 10minus8 cm2 sminus1 where 119879 = 25∘C 119877 = 8314 Jmolminus1 Kminus1 119865 =
Journal of Chemistry 5
minus75
minus50
minus25
00
25
50
75
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus585 plusmn 5mVEpa
(plusmn) minus650 plusmn 5mVEpc
(a)
minus12
minus10
minus8
minus6
minus4
minus2
0
2
4
6
8
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus631mVEpa
(plusmn) minus620mVEpc
(b)
Figure 4 Cyclic (a) and square wave (SW) (b) voltammograms of CYP3A4-based biosensor in argon-saturated PBS (pH 74 100mM KCl)containing 1mM Co(Sep)3+ in solution The biosensor contained 163120583MCYP3A4
Anodic Ipa
Cathodic Ipc
10mV sminus1
100mV sminus1
minus800 minus600 minus400 minus200minus1000
minus8
minus6
minus4
minus2
0
2
4
6
I(120583
A) 4 6 8 102
12 (mV)12
minus8minus6minus4minus2
0246
I(120583
A)
E (mV) versus AgAgCl (3M NaCl)
Figure 5 Cyclic voltammograms of biosensor (CYP3A4|GNPs|OvOxPpy||GCE) in argon-saturated PBS (50mM 100mMKCl) containing 1mM CoSep3+
96486Cmolminus1 119862 = 1 times 10minus5mol cmminus3 119899 = 1 119860 = 0071 cm2and slope = 467 times 10minus3
119868119901
= 04463119899119865119860[119899119865
119877119879]
12
11986312]12119862analyte
(2)
Concomitantly the surface concentration Γlowast(mol cmminus2) of the adsorbed electroactive species maybe estimated accordingly
119868119901119886
]=11989921198652119860Γlowast
4119877119879 (3)
where 119877 119865 119879 and 119899 are the gas constant Faraday constanttemperature and number of electrons transferred respec-tivelyThe ion exchange behaviour of conducting polypyrroleis associated by a one-electron transfer process in which themonomers are oxidized by oxidizing agents or catalysts toproduce the radical polymer cationThus for the one electrontransfer process at 25∘C Γlowast value evaluated from the slope ofthe anodic peak is 699 times 10minus7mol cmminus2
35 Mechanism of Biosensor Response to Indinavir Voltam-metric responses of the biosensor were recorded at a scanrate of 10mVs in the absence and presence of oxygen(O2) andor Indinavir in order to observe enzyme-oxygen-
substrate interactions and are shown in Figures 6(a) 6(b)and 6(c) respectively In the presence of O
2 there was a
significant increase in 119868119901119888 This is a common phenomenon
which has been recorded in literature reports with regardto biosensor studies with P450 [25] O
2is the natural
cosubstrate of CYP3A4 and P450 enzymes in general andbinds to the ferrous ion of the enzyme heme group at avery fast rate therefore the distinctive 119868
119901119888increase can be
regarded as a chemical reaction which immediately followsFe3+Fe2+ electron transfer process a phenomenon which inthis case may be described as the electrocatalytic responseof the enzyme to O
2 The O
2-CYP3A4 interaction is greatly
enhanced in the presence of substrate and as such in thepresence of Indinavir (25 120583M) there is a strong amplificationin 119868119901119888
6 Journal of Chemistry
Biosensor in oxygen-saturated
Biosensor in oxygen-saturated
c
b
a Biosensor in argon-saturated
minus8
minus7
minus6
minus5
minus4
minus3
minus2
minus1
0
1
I(120583
A)
minus800 minus600 minus400 minus200minus1000
PBS (0120583M Indinavir)
PBS (0120583M Indinavir)
PBS (25 120583M Indinavir)
E (mV) versus AgAgCl (3M NaCl)
Figure 6 Biosensor in argon-saturated PBS (0 120583M Indinavir) (a)oxygen-saturated PBS (0 120583M Indinavir) (b) and oxygen-saturatedPBS containing 25120583M Indinavir (c)
Indinavir
minus10
minus8
minus6
minus4
minus2
0
I(120583
A)
minus800 minus600 minus400 minus200minus1000
02 120583M
10120583M minus10minus9minus8minus7minus6minus5minus4minus3minus2
I(120583
A)
04 06 08 1002Indinavir (120583M)
E (mV) versus AgAgCl (3M NaCl)
Figure 7 CYP3A4-based biosensor in the presence of increasingIndinavir concentrations (02 to 10 120583M) Inset cathodic peak currentversus Indinavir concentration
Figure 7 shows the voltammetric response of the biosen-sor in oxygen-saturated PBS (pH 74 100mM KCl) con-taining 1mM Co(Sep)3+ diffusional mediator at a scan rateof 10mVs The CVs reveal that the biosensor exhibits aconcentration-dependent increase in 119868
119901119888with each addition
of Indinavir up to the final concentration of 10 120583M Thesefindings directly correspond with previous investigations[26 27] and thus it may be deduced that the substrateIndinavir is metabolised by the reduced CYP3A4 which inturn is mediated by the reduced Co(Sep)2+ The increasingrate of dioxygen to the heme in the presence of substrate isexemplified by the increase in the reduction current (119868
119901119888) as
shown in the cyclic voltammograms The response profile ofthe biosensor as shown by CV is typical of cytochrome P450enzyme in particular and heme-enzyme in general therebyadequately proving the strong catalytic effect andmetabolism
of Indinavir by CYP3A4 These favourable features of themodified electrode offer great promise for its sensing appli-cations The inset shows that the catalytic current is linearversus Indinavir concentration in the range of 02ndash10 120583Manda correlation coefficient of 09908Thedetection limit (signal-to-noise ratio is 3) is 0001120583mol Lminus1 The detection limit iswell below the plasma concentration of Indinavir (8 h afterintake) which ranges from 013 to 86mg Lminus1
4 Conclusions
An ideal nanocomposite support material consisting of GNPinfused OvOxPpy was successfully constructed based ona rapid electrochemical technique The obtained platformserved as an ideal host for enzyme immobilization for thedevelopment of a user-friendly and low-cost biosensor forIndinavir The results demonstrated that Au nanoparticlesenhanced the catalytic activity ofOvOxPpy by giving negativeshifts of CV peak potentials and increasing peak current ofthe enzyme Due to the synergy of OvOxPpy and the GNPsGNP|OvOxPpy||GCE had good stability sensitivity andselectivity to determine Indinavir in a large presence of PBS(pH 721) The biosensor response to Indinavir in oxygenatedPBS also showed significant increase GNP|OvOxPpy||GCEdisplayed strong catalytic activity towards the oxidation ofIndinavir and reveals the response of the active specieswith distinctive voltammetric peaks Low concentrationsrsquodetection of Indinavir demonstrates the ability of biosensorplatform to perform effective point-of-care diagnostics Thebiosensor constructionmethod developed in this study holdsgreat promise for ARV drugsrsquo (Indinavir) determination inreal samples and use for effective therapeutic drug monitor-ing in real time
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This research was financially supported by South AfricarsquosNational Research Foundation (NRF)
References
[1] N A Thompson ldquoLatin Postrdquo 2015 httpwwwlatinpostcomarticles47066
[2] H F Gunthard J A Aberg J J Eron et al ldquoAntiretroviraltreatment of adult HIV infection 2014 recommendations of theInternational Antiviral Society-USA panelrdquo The Journal of theAmericanMedical Association vol 312 no 4 pp 410ndash425 2014
[3] H Namme Luma M-S Doualla S-P Choukem et alldquoAdverse drug reactions ofHighlyActiveAntiretroviralTherapy(HAART) in HIV infected patients at the General HospitalDouala Cameroon a cross sectional studyrdquo The Pan AfricanMedical Journal vol 12 p 87 2012
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
4 Journal of Chemistry
3002001000 400Time (sec)
minus300
minus250
minus200
minus150
minus100
minus50
I(120583
A)
Figure 2 Current time curve for polypyrrole overoxidation(OvOxPpy||GCE)
(a)
(b) (c)
minus150
minus100
minus50
0
50
100
150
I(120583
A)
minus1200 minus1000 minus800 minus600 minus400 minus200 0 200minus1400E (mV) versus AgAgCl (3M NaCl)
(a) Bare GCE in PBS
minus35
0
minus10
50minus
700
350
700
10500
(b) OvOxPpyGCE in PBS(c) GNP|OvOxPpyGCE in PBS
120583A OvOxPPyGCEEpa
GNP|OvOxPPyGCEEpc
GNP|OvOxPPyGCEEpa
OvOxPPyGCEEpc
Figure 3 Cyclic voltammogram of GCE (a) OvOxPpy||GCE(b) and GNP|OvOxPpy||GCE (c) in PBS (pH 74) at 70mVsversus AgAgCl (inset) SWV of OvOxPpy||GCE and theGNP|OvOxPpy||GCE composite film
ions takes place resulting in loss of the conjugated structureand electrical conductivity Expulsion of the ClO
4
minus dopantions results in the uniformly sized nanopores within thepolymer film
32 Electron Transfer Systems The modification of theglassy carbon electrode during the film assembly waselectrochemically characterized by cyclic voltammetry andsquare wave voltammetry From Figure 3 it is evidentthat the bare glassy carbon electrode shows no observableelectrochemistry whereas the background current of theGNP|OvOxPpy||GCE composite is two orders of magnitudehigher as compared to that of the overoxidized-polypyrrole
film Moreover the forward and reverse square waves in theinset exhibit a similar trend with regard to magnitude ofthe background current of GNP|OvOxPpy||GCE compositefilm as compared to that of the OvOxPpy||GCE film and assuch corroborate the findings shown in cyclic voltammetricresults
33 Effect of Cobalt(III) Sepulchrate Trichloride As statedin previous sections all of the electrochemical studies per-taining to the fabricated CYP3A4-based biosensor wereconducted in the presence of diffusional electron transfermediator Co(Sep)3+ The main requirement for a goodmediator is that it exhibits fast reversible electrochemistrywhile also showing a redox response sufficiently negativein order to in turn reduce the enzyme heme group Priorinvestigations have established Co(Sep)3+ as superior choiceto facilitate electrical communication between redox centresof P450 enzymes and the electrode [24] Figure 4(a) showsthe CV taken at 100mVs and SWV recorded at a frequencyof 5Hz This revealed that as the potential was scanned fromminus200 to minus800mV the reduction current steadily increased toa maximum value after which the current decreased untilthe switching potentialmdashcathodic and anodic peak currents(119868119901119888
and 119868119901119886) shown to be fairly similar in magnitude The
CV exhibited well defined corresponding cathodic (119864119901119888)
and anodic peaks (119864119901119886) for which peak potentials were
determined as minus650- (plusmn5) and minus589 (plusmn5)mV respectivelywith corresponding 119864∘1015840 determined as 620 plusmn 5mV Thepeak potential difference (Δ119864) determined from CV resultswas approximately 61 plusmn 5mV Square wave voltammetrywas used to corroborate CV results The SW in Figure 4(b)shows well defined forward (cathodic) and reverse (anodic)square waves with 119868
119901119888shown to be slightly higher than
119868119901119886 by approximately half an order of magnitude The
peak potentials corresponded to values of minus631 and minus620 plusmn5mV for cathodic and anodic square waves respectively Incomparing CV and SW results it is well known that squarewave voltammetry has a higher signal-to-noise ratio andas such differences are considered well within experimentalvariability
34 Monooxygenation of CYP3A4 Figure 5 shows the cyclicvoltammograms of the biosensor containing the immobilizedredox species at different scan rates varying from 10 to100mVs The inset shows the linear variation of anodic andcathodic peak currents (119868
119901119886and 119868119901119888) with square root of scan
rate (]12) On increasing the scan rates the biosensor CVpeak currents increased gradually and peak potentials stayedfairly stable concomitantly resulting in little change in peak-to-peak separation with each consecutive scan rate change119868119901119888
and 119868119901119886
also increase linearly with ]12 Based on theseresults it may be deduced that the CYP3A4-film was stableand exhibited reversible electrochemistry constituted by adiffusion-controlled electron transfer process The diffusioncoefficient 119863 calculated using the Randle Sevcik equation is25 times 10minus8 cm2 sminus1 where 119879 = 25∘C 119877 = 8314 Jmolminus1 Kminus1 119865 =
Journal of Chemistry 5
minus75
minus50
minus25
00
25
50
75
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus585 plusmn 5mVEpa
(plusmn) minus650 plusmn 5mVEpc
(a)
minus12
minus10
minus8
minus6
minus4
minus2
0
2
4
6
8
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus631mVEpa
(plusmn) minus620mVEpc
(b)
Figure 4 Cyclic (a) and square wave (SW) (b) voltammograms of CYP3A4-based biosensor in argon-saturated PBS (pH 74 100mM KCl)containing 1mM Co(Sep)3+ in solution The biosensor contained 163120583MCYP3A4
Anodic Ipa
Cathodic Ipc
10mV sminus1
100mV sminus1
minus800 minus600 minus400 minus200minus1000
minus8
minus6
minus4
minus2
0
2
4
6
I(120583
A) 4 6 8 102
12 (mV)12
minus8minus6minus4minus2
0246
I(120583
A)
E (mV) versus AgAgCl (3M NaCl)
Figure 5 Cyclic voltammograms of biosensor (CYP3A4|GNPs|OvOxPpy||GCE) in argon-saturated PBS (50mM 100mMKCl) containing 1mM CoSep3+
96486Cmolminus1 119862 = 1 times 10minus5mol cmminus3 119899 = 1 119860 = 0071 cm2and slope = 467 times 10minus3
119868119901
= 04463119899119865119860[119899119865
119877119879]
12
11986312]12119862analyte
(2)
Concomitantly the surface concentration Γlowast(mol cmminus2) of the adsorbed electroactive species maybe estimated accordingly
119868119901119886
]=11989921198652119860Γlowast
4119877119879 (3)
where 119877 119865 119879 and 119899 are the gas constant Faraday constanttemperature and number of electrons transferred respec-tivelyThe ion exchange behaviour of conducting polypyrroleis associated by a one-electron transfer process in which themonomers are oxidized by oxidizing agents or catalysts toproduce the radical polymer cationThus for the one electrontransfer process at 25∘C Γlowast value evaluated from the slope ofthe anodic peak is 699 times 10minus7mol cmminus2
35 Mechanism of Biosensor Response to Indinavir Voltam-metric responses of the biosensor were recorded at a scanrate of 10mVs in the absence and presence of oxygen(O2) andor Indinavir in order to observe enzyme-oxygen-
substrate interactions and are shown in Figures 6(a) 6(b)and 6(c) respectively In the presence of O
2 there was a
significant increase in 119868119901119888 This is a common phenomenon
which has been recorded in literature reports with regardto biosensor studies with P450 [25] O
2is the natural
cosubstrate of CYP3A4 and P450 enzymes in general andbinds to the ferrous ion of the enzyme heme group at avery fast rate therefore the distinctive 119868
119901119888increase can be
regarded as a chemical reaction which immediately followsFe3+Fe2+ electron transfer process a phenomenon which inthis case may be described as the electrocatalytic responseof the enzyme to O
2 The O
2-CYP3A4 interaction is greatly
enhanced in the presence of substrate and as such in thepresence of Indinavir (25 120583M) there is a strong amplificationin 119868119901119888
6 Journal of Chemistry
Biosensor in oxygen-saturated
Biosensor in oxygen-saturated
c
b
a Biosensor in argon-saturated
minus8
minus7
minus6
minus5
minus4
minus3
minus2
minus1
0
1
I(120583
A)
minus800 minus600 minus400 minus200minus1000
PBS (0120583M Indinavir)
PBS (0120583M Indinavir)
PBS (25 120583M Indinavir)
E (mV) versus AgAgCl (3M NaCl)
Figure 6 Biosensor in argon-saturated PBS (0 120583M Indinavir) (a)oxygen-saturated PBS (0 120583M Indinavir) (b) and oxygen-saturatedPBS containing 25120583M Indinavir (c)
Indinavir
minus10
minus8
minus6
minus4
minus2
0
I(120583
A)
minus800 minus600 minus400 minus200minus1000
02 120583M
10120583M minus10minus9minus8minus7minus6minus5minus4minus3minus2
I(120583
A)
04 06 08 1002Indinavir (120583M)
E (mV) versus AgAgCl (3M NaCl)
Figure 7 CYP3A4-based biosensor in the presence of increasingIndinavir concentrations (02 to 10 120583M) Inset cathodic peak currentversus Indinavir concentration
Figure 7 shows the voltammetric response of the biosen-sor in oxygen-saturated PBS (pH 74 100mM KCl) con-taining 1mM Co(Sep)3+ diffusional mediator at a scan rateof 10mVs The CVs reveal that the biosensor exhibits aconcentration-dependent increase in 119868
119901119888with each addition
of Indinavir up to the final concentration of 10 120583M Thesefindings directly correspond with previous investigations[26 27] and thus it may be deduced that the substrateIndinavir is metabolised by the reduced CYP3A4 which inturn is mediated by the reduced Co(Sep)2+ The increasingrate of dioxygen to the heme in the presence of substrate isexemplified by the increase in the reduction current (119868
119901119888) as
shown in the cyclic voltammograms The response profile ofthe biosensor as shown by CV is typical of cytochrome P450enzyme in particular and heme-enzyme in general therebyadequately proving the strong catalytic effect andmetabolism
of Indinavir by CYP3A4 These favourable features of themodified electrode offer great promise for its sensing appli-cations The inset shows that the catalytic current is linearversus Indinavir concentration in the range of 02ndash10 120583Manda correlation coefficient of 09908Thedetection limit (signal-to-noise ratio is 3) is 0001120583mol Lminus1 The detection limit iswell below the plasma concentration of Indinavir (8 h afterintake) which ranges from 013 to 86mg Lminus1
4 Conclusions
An ideal nanocomposite support material consisting of GNPinfused OvOxPpy was successfully constructed based ona rapid electrochemical technique The obtained platformserved as an ideal host for enzyme immobilization for thedevelopment of a user-friendly and low-cost biosensor forIndinavir The results demonstrated that Au nanoparticlesenhanced the catalytic activity ofOvOxPpy by giving negativeshifts of CV peak potentials and increasing peak current ofthe enzyme Due to the synergy of OvOxPpy and the GNPsGNP|OvOxPpy||GCE had good stability sensitivity andselectivity to determine Indinavir in a large presence of PBS(pH 721) The biosensor response to Indinavir in oxygenatedPBS also showed significant increase GNP|OvOxPpy||GCEdisplayed strong catalytic activity towards the oxidation ofIndinavir and reveals the response of the active specieswith distinctive voltammetric peaks Low concentrationsrsquodetection of Indinavir demonstrates the ability of biosensorplatform to perform effective point-of-care diagnostics Thebiosensor constructionmethod developed in this study holdsgreat promise for ARV drugsrsquo (Indinavir) determination inreal samples and use for effective therapeutic drug monitor-ing in real time
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This research was financially supported by South AfricarsquosNational Research Foundation (NRF)
References
[1] N A Thompson ldquoLatin Postrdquo 2015 httpwwwlatinpostcomarticles47066
[2] H F Gunthard J A Aberg J J Eron et al ldquoAntiretroviraltreatment of adult HIV infection 2014 recommendations of theInternational Antiviral Society-USA panelrdquo The Journal of theAmericanMedical Association vol 312 no 4 pp 410ndash425 2014
[3] H Namme Luma M-S Doualla S-P Choukem et alldquoAdverse drug reactions ofHighlyActiveAntiretroviralTherapy(HAART) in HIV infected patients at the General HospitalDouala Cameroon a cross sectional studyrdquo The Pan AfricanMedical Journal vol 12 p 87 2012
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 5
minus75
minus50
minus25
00
25
50
75
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus585 plusmn 5mVEpa
(plusmn) minus650 plusmn 5mVEpc
(a)
minus12
minus10
minus8
minus6
minus4
minus2
0
2
4
6
8
I(120583
A)
minus800 minus600 minus400 minus200minus1000E (mV) versus AgAgCl (3M NaCl)
(plusmn) minus631mVEpa
(plusmn) minus620mVEpc
(b)
Figure 4 Cyclic (a) and square wave (SW) (b) voltammograms of CYP3A4-based biosensor in argon-saturated PBS (pH 74 100mM KCl)containing 1mM Co(Sep)3+ in solution The biosensor contained 163120583MCYP3A4
Anodic Ipa
Cathodic Ipc
10mV sminus1
100mV sminus1
minus800 minus600 minus400 minus200minus1000
minus8
minus6
minus4
minus2
0
2
4
6
I(120583
A) 4 6 8 102
12 (mV)12
minus8minus6minus4minus2
0246
I(120583
A)
E (mV) versus AgAgCl (3M NaCl)
Figure 5 Cyclic voltammograms of biosensor (CYP3A4|GNPs|OvOxPpy||GCE) in argon-saturated PBS (50mM 100mMKCl) containing 1mM CoSep3+
96486Cmolminus1 119862 = 1 times 10minus5mol cmminus3 119899 = 1 119860 = 0071 cm2and slope = 467 times 10minus3
119868119901
= 04463119899119865119860[119899119865
119877119879]
12
11986312]12119862analyte
(2)
Concomitantly the surface concentration Γlowast(mol cmminus2) of the adsorbed electroactive species maybe estimated accordingly
119868119901119886
]=11989921198652119860Γlowast
4119877119879 (3)
where 119877 119865 119879 and 119899 are the gas constant Faraday constanttemperature and number of electrons transferred respec-tivelyThe ion exchange behaviour of conducting polypyrroleis associated by a one-electron transfer process in which themonomers are oxidized by oxidizing agents or catalysts toproduce the radical polymer cationThus for the one electrontransfer process at 25∘C Γlowast value evaluated from the slope ofthe anodic peak is 699 times 10minus7mol cmminus2
35 Mechanism of Biosensor Response to Indinavir Voltam-metric responses of the biosensor were recorded at a scanrate of 10mVs in the absence and presence of oxygen(O2) andor Indinavir in order to observe enzyme-oxygen-
substrate interactions and are shown in Figures 6(a) 6(b)and 6(c) respectively In the presence of O
2 there was a
significant increase in 119868119901119888 This is a common phenomenon
which has been recorded in literature reports with regardto biosensor studies with P450 [25] O
2is the natural
cosubstrate of CYP3A4 and P450 enzymes in general andbinds to the ferrous ion of the enzyme heme group at avery fast rate therefore the distinctive 119868
119901119888increase can be
regarded as a chemical reaction which immediately followsFe3+Fe2+ electron transfer process a phenomenon which inthis case may be described as the electrocatalytic responseof the enzyme to O
2 The O
2-CYP3A4 interaction is greatly
enhanced in the presence of substrate and as such in thepresence of Indinavir (25 120583M) there is a strong amplificationin 119868119901119888
6 Journal of Chemistry
Biosensor in oxygen-saturated
Biosensor in oxygen-saturated
c
b
a Biosensor in argon-saturated
minus8
minus7
minus6
minus5
minus4
minus3
minus2
minus1
0
1
I(120583
A)
minus800 minus600 minus400 minus200minus1000
PBS (0120583M Indinavir)
PBS (0120583M Indinavir)
PBS (25 120583M Indinavir)
E (mV) versus AgAgCl (3M NaCl)
Figure 6 Biosensor in argon-saturated PBS (0 120583M Indinavir) (a)oxygen-saturated PBS (0 120583M Indinavir) (b) and oxygen-saturatedPBS containing 25120583M Indinavir (c)
Indinavir
minus10
minus8
minus6
minus4
minus2
0
I(120583
A)
minus800 minus600 minus400 minus200minus1000
02 120583M
10120583M minus10minus9minus8minus7minus6minus5minus4minus3minus2
I(120583
A)
04 06 08 1002Indinavir (120583M)
E (mV) versus AgAgCl (3M NaCl)
Figure 7 CYP3A4-based biosensor in the presence of increasingIndinavir concentrations (02 to 10 120583M) Inset cathodic peak currentversus Indinavir concentration
Figure 7 shows the voltammetric response of the biosen-sor in oxygen-saturated PBS (pH 74 100mM KCl) con-taining 1mM Co(Sep)3+ diffusional mediator at a scan rateof 10mVs The CVs reveal that the biosensor exhibits aconcentration-dependent increase in 119868
119901119888with each addition
of Indinavir up to the final concentration of 10 120583M Thesefindings directly correspond with previous investigations[26 27] and thus it may be deduced that the substrateIndinavir is metabolised by the reduced CYP3A4 which inturn is mediated by the reduced Co(Sep)2+ The increasingrate of dioxygen to the heme in the presence of substrate isexemplified by the increase in the reduction current (119868
119901119888) as
shown in the cyclic voltammograms The response profile ofthe biosensor as shown by CV is typical of cytochrome P450enzyme in particular and heme-enzyme in general therebyadequately proving the strong catalytic effect andmetabolism
of Indinavir by CYP3A4 These favourable features of themodified electrode offer great promise for its sensing appli-cations The inset shows that the catalytic current is linearversus Indinavir concentration in the range of 02ndash10 120583Manda correlation coefficient of 09908Thedetection limit (signal-to-noise ratio is 3) is 0001120583mol Lminus1 The detection limit iswell below the plasma concentration of Indinavir (8 h afterintake) which ranges from 013 to 86mg Lminus1
4 Conclusions
An ideal nanocomposite support material consisting of GNPinfused OvOxPpy was successfully constructed based ona rapid electrochemical technique The obtained platformserved as an ideal host for enzyme immobilization for thedevelopment of a user-friendly and low-cost biosensor forIndinavir The results demonstrated that Au nanoparticlesenhanced the catalytic activity ofOvOxPpy by giving negativeshifts of CV peak potentials and increasing peak current ofthe enzyme Due to the synergy of OvOxPpy and the GNPsGNP|OvOxPpy||GCE had good stability sensitivity andselectivity to determine Indinavir in a large presence of PBS(pH 721) The biosensor response to Indinavir in oxygenatedPBS also showed significant increase GNP|OvOxPpy||GCEdisplayed strong catalytic activity towards the oxidation ofIndinavir and reveals the response of the active specieswith distinctive voltammetric peaks Low concentrationsrsquodetection of Indinavir demonstrates the ability of biosensorplatform to perform effective point-of-care diagnostics Thebiosensor constructionmethod developed in this study holdsgreat promise for ARV drugsrsquo (Indinavir) determination inreal samples and use for effective therapeutic drug monitor-ing in real time
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This research was financially supported by South AfricarsquosNational Research Foundation (NRF)
References
[1] N A Thompson ldquoLatin Postrdquo 2015 httpwwwlatinpostcomarticles47066
[2] H F Gunthard J A Aberg J J Eron et al ldquoAntiretroviraltreatment of adult HIV infection 2014 recommendations of theInternational Antiviral Society-USA panelrdquo The Journal of theAmericanMedical Association vol 312 no 4 pp 410ndash425 2014
[3] H Namme Luma M-S Doualla S-P Choukem et alldquoAdverse drug reactions ofHighlyActiveAntiretroviralTherapy(HAART) in HIV infected patients at the General HospitalDouala Cameroon a cross sectional studyrdquo The Pan AfricanMedical Journal vol 12 p 87 2012
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 Journal of Chemistry
Biosensor in oxygen-saturated
Biosensor in oxygen-saturated
c
b
a Biosensor in argon-saturated
minus8
minus7
minus6
minus5
minus4
minus3
minus2
minus1
0
1
I(120583
A)
minus800 minus600 minus400 minus200minus1000
PBS (0120583M Indinavir)
PBS (0120583M Indinavir)
PBS (25 120583M Indinavir)
E (mV) versus AgAgCl (3M NaCl)
Figure 6 Biosensor in argon-saturated PBS (0 120583M Indinavir) (a)oxygen-saturated PBS (0 120583M Indinavir) (b) and oxygen-saturatedPBS containing 25120583M Indinavir (c)
Indinavir
minus10
minus8
minus6
minus4
minus2
0
I(120583
A)
minus800 minus600 minus400 minus200minus1000
02 120583M
10120583M minus10minus9minus8minus7minus6minus5minus4minus3minus2
I(120583
A)
04 06 08 1002Indinavir (120583M)
E (mV) versus AgAgCl (3M NaCl)
Figure 7 CYP3A4-based biosensor in the presence of increasingIndinavir concentrations (02 to 10 120583M) Inset cathodic peak currentversus Indinavir concentration
Figure 7 shows the voltammetric response of the biosen-sor in oxygen-saturated PBS (pH 74 100mM KCl) con-taining 1mM Co(Sep)3+ diffusional mediator at a scan rateof 10mVs The CVs reveal that the biosensor exhibits aconcentration-dependent increase in 119868
119901119888with each addition
of Indinavir up to the final concentration of 10 120583M Thesefindings directly correspond with previous investigations[26 27] and thus it may be deduced that the substrateIndinavir is metabolised by the reduced CYP3A4 which inturn is mediated by the reduced Co(Sep)2+ The increasingrate of dioxygen to the heme in the presence of substrate isexemplified by the increase in the reduction current (119868
119901119888) as
shown in the cyclic voltammograms The response profile ofthe biosensor as shown by CV is typical of cytochrome P450enzyme in particular and heme-enzyme in general therebyadequately proving the strong catalytic effect andmetabolism
of Indinavir by CYP3A4 These favourable features of themodified electrode offer great promise for its sensing appli-cations The inset shows that the catalytic current is linearversus Indinavir concentration in the range of 02ndash10 120583Manda correlation coefficient of 09908Thedetection limit (signal-to-noise ratio is 3) is 0001120583mol Lminus1 The detection limit iswell below the plasma concentration of Indinavir (8 h afterintake) which ranges from 013 to 86mg Lminus1
4 Conclusions
An ideal nanocomposite support material consisting of GNPinfused OvOxPpy was successfully constructed based ona rapid electrochemical technique The obtained platformserved as an ideal host for enzyme immobilization for thedevelopment of a user-friendly and low-cost biosensor forIndinavir The results demonstrated that Au nanoparticlesenhanced the catalytic activity ofOvOxPpy by giving negativeshifts of CV peak potentials and increasing peak current ofthe enzyme Due to the synergy of OvOxPpy and the GNPsGNP|OvOxPpy||GCE had good stability sensitivity andselectivity to determine Indinavir in a large presence of PBS(pH 721) The biosensor response to Indinavir in oxygenatedPBS also showed significant increase GNP|OvOxPpy||GCEdisplayed strong catalytic activity towards the oxidation ofIndinavir and reveals the response of the active specieswith distinctive voltammetric peaks Low concentrationsrsquodetection of Indinavir demonstrates the ability of biosensorplatform to perform effective point-of-care diagnostics Thebiosensor constructionmethod developed in this study holdsgreat promise for ARV drugsrsquo (Indinavir) determination inreal samples and use for effective therapeutic drug monitor-ing in real time
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
This research was financially supported by South AfricarsquosNational Research Foundation (NRF)
References
[1] N A Thompson ldquoLatin Postrdquo 2015 httpwwwlatinpostcomarticles47066
[2] H F Gunthard J A Aberg J J Eron et al ldquoAntiretroviraltreatment of adult HIV infection 2014 recommendations of theInternational Antiviral Society-USA panelrdquo The Journal of theAmericanMedical Association vol 312 no 4 pp 410ndash425 2014
[3] H Namme Luma M-S Doualla S-P Choukem et alldquoAdverse drug reactions ofHighlyActiveAntiretroviralTherapy(HAART) in HIV infected patients at the General HospitalDouala Cameroon a cross sectional studyrdquo The Pan AfricanMedical Journal vol 12 p 87 2012
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Chemistry 7
[4] V Perrone D Cattaneo S Radice et al ldquoImpact of therapeuticdrugmonitoring of antiretroviral drugs in routine clinical man-agement of patients infected with human immunodeficiencyvirus and related health care costs a real-life study in a largecohort of patientsrdquo ClinicoEconomics and Outcomes Researchvol 6 no 1 pp 341ndash348 2014
[5] A I Cederbaum ldquoMolecular mechanisms of the microsomalmixed function oxidases and biological and pathological impli-cationsrdquo Redox Biology vol 4 pp 60ndash73 2015
[6] R Fasan ldquoTuning P450 enzymes as oxidation catalystsrdquo ACSCatalysis vol 2 no 4 pp 647ndash666 2012
[7] D-H Zhang L-X Yuwen and L-J Peng ldquoParameters affectingthe performance of immobilized enzymerdquo Journal of Chemistryvol 2013 Article ID 946248 7 pages 2013
[8] M Rahman X-B Li N S Lopa S J Ahn and J-J Lee ldquoElec-trochemical DNA hybridization sensors based on conductingpolymersrdquo Sensors vol 15 no 2 pp 3801ndash3829 2015
[9] Z Wang N Sun Y He Y Liu and J Li ldquoDNA assembledgold nanoparticles polymeric network blocks modular highlysensitive electrochemical biosensors for protein kinase activityanalysis and inhibitionrdquoAnalytical Chemistry vol 86 no 12 pp6153ndash6159 2014
[10] F Valentini and G Palleschi ldquoNanomaterials and analyticalchemistryrdquo Analytical Letters vol 41 no 4 pp 479ndash520 2008
[11] Y Okawa N Yokoyama Y Sakai and F Shiba ldquoDirectelectron transfer biosensor for hydrogen peroxide carryingnanocomplex composed of horseradish peroxidase and Au-nanoparticlemdashcharacterization and application to bienzymesystemsrdquo Analytical Chemistry Research vol 5 pp 1ndash8 2015
[12] W Putzbach and N J Ronkainen ldquoImmobilization tech-niques in the fabrication of nanomaterial-based electrochem-ical biosensors a reviewrdquo Sensors vol 13 no 4 pp 4811ndash48402013
[13] H Mallin J Muschiol E Bystrom and U T BornscheuerldquoEfficient biocatalysis with immobilized enzymes or encapsu-lated whole cell microorganism by using the SpinChem reactorsystemrdquo ChemCatChem vol 5 no 12 pp 3529ndash3532 2013
[14] R A Sheldon M J Sorgedrager and M H A Janssen ldquoUseof cross-linked enzyme aggregates (CLEAs) for performingbiotransformationsrdquo Chemistry Today vol 25 pp 62ndash67 2007
[15] N R Mohamad N H Marzuki N A Buang F Huyop andR A Wahab ldquoAn overview of technologies for immobilizationof enzymes and surface analysis techniques for immobilizedenzymesrdquo Biotechnology amp Biotechnological Equipment vol 29no 2 pp 205ndash220 2015
[16] P Zucca and E Sanjust ldquoInorganic materials as supports forcovalent enzyme immobilization methods and mechanismsrdquoMolecules vol 19 no 9 pp 14139ndash14194 2014
[17] I Ince C A J Knibbe M Danhof et al ldquoA novel maturationfunction for clearance of the cytochrome P450 3A substratemidazolam from preterm neonates to adultsrdquo Clinical Pharma-cokinetics vol 52 no 7 pp 555ndash565 2013
[18] K Ariga Q Ji T Mori et al ldquoEnzyme nanoarchitectonicsorganization and device applicationrdquo Chemical Society Reviewsvol 42 no 15 pp 6322ndash6345 2013
[19] L Sasso A Heiskanen F Diazzi et al ldquoDoped overoxidizedpolypyrrole microelectrodes as sensors for the detection ofdopamine released from cell populationsrdquo Analyst vol 138 no13 pp 3651ndash3659 2013
[20] B G Kalzung S B Masters and A J Trevor Eds Basic ampClinical Pharmacology McGraw-Hill Medical New York NYUSA 11th edition 2010
[21] A Ignaszak N Hendricks T Waryo et al ldquoNovel therapeuticbiosensor for indinavirmdasha protease inhibitor antiretroviraldrugrdquo Journal of Pharmaceutical and Biomedical Analysis vol49 no 2 pp 498ndash501 2009
[22] M Bjorninen A Siljander J Pelto et al ldquoComparison ofchondroitin sulfate and hyaluronic acid doped conductivepolypyrrole films for adipose stem cellsrdquo Annals of BiomedicalEngineering vol 42 no 9 pp 1889ndash1900 2014
[23] A Fakhry H Cachet and C D Chouvy ldquoElectrochemicalcharacterisations of ultra thin overoxidized polypyrrole filmsobtained by one-step electrosynthesisrdquo Journal of the Electro-chemical Society vol 160 no 10 pp D465ndashD470 2013
[24] E A Schneider Oriented attachment of cytochrome P450 2C9to a self-assembled monolayer on a gold electrode as a biosensordesign [PhD thesis] University of California San FranciscoClaif USA 2011
[25] C Baj-Rossi G De Micheli and S Carrara ldquoElectrochemicaldetection of anti-breast-cancer agents in human serum bycytochrome p450-coated carbon nanotubesrdquo Sensors vol 12no 5 pp 6520ndash6537 2012
[26] N R Hendricks T T Waryo O Arotiba N Jahed P GL Baker and E I Iwuoha ldquoMicrosomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 24-dichlorophenolmdashan endocrine disruptor compoundrdquo Elec-trochimica Acta vol 54 no 7 pp 1925ndash1931 2009
[27] E I Iwuoha R Ngece M Klink and P Baker ldquoAmpero-metric responses of CYP2D6 drug metabolism nanobiosensorfor sertraline a selective serotonin reuptake inhibitorrdquo IETNanobiotechnology vol 1 no 4 pp 62ndash67 2007
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of