polypyrrole modified pencil graphite electrode

Accepted Manuscript Title: L-Glutamate biosensor based on L-glutamate oxidase immobilized onto ZnO nanorods/polypyrrole modified pencil graphite electrode Author: Bhawna Batra Monika Yadav Chandra Shekhar Pundir PII: DOI: Reference:

S1369-703X(15)30083-8 http://dx.doi.org/doi:10.1016/j.bej.2015.10.012 BEJ 6316

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

28-7-2015 16-10-2015 17-10-2015

Please cite this article as: Bhawna Batra, Monika Yadav, Chandra Shekhar Pundir, L-Glutamate biosensor based on L-glutamate oxidase immobilized onto ZnO nanorods/polypyrrole modified pencil graphite electrode, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2015.10.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 L-Glutamate biosensor based on L-glutamate oxidase immobilized onto ZnO nanorods/polypyrrole modified pencil graphite electrode

Bhawna Batra, Monika Yadav and Chandra Shekhar Pundir* Department of Biochemistry, M.D.University, Rohtak-124 001, India

Running Title: Amperometric L-glutamate biosensor

*Corresponding author at: Department of Biochemistry, M D University, Rohtak-124001, Haryana, India. Fax: 91-126274640, Tel.: +91 9416492413 E-mail address: [email protected]

2 Highlights  Fabricated GluOx/ZnO nanorods/PPy modified pencil graphite (PG) electrode 

Constructed

an

improved

L-glutamate

biosensor

based

on

GluOx/ZnO

nanorods/PPy/PG 

Biosensor showed low detection limit, wide linear range and fast response time



Biosensor measured L-glutamate content in various food stuffs

3 Abstract A method is described for the construction of a highly sensitive electrochemical biosensor for the detection of L-glutamate. Such a biosensor is based on immobilization of glutamate oxidase (GluOx) onto zinc oxide nanorods (ZnONRs)/polypyrrole (PPy) composite. This composite was electro-deposited onto a pencil graphite (PG) electrode. The enzyme electrode was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), Fourier transform Infra-red spectroscopy (FTIR) and cyclic voltammetry (CV). The biosensor showed optimum response at pH 8.5 (0.1 M Tris-HCl buffer) and 30°C, when operated at 20 mV s−1. The biosensor exhibited excellent sensitivity (detection limit as 0.18 nM), fast response time (less than 5s) and wide linear range (0.02500 µM). Analytical recovery of added L-glutamate (20 and 40 µM) in commercial Chinese soup were 95.40 and 97.56 % respectively .The within batch and between batch coefficients of variation (CV) in measurement of L-glutamate in commercial Chinese soup were 2.35 % and 4.5 % respectively. The enzyme electrode lost 30 % of its initial activity after 100 uses over a period of 90 days, when stored at 4°C. Keywords: Biosensor, Enzymes, Immobilized enzymes, Immobilization, Zinc oxide nanorods; Polypyrrole

4

1. Introduction L-Glutamate, an amino acid, occurs naturally in protein-rich foods such as cheese, milk, mushrooms, meat, fish, and many vegetables. It has widespread use as a flavour enhancing food additive and also linked to Chinese Restaurant Syndrome (CRS), being a common ingradient in Chinese food [1]. The main motivation for the ongoing world wide research on glutamate is due to its role in the signal transduction of nervous systems in all complex living organisms, including humans. L-Glutamate is considered to be the major mediator of excitatory signals in the mammalian central nervous system (CNS) and involved in normal brain functions including cognition, memory and learning. Both too much and too little Lglutamate is harmful. This implies that glutamate is both essential and highly toxic at the same time. Thus measurement of L-glutamate in biological materials has great significance. Different methods have been employed to determine L-glutamate, e.g. potentiometric titration [2], chromatographic [3-7], spectrophotometric [8-10] and fluorimetric [11-14]. However, these methods require time consuming sample preparation, costly equipment and skilled persons to operate. Biosensing methods overcome these drawbacks, as these are simple, sensitive, rapid and specific. Recently, the combinations of different nanomaterials and conducting polymers have been used in construction of enzyme electrodes to improve the analytic performance of biosensors. Various kinds of ZnO nanostructures have been prepared, such as nanodots, nanorods, nanowires, nanobelts, nanotubes, nanobridges and nanonails, nanowalls, nanohelixes, seamless nanorings, mesoporous single-crystal nanowires, and polyhedral cages [15-17]. Among the 1D nanostructures, ZnO nanorods(ZnONR) and ZnO nanowires have been

studied widely, because of their easy preparation and device applications.

ZnONR are known for their fast electron transfer kinetics, large surface area and thus

5 expected to improve the analytic performance of a biosensor. Polypyrrole (PPy), one of the important conducting polymers, has been applied in drug delivery [18, 19], sensors [20] and corrosion protection [21], due to its semiconducting properties. PPy has also been exploited as an electronic component in electronic devices, e.g., photoelectrochemical devices [22], organic light-emitting diodes [23], and rectifying devices [24]. Due to the special physicochemical properties, PPy has been applied in DNA sensors [25], actuators [26] and immunosensors [27]. The present work describes a unique approach of immobilizing Lglutamte oxidase(GluOx) onto ZnONR/PPy modified pencil graphite electrode (PGE) and its applications in construction of an improved amperometric L-glutamate biosensor . 2. Experimental 2.1.

Materials

L- Glutamate oxidase (GluOx) from Sigma–Aldrich, St.Louis, USA, Zn(NO3)2 potassium ferro-cyanide

(K2Fe2CN2),

potassium

ferricynide

(K2(FeCN)6.3H2O),

ammonium

persulphate ((NH4)2S2O8)(APS), pyrrole , potassium chloride (KCl) and L-glutamate from SISCO Research Lab., Mumbai, India, and glutaraldehyde from LOBA Cheme. Pvt. Ltd. Mumbai, were used. HB Pencil (used as PGE), tomato fruits, Ching’s noodles and Chinese soup manufactured by M/S Capital Food Pvt. Ltd., Mumbai were purchased from local market. DdH2O (ddH2O) was used throughout the experimental studies. 2.2.

Apparatus

Potentiostat/Galvanostat (Make: Autolab, model: PGT83785, Eco Chemie, The Netherland) with a three electrode system consisting of a Pt wire as an auxillary electrode, an Ag/AgCl electrode as reference electrode and GluOx/ZnONRs/PPy modified PG electrode as a working electrode, Transmission electron microscope (TEM) (JEOL 2100 F) and Scanning electron microscope (SEM) (Zeiss EV040), UV Spectrophotometer (Make: Shimadzu, Model 1700), X-ray diffractometer (XRD), (Make: 122 Rigaku, D/Max2550,

6 Tokyo, Japan), Fourier transform Infra-red spectrometer (FTIR) (Thermo Scientific, USA) were used. 2.3 Assay of free GluOx The assay of free GluOx was carried out as described in ref.[28] with modification. The assay was based on quantification of H2O2 which was generated from oxidation of Lglutamic acid catalyzed by L-glutamate oxidase. The H2O2 was measured using a color reaction consisting of 4-aminophenazone, phenol and peroxidase as chromogenic system [29]. The reaction mixture contained 1.8 ml of sodium phosphate buffer pH 7.4 (0.1 M), 0.1 ml of L-glutamate solution (1 mM) and 0.1ml of GluOx solution (5 U/ml). It was incubated at 37° C for 10 min. One ml of colour reagent was added and incubated it in dark at 37°C for 20 min to develop the color, A520 was read and H2O2 concentration was extrapolated from its standard curve. One unit of enzyme was defined as the µmol of H2O2 generated from the aerobic oxidation of L-glutamate by GluOx per min/ml during the assay, under standard assay conditions (pH 7.4, Temp. 37° C). 2.4 Construction of GluOx/ZnONRs/PPy modified PG electrode 2.4.1. Preparation of Zinc oxide nanorods (ZnO-NRs) ZnONRs were prepared according to the method of Zhang et al., 2007 [30]. Under continuous stirring 0.02 mol Zn (NO3)2.6H2O. was dissolved in 750 ml distilled water. The mixture consisting of polyethylene glycol and 0.15 mol of NH3.2H2O was added dropwise into the solution at room temperature, resulting in a white solution. The final mixture was rapidly heated to 60-80°C and stirred vigorously until the precipitates were formed . The precipitates (ZnO-NRs) were filtered and washed several times with ddH2O water followed by ethyl alcohol, and then dried at 60°C under atmospheric pressure overnight. 2.4.2. TEM of ZnO-NRs

7 The morphological characterization of the Zinc Oxide nanorods was carried out in a Transmission electron microscope, at J.N. University, New Delhi, on payment basis. 2.4.3. Electrodeposition of ZnO-NR/PPy onto PG electrode The surface of PG electrode (2cm × 2mm) was polished manually using alumina slurry (diameter 0.05µm) and a polishing cloth, followed by thorough washing with DW. The polished electrode was sonicated in ethanol to remove adsorbed particles and finally washed thorougly with ddH2O. A mixture of ZnO-NR/Py suspension (200 µl) and 25 ml of 1M KCl was electropolymerized onto this PGE through cyclic voltammetry in a PotentiosatatGalvanostat by applying 20 successive polymerization cycles between - 0.25 to 0.8 V at a scan rate of 20 mVs-1 (Fig. 1). Such a electrodeposited layer has remote chances of leaching during repeated use, which provides longer stability to the electrode. The resulting ZnONR/PPy modified PG electrode was washed thoroughly with DW to remove unbound matter and kept it in a dry Petri-plate at 4°C. 2.4.4. Immobilization of GluOx onto ZnO-NR/PPy modified PG electrode A

schematic

representation

of

preparation

of

ZnO-NR/PPy/PG

electrode

and

immobilization of GluOx onto this electrode is shown in Scheme 1. ZnO-NRs/PPy/PG electrode was dipped into 1.5 ml of GluOx solution (5 U/ml) in 0.1 M sodium phosphate buffer (pH 7.5) and kept overnight at room temperature for immobilization. The resulting enzyme electrode (GluOx/ZnO-NR/PPy/PGE) was washed 3-4 times with 0.1M sodium phosphate buffer (pH 7.5), to remove residual unbound enzyme. The resulting GluOx/ZnONRs/PPy/PG electrode was used as working electrode and stored at 4oC when not in use. 2.4.5. Scanning electron microscopy The SEM images of bare PG electrode, ZnONRs/PPy/PG and GluOx/ZnONRs/PPy/PG electrode were taken in a scanning electron microscope at Jawahar Lal University, New Delhi on commercial basis.

8 2.5.Cyclic voltametric measurement and testing of L-glutamate biosensor Cyclic voltammetry (CV) of GluOx/ZnONRs/PPy/PG electrode was recorded in Potentiostat–Galvanostat between the potential range -0.05 to 0.5V vs Ag/AgCl as reference and Pt wire as counter electrode in a 25 ml of 0.2 M sodium phosphate buffer (pH 7.5) containing 100 µl of 1mM L-glutamate. 2.6. Optimization of L-glutamate biosensor To optimize working conditions of L-glutamate biosensor, effects of pH, incubation temperature, time and substrate (L-glutamate) concentration on biosensor response were studied. To determine optimum pH, the pH was varied between pH 5.5 to 9.5 at an interval of pH 0.5 using the following buffer, each at a final concentration of 0.1M: pH 7.0 to 8.0 sodium phosphate buffers and pH 8.5 to 10.0 Tris HCl buffer. Similarly to determine optimum temperature the reaction mixture was incubated at different temperatures (20– 50ºC) at an interval of 5°C. The effect of L-glutamate concentration on biosensor response was determined by varying the concentration of L-glutamate in the range 0.02-550 µM. 2.7. Application of L-glutamate biosensor in food stuffs Food samples (1.0 ml each) were drawn from crushed tomato fruit, noodles (in dd H2O) and Chinese soup. L-Glutamic acid content in these food samples was determined by the present biosensor in the similar manner as described above for its response measurement, under its optimal working conditions except that L-glutamic acid was replaced by food sample. Lglutamic acid content in food was interpolated from standard curve between glutamic acid concentration vs current in µA prepared under optimal assay conditions of GluOx/ZnONRs/PPy/PG electrode (Fig. 2). 2.8. Evaluation The analytic performance of the biosensor was tested by studying its analytical recovery, detection limit, precision and correlation. Analytical recovery was studied using Chinese

9 soup with two different L-glutamate concentrations (20 and 40 µM). To study the reproducibility and reliability of the present biosensor, L-glutamate level in chinese soup was measured five times on single day (within batch) and five times again after their storage at -20 °C for 1 week. To study the correlation, the L-glutamate level was measured in 10 samples of chinese soup by the standard enzyme colorimetric method (x) and the present method (y) and their correlation was studied using regression equation. 2.9. Storage stability of GluOx/ZnONRs/PPy/PG electrode The stability of the enzyme/working electrode was tested every week under its optimum conditions for 3 months. The electrode was stored in dried condition at 4ºC, when not in use. 3. Results and discussion 3.1.

Characterization of ZnONR/PPy

The typical TEM images of ZnONRs showed its rod shape with an average diameter of 34.0± 3.7 nm (Fig. 3 A). The XRD patterns of the ZnONRs clearly showed its characteristics peaks. All the diffraction peaks of the samples were indexed to the hexagonal phase of ZnO having lattice parameters a = 3.249 and c = 5.206 Ǻ card No (JCPD file No. 36-1451), which were in good agreement with the data from the Joint Committee of Powder Diffraction Standards (JPDS). No characteristic peaks of impurities were observed, revealing the high purity of the ZnONR/PPy (Fig. 3 B). The peak at 1382 cm−1 were due to the bridging mode of zinc at the ZnO surface (Fig. 3 C). 3.2.

SEM studies of PG electrode during its modification

The SEM images of the surface of bare PG electrode, ZnONRs/PPy/PG electrode and GluOx/ZnONRs/PPy/PG electrode with are shown in Fig. 4 (A), (B) and (C) respectively. The stepwise modification of electrode could be seen clearly from these SEM images. The SEM image of the bare PG electrode showed a smooth and featureless morphology (Fig. 4 A). The ZnONRs/PPy/PG composite film exhibited rod like structures, confirming the

10 electrodeposition of ZnONRs and PPy film (Fig. 4 B), hence effective surface area is larger. On immobilization of GOx, the globular structural morphology appeared, due to the interaction between ZnONRs/PPy/PG Plate with GluOx (Fig. 4 C). 3.3.

Electrochemical impedance measurements (EIS)

Fig. 5 showed electrochemical impedance spectra (EIS) of (i) bare PG electrode (ii) ZnONRs/PPy/PG electrode and (iii) GluOx/ZnONRs/PPy/PG electrode in 5 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) as a redox probe. EIS provided an effective method to probe electronic features of surface-modified electrodes. The RCT values (semicircle diameter) for bare PG electrode, ZnONRs/PPy/PG electrode and GluOx/ZnONRs/PPy/PG electrodes were 630Ω, 400Ω and 580Ω respectively. The RCT of GluOx/ZnONRs/PPy/PG (iii) bioelectrode was higher compared with that of ZnONRs/PPy/PG (ii) electrode. This increase in RCT can be attributed to the fact that most biological molecules, including enzymes, are poor electrical conductors and cause hindrance to electron transfer. These results also indicate the binding of enzymes onto ZnONRs/PPy/PG composite. 3.4. Fig.6.

FTIR spectra showed

FTIR

spectra

of

ZnONR/PPy/PG

electrode

(curve

i)

andGluOx/ZnONR/PPy/PG electrode (curve ii). FTIR spectrum of ZnONR/PPy composite on PG electrode, exhibits absorption peaks at 1454.9 and 1632.6 cm−1 for typical pyrrole ring vibration and that of metal oxide band at 523.7 cm−1, while peak at 1549 cm-1 is attributed to amide I group (C-O stretching along with N-H deformation mode) as shown in curve ii. This confirmed the immobilization of GluOx onto ZnONR/PPy/PG electrode. 3.5.

Responses measurements of glutamate biosensor

The maximum response (current in mA) was observed at 0.065 V (Fig.7) and hence subsequent studies were carried out at this voltage. Amperometric response of GOx/ZnONRs/PPy/PGE gets increased by the addition of 100 μl (0.5 mM) L-glutamate at

11 the applied potential of 0.065 V. When the glutamate was added into the buffer solution, the oxidation current rose steeply to reach a stable value. 3.6. Optimization of biosensor The experimental conditions affecting the biosensor response were studied in terms of effect of pH, incubation temperature, time and substrate (L-glutamate) concentration. The optimum current was obtained at pH 8.5, which is near to that of earlier reported Lglutamate biosensors (Table 1). The optimum temperature of the present biosensor was at 30ºC, which is higher than earlier reported biosensors but similar to one biosensor (Table 1). The current response decreased gradually after 30 °C but rapidly after 50°C, due to thermal inactivation of the enzyme. Hence, the subsequent experiments were performed at 30°C. The biosensor showed optimum response within 5 s, which is lower than earlier L-glutamate biosensors (Table 1). There was a hyperbolic relationship between biosensor response and L-glutamic acid concentration range 0.02-600 µM with the linearity up to 500 µM. 3.7. Evaluation of biosensor 3.7.1. Linearity There was a linear relationship between the current (in μA) and the L-glutamate concentration in the range 0.02-500 µM. 3.7.2. Detection limit The limit of detection (LOD) of the present biosensor was 0.18nM (S/N = 3) as calculated using the formula LOD = 3.3(SD/S) where SD = Standard deviation of the response, S = Slope of the calibration curve, which is lower than that of earlier biosensor (Table 1). 3.7.3. Precision Within-sample and between-sample coefficients of variation (CVs) for the determination of L-glutamate in Chinese soup on the same day and after one week of storage were 2.35 % and 4.5 % respectively. These results highlight the good reproducibility and consistency of

12 the present method, which can be attributed to the excellent immobilization of GluOx onto the ZnONR/PPy/PG electrode. 3.7.4. Analytical recovery The average recoveries of L-glutamate added to Chinese soup (at levels of 20 and 40 µM) were 95.40 and 97.56 % respectively, demonstrating the good accuracy of the present biosensor. 3.7.5. Application of L-glutamate biosensor L-Glutamic acid level in following food stuffs such as crushed tomato, noodles and Chinese soup was measured by the present biosensor and found to be 250 µM, 310 µM and 360 µM respectively (range 250 to 360 µM). There was a good correlation (r = 0.99) between these values and the values obtained by standard colorimetric method, (Fig. 8). 3.7.6. Long-term stability of enzyme electrode The enzyme electrode lost only 30% of its initial activity after 100 applications/uses/ over a period of 90 days. These observations indicate the better stability of enzyme electrode than earlier enzyme electrodes (Table 1). 4. Conclusion An improved amperometric glutamate biosensor was constructed by immobilizing GluOx onto ZnONR/PPy electrodeposited onto PG electrode, which exhibited relatively rapid response (5 s), broad linear range (0.02-500 µM), low detection limit (0.18 nM), good reproducibility and long stability (80 days at 4°C). Acknowledgement Author (Bhawna Batra) is thankful to the Council of Scientific and Industrial Research (CSIR), India, for the award of Junior Research Fellowship during this study.

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18 Figure captions Fig.1. Cyclic voltamogram for electrodeposition of

ZnONR/PPy composite film.

Supporting electrolyte: 1M KCl solution; Scan rate: 20 mV/s. Fig.2. Standard curve for glutamate biosensor for effect of glutamate concentration on response of glutamate biosensor based on ZnONR/PPy electrode bound glutamate oxidase. Fig.3. (A) Transmission electron microscopic (TEM) image of ZnONR (B) X-ray diffraction (XRD) pattern of ZnONR (C) FTIR spectra of ZnONR Fig.4.

SEM

images

of

(a)

bare

PG

electrode,

(b)

ZnONR/PPy/PG

(c)

GluOx/ZnONR/PPy/PG electrode Fig.5. Impedance spectroscopy study of (i) Bare PG (ii) ZnONR/PPy/PG (iii) GluOx/ ZnONR/PPy/PG in 5 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) as a redox probe Fig.6. FTIR spectra of (i) ZnONR/PPy/PG and (ii) GluOx/ZnONR/PPy/PG electrode Fig.7. Cyclic voltammetry response of GluOx/ZnONR/PPy/PG on successive addition of 100 μl (0.5 mM) glutmate in 25 ml 0.1M sodium phosphate buffer (pH=7.5) in the potential range -0.100 V to 0.125 V at a scan rate of 50 mV/s. Fig.8. Correlation between glutamate values measured by chemical photochlorometric method (x axis) and the current method (y axis) employing the glutamate biosensor based on GluOx/ ZnONR/PPy/PG Scheme1. Schematic representation of chemical reaction involved in the fabrication of GluOx/ ZnONR/PPy/PG electrode

19

Fig.1.

20

Fig. 2.

21

Fig. 3.

22 A

B

C

Fig.4.

23

Fig. 5.

24

Fig. 6.

25

Fig. 7.

26

Fig. 8.

27

Scheme 1.

28 Table 1. A comparison of analytical characteristics of various glutamate biosensors Properti es Source of enzyme Support of immobili zation

Type of transduc er Methods for immobili zation Optimu m pH Tempera ture Linearity

[30]

Teflo ncoate d Pt wire

[32]

[33]

[34]

Strepto myces sp. Graphi te electro de

-

-

Nafio n film on MnO2 bulkmodifi ed carbo n electr odes

Multil ayer of polym er films

Strept omyce s sp. Polyca rbonat e membr ane

-

[36]

[37]

[38]

[39]

[40]

[41]

-

-

-

-

Pt nanop article modifi ed ordere d threedimen sional gold nanow ire arrays (Pt NP/N AEs)

Pt electrod e modifie d with PPy and MWCN T

Tetraful valenetetracya noquino dimetha ne (TTFTCNQ) paste

Networ ks of SWCN Ts enhance d with Pd nanocu bes and Pt nanosph eres

Strepto myces sp. cMW CNT/ AuNP/ CHIT/ Au

Ampero metric

Ampero metric

Ampero metric

DO metric

Ampero metric

-

Crosslinking

-

Crosslinking

Crosslinking

Amper ometri c Covale nt

Ampero metric

Crosslinking

Amper ometric Crosslinking

N-(3dimethy laminop ropyl)N'ethylcar bodiimi de hydroch loride (EDC) activate d thioglyc olic acid (TGA) selfassembl ed monola yer (SAM) -

Strepto myc-es sp. Polyca rbonate membr ane

Crosslinking

Adsorpti on

-

7.0

-

7.4

7.0

7.5

-

8.5

25

-

25

35

-

30

-

0.1 microM -10.0 mM 0.089 µM -

50 nM - 1.6 mM

0.136138 µM

5-500 µM

0.5-50 µM

0.025000 µM

4.6 nM

0.68 µM 120 s -

0.35 µM

0.18 nM

4s 21 days

5s 80 days

-

1.6 µM 2s 120 days Serum

-

Food stuffs

-

Amper ometri c Crosslinking

Entrap ment

Amper ometri -c Entrap ment

7.4

-

7.75

7.4

6.0

7.4

-

-

-

24

-

-

-

DO metric

[35]

Amp erom et-ric Adso rptio n

Detectio 0.3 n limit µM Response T Storage stab Application

[31]

1-250 µM

53855 µM

0.5– 8.0 mM

681271 µM

-

Up to 140 µM

0.7 µM -

9.12 µM -

-

68 µM

-

0.3 µM 7s -

-

120 s 60 days Food sample s

-

-

16 days Soy sauce

0.05 mM 20-50 s 10 days

-

-

Tomato

-

-

[42] Streptom yces sp. BDD/Pt/ PPD (boron-

ZnONR/ PPy/PG

doped diamon d/platin um/poly diallyldi methyl ammoni um chloride )

ZnONR: Zinc oxide nanorods; PGE: pencil graphite electrode; cMWCNT: carboxylated multiwalled carbon naotubes; AuNP: gold nanoparticles; CHIT: chitosan; SWCNT: single walled carbon nanotubes; PPyNPs:Polypyrrole nanoparticles; PANI: Polyaniline

Present work Streptom yces sp.