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Process Biochemistry 47 (2012) 2131–2138

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epage: www.elsev ier .com/ locate /procbio

onstruction of d-amino acid biosensor based on d-amino acid oxidasemmobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticlesybrid film

uman Lata, Bhawna Batra, C.S. Pundir ∗

epartment of Biochemistry, M.D. University, Rohtak 124 001, Haryana, India

r t i c l e i n f o

rticle history:eceived 12 June 2012eceived in revised form 30 July 2012ccepted 31 July 2012vailable online 8 August 2012

eywords:-Amino acid

a b s t r a c t

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto polyindole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodepositedon surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using thisenzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary elec-trode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5and 35 ◦C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor

-Amino acid biosensor-Amino acid oxidasenS nanoparticlesoly (indole-5-carboxylic acid)

response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosen-sor was 58.85 �A cm−2 mM−1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode wasused 120 times over a period of 2 months when stored at 4 ◦C. The biosensor has an advantage overearlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the exter-nal environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor wasevaluated and employed for measurement of d-amino acid level in fruits and vegetables.

. Introduction

d-Amino acids level is increased in foods under high tem-erature or extreme pH or due to adulteration or microbialontamination as d-amino acids are major components of the bac-erial cell wall [1–3]. Changes in d-amino acids level in brain aressociated with several neurological and psychiatric diseases andhus have a major impact on the organism as a whole. Therefore,he determination of d-amino acid in biological materials is impor-ant [4–7]. A number of methods are available for determinationf d-amino acids such as high performance liquid chromatogra-hy (HPLC), gas chromatography (GC), or capillary electrophoresis8–10]. The analysis of d-amino acids by GC/HPLC is cumbersomeor samples in small quantity, due to the multiple steps necessaryor sample cleanup and derivatization. Thus, for routine and qual-ty control measurements, a simple and easily applicable analytical

ethod is required. Biosensing methods meet these requirements.

number of d-amino acid biosensors have been reported basedn Prussian blue (PB) film electrodeposited onto gold electrode11], gel matrix of hydroxyethyl cellulose [12], l- and/or d-amino

∗ Corresponding author. Tel.: +91 9416492413; fax: +91 126274640.E-mail addresses: chandraspundir@gmail.com, pundircs@rediffmail.com

C.S. Pundir).

359-5113/$ – see front matter © 2012 Published by Elsevier Ltd.ttp://dx.doi.org/10.1016/j.procbio.2012.07.034

© 2012 Published by Elsevier Ltd.

acid oxidase immobilized onto rhodonised carbon electrode [13],amine-modified silica gel [14], activated controlled pore glass [15],eggshell membrane [16], Prussian blue and SWCNT [17], graphiteworking electrode [18], purified Rodotorrula gracillis DAAO mutantswere covalently immobilized on an Amberzyme Oxirane support bya coupling procedure involving protein-free amino groups [19] andd-amino acid oxidase on a poly (o-phenylenediamine) and Nafion-modified platinum–iridium disk electrode [20]. In these biosensors,the enzyme electrode brings about the stereospecific oxidativedeamination of d-amino acids catalyzed by immobilized DAAO asfollows:

D-Amino acid + O2 + H2ODAAO−→ �-keto acid + NH3 + H2O2 (1)

The H2O2 generated is measured electrochemically, which isdirectly proportional to the concentration of d-amino acid. How-ever, these biosensors suffer from low stability, reusability andsensitivity and thus require to be improved. The use of nano-materials in construction of enzyme electrodes has led to theimprovement in their stability, reusability, sensitivity and anti-interference ability. Among the various metal oxide nanoparticles

available, zinc sulphide nanoparticles (ZnSNPs) are chemicallymore stable and technically better than other chalcogenides (suchas ZnSe) [21,22]. These nanomaterials are considered as a promisinghost material due to their excellent role in catalysis owing to their

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uantum size and magnetic functionality [23–25]. Further theseanoparticles provide a favorable microenvironment for enzymeso exchange electrons directly with an electrode, thus improvinghe sensitivity of amperometric biosensor.

Among the various conducting polymers employed in construc-ion of enzyme electrodes, such as polyacetylene, polythiophenend polypyrrole, polyaniline, polyindole (Pin) is a better polymer,s it can be easily polymerized into a film which possesses highonductivity and chemical stability. Further, the polyindole filmas fairly better thermal stability, higher redox activity and loweregradation rate compared to polypyrrole and polyaniline film26,27].

We describe herein the construction of an improved ampero-etric biosensor for determination of d-amino acid in fruits and

egetables by immobilizing covalently a DAAO (purified from goatidney) onto polyindole-5-carboxylic acid/zinc sulphide nanopar-icles (Pin5-COOH/ZnSNPs) hybrid film electrodeposited on theurface of Au electrode.

. Experimental design

.1. Analytical methods

Cyclic voltammetry was performed using modular electrochemical systemAutolab, model: AUT83785, manufactured by Eco Chemie, The Netherlands)quipped with PSTAT10 module and driven by a GPES software. A three electrodeonfiguration was used with DAAO/Pin5-COOH/ZnSNPs modified Au electrode as aorking electrode, Ag/AgCl as reference electrode and a Pt wire as a counter elec-

rode. Scanning electron microscope (SEM) (Zeiss EV040) for morphological studies,V–visible spectrophotometer (Shimadzu, Model 1700) for UV–visible spectra,

ransmission electron microscope (TEM) (JEOL 2100 F) for TEM study, X-ray diffrac-ometer (XRD), (122 Rigaku, D/Max2550, Tokyo, Japan) for X-ray diffraction studiesnd Fourier transform infra-red spectrometer (FTIR) (Thermo Scientific, USA) forTIR spectra were used.

.2. Reagents

Sephadex G-100 and DEAE–Sephacel from Sigma–Aldrich, USA, d-alanine, fer-ous chloride and ferric chloride from SRL, Mumbai, India were used. All otherhemicals used were of analytical reagent grade. Gold electrode, fruit juices andoat kidney were purchased from local market. Double distilled water (DW) wassed in all experimental studies.

.3. Extraction and purification of d-amino acid oxidase

DAAO was purified from the goat kidney cortex region as previously described28]. The cortex tissue was homogenized with cold 0.01 M Tris HCl (pH 8.0) in a 3:1atio (w/v) in a chilled pestle and mortar. The extract was filtered through a muslinloth and the filtrate was centrifuged at 10,000 × g for 30 min at 4 ◦C. The pelletas discarded and the supernatant was collected and treated as crude enzyme.

t was tested for DAAO activity as described [28] with modification and proteinontent by Lowry method. The enzyme was purified as previously described [28]sing ammonium sulfate precipitation (0–80%), gel filtration on Sephadex G-100nd ion-exchange chromatography on DEAE Sephacel using a linear gradient of KCl0.1–0.6 M). This resulted into 57-fold purification of enzyme with 19.5% yield. Theurified enzyme had a specific activity of 184.6 units/mg.

.4. Assay of d-amino acid oxidase

The assay of DAAO was carried out in dark as described [28] with modification.he reaction mixture containing 1.7 ml of 0.01 M Tris HCl buffer (pH 8.0), 0.1 mlf FAD (10−3 M), 0.1 ml of CuSO4 (10−2 M) and 0.1 ml enzyme was pre-incubatedt 37 ◦C for 2 min. The reaction was started by adding 0.1 ml d-alanine solution10−4 M). After incubating it at 37 ◦C for 5 min, 1.0 ml color reagent was added andept at room temperature for 15 min to develop the color. Absorbance was read at20 nm against control and the amount of H2O2 generated during the reaction wasetermined from a standard curve of H2O2.

One unit of enzyme is defined as amount of enzyme required to catalyze theormation of 1.0 micromole of H2O2 from oxidation of d-alanine per min/ml under

tandard assay conditions.

The color reagent consisted of 50 mg 4-aminophenazone, 100 mg solid phenolnd 1.0 mg horseradish peroxidase (RZ = 1.0) per 100 ml of 0.4 M sodium phosphateuffer pH 7.0. It was stored in amber colored bottle at 4 ◦C and prepared fresh aftervery week.

try 47 (2012) 2131–2138

2.5. Construction of DAAO/Pin5-COOH/ZnSNPs modified Au electrode

2.5.1. Preparation of ZnSNPsZnSNPs were synthesized by chemical precipitation method [24]. Aqueous solu-

tions of 0.5 M zinc acetate (Zn(CH3COO)2·H2O) and 0.5 M sodium sulphide (Na2S)were used for synthesis of ZnSNPs. To 100 ml zinc acetate solution, sodium sulphidesolution was added drop-wise under constant stirring until white colored precip-itates appeared. The stirring was allowed further for 15 min at room temperature.ZnSNPs were washed several times with DW. The prepared nanoparticles were keptat 60 ◦C for drying.

2.5.2. Preparation of Pin5-COOH/ZnS nanocompositesIt was prepared as described [24] with modification. Dispersion was prepared by

mixing of 10.0 g ZnSNPs and 1.0 ml indole monomers in 50.0 ml distilled water. Then4.5 g FeCl3 (as oxidizing agent for polyindole synthesis) was added to the ZnSNPsdispersion under continuous stirring. After 2 h of stirring, the particles were cleanedby DW, filtered, extracted for 10 h and dried at 50 ◦C.

2.5.3. Electrodeposition of Pin5-COOH/ZnSNPs onto Au electrodePrior to the surface modification, the Au electrode (0.2 cm2) was cleaned

with piranha solution [H2SO4:H2O2 in 3:1 ratio (v/v)] for 20 min and then rinsedthoroughly with DW. Then the electrode was polished with alumina slurry. To elec-trodeposited nanocomposite film of Pin5-COOH/ZnSNPs on surface of Au electrode,the polished Au electrode was immersed into 25 ml, 0.01 M Tris HCl buffer pH 7.5containing 0.1 g Pin5-COOH/ZnSNPs nanocomposite and then applied 10 polymer-ization cycles in the potential range, −0.1 V to +0.6 V with the applied scan rateof 50 mV s−1. The resulting Pin5-COOH/ZnSNPs/Au modified electrode was washedthoroughly with DW to remove unbound matter and stored in dry petri plate at 4 ◦Cuntil use.

2.5.4. Preparation of enzyme electrode (DAAO/Pin5-COOH/ZnSNPs/Au)The purified DAAO (25 IU/ml) was immobilized onto the Pin5-COOH/ZnSNPs

modified Au electrode surface through EDC/NHS activation chemistry by dippingmodified electrode in a mixture of 0.5 ml of 0.2 M EDC and 0.5 ml 0.2 M NHS, byadjusting pH 6.0 and keeping at room temperature for 1 h. The EDC/NHS treatedelectrode was dipped into 1.5 ml of purified enzyme and kept at 4 ◦C overnight. Theresulting enzyme electrode was washed with 0.01 M Tris HCl buffer, pH 7.5, 4 timesto remove residual/unbound enzyme. The resulting DAAO/Pin5-COOH/ZnSNPs/Auelectrode was used as working electrode. This working electrode was characterizedby SEM, FTIR and EIS at different stages of its construction.

2.5.5. Electrochemical measurementElectrochemical measurements were carried out in a potentiostat/galvanostat

with a three electrode system. Prior to response measurements, the steady statecurrent was achieved by polarizing the working electrode at 0.15 V in the reac-tion buffer. On the addition of 100 �l (10 mM d-alanine), it was oxidized to �-ketoacid, producing an electroactive H2O2, which was split into 2H+ + O2 + 2e− under apotential of 0.15 V. The flow of e− , i.e. current, was measured in mA.

2.6. Response measurement of DAAO/Pin5-COOH/ZnSNPs/Au electrode and itsoptimization

The measurements of d-amino acid were performed by employing cyclicvoltammetry. The biosensor was optimized by studying its optimum pH, tempera-ture and response time. To determine optimum pH, the reaction buffers of differentpH in the range, pH 5.0–10.0 at an interval of 0.5 were used (0.01 M sodium succinatefor pH 5.0 and 5.5, 0.01 M sodium phosphate for pH 6.0 and 6.5 and 0.01 M Tris HClfor pH 7.0–10.0). Similarly, the optimum temperature was studied by incubating thereaction mixture at 20–50 ◦C, with an interval of 5 ◦C. Optimum response time wasdetermined by measuring the biosensor response (mA) at different time from 2 s to12 s with an interval of 2 s.

2.7. Amperometric measurement of d-amino acid in fruit juices

d-Amino acid (DAA) level in different fruit juices was measured by the presentbiosensor in the same manner as described above for response measurement underoptimal working conditions, except that d-alanine was replaced with the fruit juice.The current was recorded and the amount of DAA was interpolated from the stan-dard curve between d-alanine concentration and current (mA) prepared under theoptimal working conditions (Fig. 1a inset and b).

2.8. Storage stability of enzyme electrode

The long-term storage and stability of the biosensor was investigated over 2months, when enzyme electrode was stored dry in a refrigerator at 4 ◦C. The activityof enzyme electrode was measured after every one week.

S. Lata et al. / Process Biochemis

Fig. 1. (a) Effect of substrate concentration on response of DAAO/Pin5-COOH/ZnSNPs/Au electrode. (inset) The linear calibration plot corresponding to thecurrent response of different concentration of d-alanine. (b) Cyclic voltammetry netcurrent responses of biosensor in 0.01 M Tris HCl buffer (pH 7.5) containing differentc

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oncentrations (0.001–2.0 mM) of d-alanine at a scan rate of 50 mV s−1.

. Results and discussion

.1. Characterization of ZnSNPs

UV–visible spectra of ZnSNPs showed a strong absorbance peakround 500 nm (Fig. 2a), confirming the synthesis of ZnSNPs. Theypical TEM images of ZnSNPs showed its spherical shape with

diameter of 20 nm (Fig. 2b). The XRD patterns of the preparednSNPs (Fig. 2c) showed the characteristics peaks of ZnSNPs cor-esponding to the (8 2), (9 3), (1 1 1), (2 2 0), (3 1 1) and (3 3 0).rystallite size of ZnS nanoparticles was calculated by followingcherrer’s equation:

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here � = 0.9, D is the crystallite size (Å), � (Å) = 1.54 be the wave-ength of Cu K� radiation and is the corrected half width of theiffraction peak. The average crystallite size calculated from theseeaks using Scherer’s formula was 20 nm. FTIR spectra of ZnSNPshowed the absorption peaks at 3448.33 cm−1 and 1632.00 cm−1

Fig. 2d), assigned to the ZnSNPs stretching vibration mode. Thesebservations confirmed the formation of ZnSNPs.

try 47 (2012) 2131–2138 2133

3.2. SEM studies of enzyme electrode at different stages of itsconstruction

The SEM images of the surfaces of the bare Au electrode, Pin5-COOH/ZnSNPs/Au electrode and DAAO/Pin5-COOH/ZnSNPs/Auelectrode are shown in Fig. 3a–c respectively. The stepwise modi-fication of electrode could be seen clearly from these SEM images.The SEM image of the bare Au electrode showed a smooth and fea-tureless morphology (Fig. 3a), whereas Pin5-COOH/ZnSNPs showedthe net like porous structure indicating the larger effective surfacearea on the surface of Au electrode (Fig. 3b). On immobilizationof DAAO, the globular structural morphology of enzyme over themodified Pin5-COOH/ZnSNPs electrode appeared (Fig. 3c).

3.3. Construction of DAAO/Pin5-COOH/ZnSNPs/Au electrode

Fig. 4a and b) show chemical reactions/steps involved inthe fabrication of DAAO/Pin5-COOH/ZnSNPs/Au electrode. Firstly,Pin5-COOH/ZnSNPs film (thickness 0.8 �m) were deposited elec-trochemically on the surface of Au electrode, as this method iseasy and the layer thickness could be controlled. Our resultsshowed that Pin5-COOH/ZnSNPs provided a remarkable syner-gistic effect toward the oxidation of d-amino acid. The purifiedDAAO was immobilized covalently onto Pin5-COOH/ZnSNPs elec-trode through EDC and NHS chemistry with conjugation yieldof 869.5 �g/cm2 and retention activity of 85.33%. Incubation ofDAAO solution first with EDC leads to its coupling with EDC andthen NHS replaced the EDC to activate the carboxylic group ofPin5-COOH/ZnSNPs electrode leads to effective collision between

COOH groups of Pin and NH2 groups on the surface of the DAAOto form an amide bond ( CO NH ) between enzyme and modifiedelectrode.

3.4. Cyclic voltammetric studies

Fig. 5a–c shows cyclic voltammograms (CVs) for Auelectrode, Pin5-COOH/ZnSNPs/Au electrode and DAAO/Pin5-COOH/ZnSNPs/Au bioelectrode in phosphate buffer saline (50 mM,pH 7.0, 0.9% NaCl) containing 100 �l d-alanine (0.1 mM) recordedat different scan rates (10–100 mV s−1) respectively. It wasobserved that the anodic potential shifted toward positive sideand the cathodic peak potential shifted in the reverse direc-tion. It appeared that the Pin5-COOH/ZnSNPs/Au nanocompositeelectrode provides a biocompatible environment to the enzyme,and ZnSNPs act as an electron mediator, resulting in an accel-erated electron transfer between enzyme and electrode. ThePin5-COOH/ZnSNPs/Au nanocomposite electrode provides a bet-ter biocompatible environment because of the presence of free

COOH groups of Pin5-COOH and more surface area of ZnSNPs.The higher redox potential of enzyme electrode lead to moreionization of functional groups (e.g. COOH groups) and thusbetter biocompatibility [29–31].

3.5. FTIR spectra

Fig. 6 shows FTIR spectra for Pin5-COOH/ZnSNPs/Au electrode(curve A) and DAAO/Pin5-COOH/ZnSNPs/Au electrode (curve B).The spectrum associated with the oxidized Pin were characterizedby a very large adsorption band located in the spectral domainbetween 3700 and 3100 cm−1, which is a characteristic of OHgroups belonging to residual water molecules trapped in the poly-mer matrix as well as water molecules absorbed in Pin. The peaks

tively. A minor absorption peak was found at 1690 cm−1, whichmight be due to the attachment of COOH group onto the indole.

2134 S. Lata et al. / Process Biochemistry 47 (2012) 2131–2138

Fig. 2. UV–visible spectra of (a) ZnSNPs, (b) transmission electron microscopic (TEM) image of ZnSNPs, (c) X-ray diffraction (XRD) pattern of ZnSNPs, and (d) FTIR spectra ofZnSNPs.

Fig. 3. SEM images of (a) bare gold (Au) electrode, (b) Pin5-COOH/ZnSNPs/Au electrode and (c) DAAO/Pin5-COOH/ZnSNPs/Au electrode.

S. Lata et al. / Process Biochemistry 47 (2012) 2131–2138 2135

Fig. 4. (a) Schematic representation of chemical reaction involved in the fabrication of DAAO/Pin5-COOH/ZnSNPs/Au electrode. (b) Schematic diagram of d-amino acidbiosensor.

Fig. 5. Cyclic voltammogram (CV) of (a) bare Au electrode (b) Pin5-COOH/ZnSNPs/Au electrode and (c) DAAO immobilized onto Pin5-COOH/ZnSNPs/Auelectrode in 0.01 M pH 7.5 Tris HCl buffer solution containing 100 �l d-alanine(0.1 mM) at a scan rate of 50 mV s−1.

Fig. 6. FTIR spectra of (curve A) Pin5-COOH/ZnSNPs/Au and (curve B) DAAO/Pin5-

COOH/ZnSNPs/Au electrode.

2136 S. Lata et al. / Process Biochemistry 47 (2012) 2131–2138

Fig. 7. Impedance spectra of (i) Pin5-COOH/ZnSNPs/Au electrode and (ii) DAAOimmobilized onto Pin5-COOH/ZnSNPs/Au electrode in 0.01 M pH 7.5 Tris HCl bufferc

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alanine in orange juice (5 mM and 10 mM) was 98.63% and 97.31%,

ontaining [K3Fe(CN)6] (5 mM).

n the FTIR spectrum of DAAO/Pin5-COOH/ZnSNPs/Au electrodecurve B), DAAO binding is indicated by the appearance of addi-ional absorption bands at 1640 and 1533 cm−1 assigned to thearbonyl stretch (amide I band) and N H bending (amide II band),espectively.

.6. Electrochemical impedance measurements

Fig. 7 shows electrochemical impedance spectra (EIS) of bare Aulectrode (a) Pin5-COOH/ZnSNPs/Au electrode (b) and DAAO/Pin5-OOH/ZnSNPs/Au electrode (c). The charge transfer process inAAO/Pin5COOH/ZnSNPs/Au electrode was studied by monitor-

ng charge transfer resistance (RCT) at the electrode and electrolytenterface. The value of the electron transfer resistance (semicircleiameter) (RCT) depends on the dielectric and insulating features athe electrode/electrolyte interface. The RCT values for bare Au elec-rode, Pin5-COOH/ZnSNPs/Au and DAAO/Pin5-COOH/ZnSNPs/Aulectrodes were 409 �, 258 � and 356 � respectively. This increasen RCT is attributed to the fact that most biological molecules,ncluding enzymes, are poor electrical conductors at low fre-uencies (at least <10 kHz) and cause hindrance to the electronransfer.

.7. Optimization of experimental conditions

As the proton is critical in redox behavior of DAAO-FAD,he increase of DAAO response at high pH is possibly due

ig. 8. (a) Influence of applied pH on the current response of DAAO/Pin5-COOH/ZnSNPAAO/Pin5-COOH/ZnSNPs/Au electrode.

trophotometric method (x-axis) and the current method (y-axis) employing thed-amino acid biosensor based on Pin5-COOH/ZnSNPs/Au composite film.

to the decrease of proton concentration and bioactivity of theimmobilized DAAO. The current response resulting from theenzyme-catalyzed reaction achieved a maximum value at pH 7.5(Fig. 8a). Therefore, a pH of 7.5 was used in further experiments.The current response of the enzyme electrode was enhanced, asthe incubation temperature increased upto 35 ◦C after which itwas declined (Fig. 8b), due to heat denaturation of protein/enzyme.This optimum temperature of the biosensor (35 ◦C), was compara-ble to that of earlier biosensors [11–20]. There was a hyperbolicrelationship between sensor response (mA) and d-alanine concen-tration in the range 0.001–3.5 mM (Fig. 1a), which is comparablewith earlier biosensor [11–20]. There was a linear relationshipbetween current (mA) and d-alanine concentration ranging from0.001 mM to 2.0 mM in the reaction mixture. Km for d-alaninewas 0.8 mM, which is comparable to that of earlier reportedbiosensors [11–20].

3.8. Evaluation of d-amino acid biosensor

The detection limit of biosensor was 0.001 mM at a signal tonoise ratio of 3, which is lower than those reported for d-amino acidbiosensor [11–20]. Analytical recovery of exogenously added d-

s/Au electrode. (b) Influence of applied temperature on the current response of

respectively, showing the reliability of the method. The resultsof within and between batch coefficients of variation for orangejuice DAA determination were <2.08% and <3.07%, respectively

S. Lata et al. / Process Biochemistry 47 (2012) 2131–2138 2137

Table 1d-Amino acid levels in different brands of fruit juices as measured by the DAAO/Pin5-COOH/ZnSNPs/Au electrode.

S. No. Fruit juices d-Amino acid (mM)Mean ± SD (n = 3)

1 Mixed fruit 1.02 ± 0.22 Pineapple 0.52 ± 0.33 Guava 0.43 ± 0.34 Mango 0.78 ± 0.25 Fruity 0.73 ± 0.36 Litchi 0.63 ± 0.37 Lime 0.65 ± 0.28 Apple* 0.62 ± 0.39 Orange 1.37 ± 0.3

10 Grapes juice* 1.01 ± 0.3

* d-Amino acid level in apple juice (0.69 mM) and grapes juice (0.96 mM) [17].

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ndicating the good reproducibility and consistency of the method.he linear plot reveals that such electrode can work well in d-lanine solution with a sensitivity of 58.85 �A cm−2 mM−1. To testhe accuracy of method, d-amino acid value in orange juice as mea-ured by present method was compared with those obtained bytandard spectrophotometric method using free enzyme [28]. Theevel of d-amino acid in orange juice as measured by both the meth-ds matched with each other with a good correlation (r = 0.979458)Fig. 9). The results showed that the data obtained by present

ethod and standard spectrophotometric method were correlatedell.

.9. Determination of d-amino acid in fruit juices

d-Amino acid levels as measured by the present biosensoranged from 0.43 mM to 1.37 mM in fruit juices (Table 1). Thesealues are comparable to those stated in earlier reports [11–20].

.10. Long-term stability of enzyme electrode

The stability of the biosensor was investigated by ampero-etric measurements in the presence of 0.1 mM d-alanine. The

urrent response of enzyme electrode was retained about 80% ofts original response after its 120 uses during the span of 2 monthsFig. 10), when stored at 4 ◦C which is much better than earlier elec-rodes [11–20]. The good stability, repeatability and reproducibilitybserved for the present biosensor could be attributed to the

ovalent immobilization of DAAO onto Pin5-COOH/ZnSNPs elec-rodeposited on Au electrode.

A comparison of analytical characteristics of present biosensorith those of earlier biosensors have been summarized in Table 2. Ta

ble

2A

com

par

iso

Prop

erti

es

Sou

rce

of

Sup

por

t

oM

eth

ods

fTy

pe

of

trW

orki

ng

oO

pti

mu

mR

esp

onse

Lin

eari

ty

Det

ecti

onSt

orag

e

stK

m

Ap

pli

cati

o

*N

R:

not

2 hemis

4

iDrgamr

A

Ca

R

[

[

[

[

[

[

[

[

[

[

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[

[

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[

[

[

[

[

138 S. Lata et al. / Process Bioc

. Conclusion

The present study revealed that Pin5-COOH/ZnSNPs hybrid films a very good candidate for the construction of a highly sensitiveAA biosensor. DAAO/Pin5-COOH/ZnSNPs showed relatively rapid

esponse, high sensitivity, broad linear range, low detection limit,ood reproducibility and long term stability. The wide detectionnd high sensitivity may be assigned to the amplification of theagnitude of current response since ZnSNPs could catalyze the

eaction of H2O2.

cknowledgements

Authors (Suman Lata) and (Bhawna Batra) are thankful to theouncil of Scientific and Industrial Research (CSIR), India, for theward of Senior and Junior Research Fellowship during this study.

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