Synthesis of albumin capped gold nanoparticles and their direct attachment on glassy carbon electrode for the determination of nitrite ion

Accepted Manuscript Synthesis of albumin capped gold nanoparticles and their direct attachment on glassy carbon electrode for the determination of nitrite ion

Sekar Shankar, N.S.K. Gowthaman, S. Abraham John PII: DOI: Reference:

S1572-6657(18)30627-1 doi:10.1016/j.jelechem.2018.09.030 JEAC 12613

To appear in:

Journal of Electroanalytical Chemistry

Received date: Revised date: Accepted date:

10 July 2018 11 September 2018 14 September 2018

Please cite this article as: Sekar Shankar, N.S.K. Gowthaman, S. Abraham John , Synthesis of albumin capped gold nanoparticles and their direct attachment on glassy carbon electrode for the determination of nitrite ion. Jeac (2018), doi:10.1016/ j.jelechem.2018.09.030

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.

ACCEPTED MANUSCRIPT Synthesis of albumin capped gold nanoparticles and their direct attachment on glassy carbon electrode for the determination of nitrite ion Sekar Shankar, N.S.K. Gowthaman and S. Abraham John*

AC

CE

PT

ED

M

AN

US

CR

IP

T

Centre for Nanoscience and Nanotechnology Department of Chemistry The Gandhigram Rural Institute – Deemed to be University Tamil Nadu– 624 302, India.

*Corresponding author: [email protected]; [email protected] 1

ACCEPTED MANUSCRIPT Abstract Development of a facile method for the detection of nitrite ion is very important due to its huge impact on environment. In the present study, water soluble bovine serum albumin capped gold nanoparticles (BSA-AuNPs) were directly attached on glassy carbon (GC) electrode for the

T

sensitive determination of nitrite ion. The BSA-AuNPs were synthesized and HR-TEM images

IP

showed that the average size was ~ 5 nm. The BSA-AuNPs were then directly attached on GC

CR

electrode without any external linker and confirmed by SEM, EDS, XPS, DRS and CV. The appearance of peak at 286 eV (C-N) in XPS confirmed that BSA-AuNPs were attached on GC

US

substrate through Michaeli’s nucleophilic addition. The GC/BSA-AuNPs electrode showed an

AN

excellent catalytic activity towards nitrite by not only shifting its oxidation potential towards less positive potential by 150 mV but also enhanced its oxidation current by 3-fold compared to bare

M

GC electrode. The amperometric current was increased linearly while increasing nitrite

ED

concentration from 10×10-9 to 1×10-6 M (R2=0.9917) and the limit of detection (LOD) by amperometry was found to be 2×10-9 M (S/N=3). The real-time application of the present electro-

PT

catalyst was evidenced by determining nitrite in water samples.

CE

Keywords: Gold nanoparticles, bovine serum albumin, nitrite ion detection, cyclic voltammetry,

AC

amperometry, real sample analysis

2

ACCEPTED MANUSCRIPT 1. Introduction Nitrite ion has been used therapeutically as medication of vasodilation and as an antidote for cyanide poisoning in doses of 30-300 mg without severe toxic effect. However, accidental addition of excessive amounts of nitrite ion to foods has led to instances of poisoning of both adult

T

and children. The proved toxicity of nitrites is primarily due to their interaction with blood pigment

IP

to produce methemoglobinemia and their possible reaction under normally encountered situations

CR

with amines or amides to form toxic nitroso compounds [1]. Higher concentration of nitrosamines has been found in nitrite preserved fish meal intended for animal feeds. Moreover, it produces

US

carcinogenic N-nitrosoamine in human body upon interaction with protein. The formed N-

AN

nitrosoamine has been shown to be carcinogenic for animals, from tobacco smoke and also some foods [2-4]. Further, excess concentration of nitrite ion present in the blood leads to haemoglobin

M

oxidation besides gastric cancer and blue baby syndrome are believed to be combined with nitrite

ED

ion intake [5,6]. The most important acute toxic effect of nitrite ion administration is the induction of methemoglobinemia by oxidation of haemoglobin from the ferrous to ferric form. Excess of

PT

nitrite ions present in drinking water can make adverse health effects, especially for infants and

CE

children [7,8]. Therefore, the U.S. environmental protection agency (EPA) recommended 1 mg/L (1 ppm or 71.4 µM) in drinking water and European community restricted the maximum allowed

AC

concentration of nitrite ion in drinking water to be 0.1 mg/L. Moreover, the world health organization (WHO) recommends 3 mg/L in ground water [9,10]. Therefore, determination of nitrite ion at trace level in water is of special interest in the current research. Spectrophotometry [11,12], surface-enhanced Raman scattering [13], chromatography [14,15], capillary electrophoresis [16], chemiluminescence [17,18] and electrochemical methods [19-21] have been used for the determination of nitrite ion. Among them, electrochemical methods 3

ACCEPTED MANUSCRIPT have several advantages which include simple instrumentation, fast response, reliability, good selectivity and sensitivity. However, stable determination of nitrite ion is not possible at a bare electrode because species present in water poison the electrode surface. In particular, the excellent properties of AuNPs encouraged exploiting them for a various applications [22,23].

T

Electrochemical sensing applications of AuNPs modified electrodes have gained significant

IP

attention for the past two decades. Few reports on the determination of nitrite ion using AuNPs

CR

modified electrodes have been published [24-26]. For example, Wang et al. reported the attachment of AuNPs on the surface of choline chloride modified GC electrode for the determination of nitrite

US

ion with 1.0×10-7 M limit of detection (LOD) [24]. Liu et al. co-deposited Au and Fe(III)

AN

nanoparticles on GC electrode and reported a LOD of 2 × 10-7 M for nitrite ion [25]. Although the reported papers showed good LOD the fabrication of electrode involves complicated procedure

M

because external linker is required to attach the AuNPs. Hence, development of an electrochemical

ED

sensor involving simple electrode modification procedure for the sensitive and selective determination of nitrite ion is very important. Thus, the aim of the present study is to directly attach

PT

AuNPs on glassy carbon (GC) electrode for the sensitive and selective determination of nitrite ion

CE

using BSA capped AuNPs (BSA-AuNPs) synthesized by wet chemical method. BSA capped Au nanoclusters have been extensively synthesized and used for optical sensing applications [27,28].

AC

However, synthesis of BSA capped AuNPs and their electrochemical sensing applications have not been reported so far.

BSA is the most abundant serum protein (66.7 kDa) present in plasma and has good property to interact in a reversible manner with number of compounds [29]. It contains large number of thiol groups (35 cysteine residues which form 17 internal disulfide bonds) and the free thiol group residue of BSA could be spontaneously attached on AuNPs surface through the 4

ACCEPTED MANUSCRIPT formation of Au-S bond. In the present study, amine groups present on the surface of BSA-AuNPs was used for the direct attachment of AuNPs without any linker on the GC electrode through Michaelis type addition reaction [30]. The BSA-AuNPs modified GC electrode shows an excellent electrocatalytic oxidation of nitrite ion. The amperometric current response of nitrite linearly

T

increases against the concentration in the range of 10×10-9 to 1×10-6 M (R2 = 0.9965) and the LOD

IP

was found to be 2×10-9 M (S/N=3). Further, the modified electrode was used for the selective

exploited to determine nitrite ion in tap water samples.

US

2. Experimental section

CR

determination of nitrite in 4000 fold-excess presence of the common interferences. It was also

AN

2.1. Chemicals

Sigma-Aldrich products of HAuCl4.3H2O and bovine serum albumin (BSA) were

M

purchased and used as received. Sodium borohydride and sodium nitrite were purchased from

ED

Merck and used as received. 0.2 M phosphate buffer (PB) solution (pH 7.2) was prepared using Na2HPO4 and NaH2PO4. Glassy carbon (GC) plates (1 mm thickness) were purchased from Alfa

PT

Aeaser (India). All other chemicals utilized in this investigation were of analar grade. Double

CE

distilled water was used to prepare all the solutions. 2.2. Synthesis of BSA-AuNPs

AC

All the glasswares were cleaned using aqua regia (3:1; HCl:HNO3). A well dispersed colloidal solution of BSA-AuNPs was prepared by the following procedure. Briefly, 0.3 ml of HAuCl4.3H2O (31.7 mM) was added into 10 ml of millipore water in a beaker. To this solution, 1.3 ml of fresh ice cold NaBH4 (0.25%) was added with constant stirring for 10-15 min at room temperature and the solution color was changed into wine red immediately after the addition of NaBH4. Then, 1 % of 0.2 ml of freshly prepared BSA (tris buffer pH 7) was added drop by drop 5

ACCEPTED MANUSCRIPT and the wine red remains same and the solution was further stirred for 30 min. The synthesized BSA-AuNPs were stored in a bottle at 4⁰C and they remained stable for several months. 2.3. Modification of GC electrode with BSA-AuNPs Prior to the electrochemical experiment, the GC working electrode of geometric area

T

0.07cm2 was mirror polished with alumina powder (0.05 µm). After sonication for 5-10 min in

IP

ethanol and water, the cleaned GC electrode was checked in 0.2 M PB solution containing 1 mM

CR

K3[Fe(CN)6]. The concentration of BSA-AuNPs was found to be 260 µM. The well cleaned GC

US

electrode was immersed into 0.5 ml of (260 µM) BSA-AuNPs solution. We kept the volume (0.5 ml) and concentration (260 µM) of BSA-AuNPs constant and varied the immersion times (4, 6, 8

AN

and 10 h). We found that higher Au oxide peak current was observed at 8 h immersion time. Hence, the BSA-AuNPs modified electrode was prepared by immersing a well cleaned GC electrode in the

M

colloidal solution of BSA-AuNPs for 8 h and then washed with water. The resulting electrode is

ED

termed as GC/BSA-AuNPs electrode (Scheme 1). For SEM, EDS, XRD and XPS measurements GC plates were used to modify BSA-AuNPs similar to modification on GC electrode. The detailed

PT

instrumentation methods were described in ESI.

CE

3. Results and discussion

3.1. Characterization of BSA-AuNPs

AC

Fig.S1 shows the UV-vis absorption spectra corresponding to the colloidal solution of bare and BSA-AuNPs. The bare AuNPs (i.e prepared in the absence of BSA) show the surface plasmon resonance (SPR) band at 519 nm (curve a). On the other hand, the colloidal solution of BSA-AuNPs exhibits the SPR band at 513 nm (curve b). The synthesized BSA-AuNPs were stable for more than six months. The obtained 6 nm blue shift indicates the interaction of BSA with AuNPs. BSA contains large number of thiol groups (35 cysteine residues which form 17 internal disulfide bonds). 6

ACCEPTED MANUSCRIPT The presence of free thiol group residue could be spontaneously attached onto AuNPs surface by means of the formation of Au-S bond (i.e. -SH of the cysteine that binds to the AuNPs surface). Recently, interaction between silver nanoparticles (AgNPs) and BSA was studied [31,32]. These studies suggested that a hydrophobic interaction (i.e. ΔH°>0, ΔS°>0) was predominant between

T

BSA and AgNPs. Further, hydrophobic interaction of protein side chains or domains of BSA with

IP

AuNPs was expected, which resulted reduction in entropy and a loss of solvation enthalpy, both of

CR

which keep the system more stable. The size and morphology of the bare AuNPs and BSA-AuNPs were examined by HR-TEM. The HR-TEM images obtained for bare AuNPs and BSA-AuNPs are

US

shown in Fig.1. The TEM images of bare AuNPs recorded at different magnifications show that the

AN

AuNPs were spherical and the size of the AuNP was found to be ~ 9.3 nm (Figs.1a and b). On the other side, TEM images of BSA-AuNPs show that they were roughly spherical shape with the size

M

of ~5 nm. This was also evidenced from the 6 nm blue shift in UV-visible spectrum for AuNPs after

ED

the addition of BSA. The obtained decrease in the particle size of BSA-AuNP was attributed to the adsorption of BSA onto the surface of AuNPs. Similar decrease in particle size of ZnO after the

PT

addition of BSA was recently reported [33]. Further, the high resolution TEM image of single

CE

AuNP shows the one dimensional lattice fringes (Inset; Fig.1c). The d spacing was estimated to be 0.22 nm, which was consistent with the gold lattice of the 111 plane. The selected area electron

AC

diffraction pattern shown in Fig.1d (inset) confirms the crystalline nature of the BSA-AuNPs. 3.2. Characterization of BSA-AuNPs modified substrate by XRD, SEM, EDS, DRS and XPS The BSA-AuNPs contains several amine groups on their surface. These free amino groups are utilized to attach the BSA-AuNPs on GC electrode through Michaeli’s type nucleophilic addition [30]. The crystalline nature of the BSA-AuNPs was investigated by XRD. The XRD pattern of the BSA-AuNPs shows well defined peaks at 38.3°, 43.3° and 77.9°, corresponding to 7

ACCEPTED MANUSCRIPT (111), (200) and (311) planes of Au, respectively (Fig.S2). The diffraction peaks were well indexed with spherical AuNPs and the reflection peaks of the AuNPs were in good agreement with JCPDS: 65-2870. The morphology of the BSA-AuNPs modified on GC substrate was investigated by SEM. Fig.2 shows the SEM images obtained for bare GC and GC/BSA-AuNPs plates. The SEM image of

T

bare GC plate does not show any particles (Fig. 2A). But, BSA-AuNPs modified GC plate shows

IP

that the AuNPs were fully covered on GC substrate (Fig. 2B). This confirms the successful

CR

modification of BSA-AuNPs on GC substrate. The EDS was used to analyze the elements present on the surface with their chemical compositions. Fig.S3 shows EDS obtained for BSA-AuNPs on

US

GC substrate. The peaks obtained at 2.1 and 9.7 keV are the characteristic peaks of Au and 0.39

AN

keV is the characteristic peak of nitrogen and the weight percentage of Au and N is 10 and 4.5%, respectively. The presence of both Au and N peaks confirms the successful modification of BSA-

M

AuNPs on GC substrate. Further, the modification of BSA-AuNPs on GC electrode was

ED

characterized by DRS. Fig.S4 shows the diffuse reflectance spectrum (DRS) of BSA-AuNPs modified GC substrate. It shows an absorption peak at 591 nm. The electromagnetic interaction of

PT

the attached AuNPs with the substrate is the cause for the observed more than 40 nm red shift in

CE

contrast to the colloidal solution of BSA-AuNPs [34]. Fig.3a shows the XPS survey spectrum of bare and BSA-AuNPs modified GC substrates. The bare GC surface shows only C1s and O1s peaks

AC

(curve i). The presence of O1s peak for the bare GC surface is likely due to the surface oxidation during the preparation. XPS survey spectrum showed that Au4f, N1s, C1s and O1s elements were appeared for the GC/BSA-AuNPs substrate (curve ii). The presence of Au4f and N1s elements for GC/BSA-AuNPs surface confirms the successful modification of BSA-AuNPs on GC substrate [35]. The nature of C, N and Au present on the BSA-AuNPs was further analyzed by deconvoluting the specific regions (Fig.3 b-d). Au4f, C1s and N1s regions were analyzed by Gaussian functions 8

ACCEPTED MANUSCRIPT after background correction. The Au4f spectrum depicts the characteristic Au4f5/2 and Au4f7/2 peaks respectively at 87.8 and 84.2 eV with a spin-orbit coupling of 3.6 eV (Fig.3b). This indicates the presence of zero valency Au. The N1s spectrum of BSA-AuNPs modified substrate was deconvoluted into two components at 398.2 and 399.6 eV and were attributed to sp2 hybridized N

T

atom linkage (=N-) and free amino group (-NH2) (Fig.3c), respectively. These results supported the

IP

direct attachment of BSA-AuNPs on substrate. Further, it reveals that few unreacted free amino

CR

groups present on their surface even after attached on GC substrate. The C1s spectrum of the BSAAuNPs modified was deconvoluted into four component peaks at 284.4, 286, 287.3 and 289.1 eV

US

(Fig.3d) and were attribution of the C=C, -C-N, -C=N and O=C-OH bonds on the BSA-AuNPs

AN

modified substrate. The obtained results reveal the presence of free carboxyl groups of BSA on the surface and it could be useful for making hydrogen bond with the targeted analytes for sensing

M

applications.

ED

3.3. Characterization of GC/BSA-AuNPs electrode by CV The modification of BSA-AuNPs on GC electrode was further confirmed by cyclic

PT

voltammetry. The electrochemical behavior of bare GC, GC/BSA, GC/bare-AuNPs and GC/BSA-

CE

AuNPs electrodes in 1 mM K3[Fe(CN)6] containing 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s-1 is shown in Fig.S5. Bare GC electrode shows a well defined redox peak for the redox probe,

AC

[Fe(CN)6]3-/4- with a peak separation of 75 mV, indicating the quasi-reversible one-electron transfer process. On the other hand, both reduction and oxidation peak currents due to [Fe(CN)6]3-/4- redox couple were significantly decreased at GC/BSA modified electrode. Further, the peak separation was increased to 225 mV when compared to bare GC electrode. This indicates the blocking nature of GC/BSA modified electrode towards the redox response of [Fe(CN)6]3-/4- couple. However, after the attachment of bare AuNPs, the electron transfer reaction was restored with 93 mV peak 9

ACCEPTED MANUSCRIPT separation and considerable increase in the peak current. Similarly, the electron transfer reaction was very facile after the attachment of BSA-AuNPs with a slight increase of peak current with a peak separation of 90 mV when compared to bare Au-NPs, indicating the efficient pathway provided by BSA-AuNPs for electron transfer. The observed electrochemical behavior exhibits that

T

the attached BSA-AuNPs act as an ‘electron antenna’ which forms a good conducting surface that

IP

facilitates the electron transfer. The obtained results suggest that BSA-AuNPs promoted the

CR

electron transfer reaction in contrast to BSA and bare AuNPs modified electrodes. As shown in Fig. S5, the redox current of [Fe(CN)6]3-/4- couple at the BSA-AuNPs

US

modified electrode was higher than that of bare and GC/BSA electrodes. This suggests that the

AN

attachment of BSA-AuNPs on GC electrode increased the effective electroactive surface area in agreement with Randles-Sevcik equation (i.e. the peak current is directly proportional to electrode

M

surface area). The effective surface area was calculated by using the Randles-Sevcik equation (1)

ED

[36].

Ip = 2.69 × 105AD1/2 n3/2 ν1/2 C --------------- (1)

PT

where Ip is maximum current, A is the effective surface area of the electrode (cm2), D is the

CE

diffusion coefficient (6.7 × 10-6cm2 s-1 for [Fe(CN)6]3-/4- redox probe), n is number of electron

AC

transferred in redox event (n=1 for [Fe(CN)6]3-/4- redox probe), ν is the scan rate (50 mV s-1) and C is the concentration of the redox probe (1.0 mM). The effective surface area was found to be 0.05, 0.04 and 0.06 cm2 for bare GC, GC/BSA and GC/BSA-AuNPs electrodes, respectively. The obtained higher effective surface area of GC/BSA-AuNPs electrode was attributed to the large surface area provided by BSA-AuNPs. The immersion time for the fabrication of BSA-AuNPs on GC electrode was optimized by varying the time (4, 6, 8 and 10 h). Fig.S6 shows the CVs obtained for the modification of BSA10

ACCEPTED MANUSCRIPT AuNPs at different time intervals in 0.2 M PB solution. No electrochemical response was observed for 4 h immersion of GC electrode in BSA-AuNPs (curve a) whereas less pronounced gold oxide oxidation and reduction peaks were appeared at +0.88 V and +0.45 V, respectively for 6 h immersion (curve b). However, when the immersion time was increased to 8 h, the Au oxidation

T

and reduction peaks were dramatically increased (curve c). Further increasing the immersion time

IP

to 10 h does not change the Au oxidation and reduction peak currents. Hence, 8 h immersion time is

CR

optimized for the modification of BSA-AuNPs on GC electrode. Since AuNPs were capped with a protein, 8 h was required to attach maximum AuNPs on the GC electrode. Here, the presence of

US

amine groups on BSA was involved in the direct attachment with GC electrode through Michaelis

AN

addition reaction [30]. The BSA-AuNPs modified electrode was also optimized with respect to pH. The pH studies are carried out from 4 to 12 using PB solution (Fig.S7). While varying the pH from

M

4 to 10, the Au oxidation current was increased and maximum peak current was observed at pH 7

ED

and after that it started to decrease from pH 8 to 10. Hence, all the experiments were carried out at a physiological pH of 7.2. Fig.S8 shows the continuous CVs (5 cycles) for GC/BSA-AuNPs electrode

PT

at a sweep rate of 50 mV s-1 in 0.2 M PB solution (pH 7.2). The oxidation and reduction peaks of

CE

gold oxide remain stable even after 5 cycles. This suggests that the BSA-AuNPs attached on GC electrode were highly stable.

AC

3.4. EIS studies of BSA-AuNPs modified electrode Fig.S9 shows the Nyquist plot for bare GC, GC/BSA and GC/BSA-AuNPs electrodes in 1 mM K3[Fe(CN)6] containing 0.2 M PB solution (pH 7.2) at 0.01 to 100000 Hz scanning frequencies. The plots obtained were best fitted with equivalent electrical circuit (Fig S9 inset), where R refer to the resistance, Q refers to the constant phase element and W refers to the Warburg element. The charge transfer resistance (RCT) values for bare GC, GC/BSA and GC/BSA-AuNPs 11

ACCEPTED MANUSCRIPT electrodes were found to be 33469, 42220 and 5689 Ω, respectively. The RCT value of BSA-AuNPs modified electrode was significantly lower than bare GC and GC/BSA electrodes, indicating high conductive nature of BSA-AuNPs modified electrode. Further, the heterogeneous electron-transfer rate constant (ket) for the redox probe

T

Fe(CN)63-/4- at the modified electrode can be calculated using equation (2)

IP

ket=RT/n2F2ARCTC° ------------ (2)

CR

The calculated ket values are 1.13 × 10-4, 9.0 × 10-5 and 6.68 × 10-4 cm s-1 for bare GC, GC/BSA and GC/BSA-AuNPs electrodes, respectively. The obtained higher ket value at GC/BSA-AuNPs

US

electrode indicates that the electron transfer reaction was faster at this electrode than bare GC and

AN

GC/BSA electrodes.

3.5. Electrochemical oxidation of nitrite ion at GC/BSA-AuNPs electrode

M

The electrochemical oxidation of nitrite ion at GC/BSA-AuNPs electrode was studied by linear

ED

sweep voltammetry (LSV). Fig.4 shows the oxidation of nitrite ion at bare GC, GC/bare AuNPs and GC/BSA-AuNPs electrodes. The oxidation of nitrite ion occurs at 0.97 V for bare GC electrode

PT

(curve b) and it was shifted to more positive potential in the subsequent cycles with decreased peak

CE

current. After 5 cycles, the oxidation potential of nitrite ion was shifted from 0.97 to 1.1 V with decreased peak current (curve b’). This is due to the adsorption of the oxidized products of nitrite

AC

on the GC electrode surface. Therefore, bare GC electrode was not suitable for the stable determination of nitrite. The GC/bare AuNPs shows the oxidation of nitrite ion at 0.99 V (curve a). After 5 cycles, the oxidation current was significantly decreased besides the oxidation potential was shifted from 0.99 to 1.1 V (curve a’). The decrease in peak current and shift in oxidation potential of nitrite ion were attributed to the leaching of bare AuNPs from the GC electrode. However, the GC/BSA-AuNPs electrode shows a sharp oxidation peak for nitrite ion at 0.82 V (curve m) with 12

ACCEPTED MANUSCRIPT three fold higher oxidation current and 150 mV less positive potential shift than bare GC electrode. The obtained higher oxidation current at GC/BSA-AuNPs electrode was ascribed to the large surface area provided by BSA-AuNPs in contrast to bare electrode (curve b). Besides, the possible hydrogen bonding interaction between nitrite ion and free carboxyl group of BSA also responsible

T

for the enhanced oxidation current. Further, the oxidation peak potential remains stable at the

IP

GC/BSA-AuNPs electrode even after five cycles (curve m’). This shows that the GC/BSA-AuNPs

CR

electrode can be used as a stable electrocatalyst for the nitrite oxidation in contrast to bare GC electrode. The mechanism for the oxidation of nitrite in neutral medium is shown below (equations

2NO2 + 2e-

-------- (3)

AN

2NO2-

US

(3) and (4)) [37]. The oxidation of nitrite gives nitrate ion.

NO3- + NO2- + 2H+ -------- (4)

2NO2 + H2O

M

3.6. Effect of scan rate

ED

Fig.S10 shows the LSVs obtained for 0.5 mM nitrite ion at GC/BSA-AuNPs electrode in pH 7.2 solution at different sweep rates. The oxidation peak current of nitrite was increased with the

PT

increase of sweep rate from 10-100 mV s-1. A good linearity between the nitrite oxidation current

CE

and square root of the sweep rate was obtained with a correlation coefficient of 0.9998, indicating that the oxidation of nitrite ion was a diffusion controlled process at GC/BSA-AuNPs electrode.

AC

3.7. Determination of nitrite ion by differential pulse voltammetry (DPV) Since GC/BSA-AuNPs electrode shows a stable response towards nitrite oxidation, sensitive detection of nitrite was carried out by DPV using GC/BSA-AuNPs electrode. Fig.5 shows the DPVs obtained for each 5 µM addition of nitrite ion in 0.2 M PB solution (pH 7.2) at BSAAuNPs modified electrode. The addition of 5 µM nitrite ion shows an oxidation peak at 0.77 V. Further increasing each 5 µM of nitrite ion from 10 to 75 µM, the current was increased without 13

ACCEPTED MANUSCRIPT shifting its oxidation potential at GC/BSA-AuNPs electrode and the sensitivity was 0.1560 μA μM-1 by DPV. 3.8. Amperometric determination of nitrite ion at GC/BSA-AuNPs electrode Fig.6 displays the amperometric i-t curve for the oxidation of nitrite ion at BSA-AuNPs

T

modified electrode in a homogeneously stirred pH 7.2 PB solution with a constant applied potential

IP

of +1.0 V. The GC/BSA-AuNPs electrode exhibit the initial current response for 10 nM nitrite ion

CR

and further addition of 30 nM nitrite into the same solution with 50 s interval, the increase in current response was observed and within 3 s the steady state current response was reached. The

US

current response was increased for further addition of 50, 100, 300, 500 and 1000 nM nitrite ion to

AN

the same solution with a time interval of 50 s. The amperometric current was increased linearly with increasing nitrite ion concentration from 10 × 10-9 M to 1 × 10-6 M with a correlation

M

coefficient of 0.9917. The limit of detection (LOD) was found to be 2×10-9 M (S/N=3) and the

ED

sensitivity was found to be 1.0 μA μM-1 by amperometry. The wide range of determination and LOD obtained for nitrite ion at GC/BSA-AuNPs electrode were compared with the reported papers

PT

[24-26, 38-47] (Table S1). It can be seen from Table S1 that the present sensor showed the lowest

CE

LOD with a wide concentration range determination of nitrite ion. Further, the modification of AuNPs in the reported papers involves tedious procedures which include a suitable linker to attach

AC

the AuNPs. On the other hand, the present modification is facile because it does not require any external linker to attach the AuNPs. 3.9. Effect of interferences The determination of nitrite ion in the presence of common and physiological interferences such as Na+, K+, NH4+, Cu2+, NO3-, CH3COO-, F-, SO42-, glucose, uric acid, ascorbic acid and hydrazine was studied at GC/BSA-AuNPs electrode by amperometry. Fig.7 displays the 14

ACCEPTED MANUSCRIPT amperometric i-t curve obtained for nitrite ion at GC/BSA-AuNPs electrode in the presence of above interferences in pH 7.2 PB solution. The current response was increased for the addition of 50 nM nitrite ion (curve a) and further addition of 50 nM nitrite ion in the next step with 50 s sample interval. While adding 200 µM each Na+, K+, NH4+ and Cu2+ (b-e) the increase in the

T

current response was not observed. However, the current response was increased similar to the early

IP

steps after the addition of 50 nM nitrite ion to the same solution. Further addition of 200 µM each

CR

NO3-, CH3COO-, F- and SO42- (f-i) to the same solution caused no change in current response. Similarly, the addition of 200 µM each glucose, uric acid, ascorbic acid and hydrazine (j-m) did not

US

change the current response. These results indicated that the determination of 50 nM nitrite ion is

AN

possible even in the presence of 4000-fold excess of common and physiological interferences. 3.10. Practical application

M

The practical application of the GC/BSA-AuNPs was demonstrated by determining the

ED

concentration of nitrite ion present in the tap water samples collected from our university campus by amperometry. Fig.S11 shows the amperometric current response for nitrite ion in tap water

PT

samples at GC/BSA-AuNPs electrode in a homogeneously stirred PB solution (pH 7.2) with an

CE

applied potential of +1.0 V. Water sample in PB solution (pH 7.2) did not show any response. This indicates that tap water sample is free from nitrite ions. However, addition of 30 µM nitrite ion to

AC

the water sample, current response was increased. Further 40 and 50 µM nitrite ion addition into the same solution increased the current response and the results are given in Table S2. The observed current response was compared with the current response of known nitrite ion concentration. As shown in Table S2, the recovery was found between 99.7 and 100.4 % and the relative standard deviation of the three measurements was between 0.39–0.76, which proved that the modified electrode can be successfully applied to determine nitrite ion in real samples. Finally, the present 15

ACCEPTED MANUSCRIPT method of determination of nitrite ion is validated with the HPLC method. As shown in Table S3, a good agreement with the HPLC method analysis was observed. 3.11. Stability of the GC/BSA-AuNPs electrode The BSA-AuNPs modified electrode was very stable while kept in 0.2 M PB solution. To

T

check the stability of the electrode towards the determination of nitrite, the LSV was carried out at

IP

every 20 min interval time. It was found that the oxidation current of nitrite remains same with

CR

2.1% relative standard deviation for 10 repetitive measurements, indicating the good durability and reproducibility of the present modified electrode. To further ascertain the repeatability of the

US

results, three different BSA-AuNPs modified electrodes were prepared and their response towards

AN

the oxidation of nitrite was tested by 10 repeated measurements. The oxidation peak potential of nitrite remains same at all the three electrodes. The above results reveal the high stability and

M

reproducibility of the present modified electrode towards the determination of nitrite ion.

ED

4. Conclusions

In this study, we have demonstrated a direct and simple strategy to fabricate BSA-AuNPs

PT

on GC electrode for the determination of nitrite ion. The BSA capped AuNPs were synthesized and

CE

were attached on GC electrode without the aid of any linker for the first time. In the XPS studies, the C 1s component at 286 eV (C-N) is ascribed to carbon attached to the nitrogen groups, which

AC

confirms the attachment of BSA-AuNPs on the GC electrode. The GC/BS-AuNPs electrode was utilized for the determination of nitrite ion. The higher electrocatalytic activity is attributed to the large surface area provided by BSA-AuNPs. Further, the LOD of nitrite ion was found to be 2×10-9 M (S/N=3). The modified electrode showed good selectivity towards nitrite ion even in the presence of 4000-fold common interferences. The practical application of the present electrocatalyst was proved by determination of nitrite ion in water samples. 16

ACCEPTED MANUSCRIPT Acknowledgement Financial support from Science and Engineering Research Board (SERB), Department of

AC

CE

PT

ED

M

AN

US

CR

IP

T

Science and Technology (EMR/2016/002898), New Delhi, India is gratefully acknowledged.

17

ACCEPTED MANUSCRIPT References [1] I.A. Wolf, A.E. Wasserman, nitrates, nitrites and nitrosamines, Science 177 (1972) 15-19. [2] W. Lijinsky, S.S. Epstein, N-nitrosamines as environmental carcinogens, Nature 225 (1970) 2123.

T

[3] N.S. Bryan, D.D. Alexander, J.R. Coughlin, A.L. Milkowski, P. Boffetta, Ingested nitrate and

IP

nitrite and stomach cancer risk: An updated review, Food. Chem. Toxicol. 50 (2012) 3646-

CR

3665.

[4] M.C.Archer, S.D. Clark, J.E. Thilly, S.R. Tannenbaum, Environmental nitroso compounds:

US

reaction of nitrite with creatine and creatinine, Science 174 (1971) 1341-1343.

AN

[5] D.M. Manassaram, L. C. Backer, D.M. Moll, A review of nitrates in drinking water: maternal exposure and adverse reproductive and developmental outcomes, Environ. Health Perspect.

M

114 (2006) 320-27.

ED

[6] D.M. Zurcher, Y.J. Adhia, J.D. Romero, A.J. Mcneil, Modifying a known gelator scaffold for nitrite detection, Chem. Commun. 50 (2014) 7813-16.

PT

[7] C. Jo, H.J. Ahn, J.H. Son, J.W. Lee, M.W. Byun, Packaging and irradiation effect on lipid

CE

oxidation, color, residual nitrite content and nitrosamine formation in cooked pork sausage, Food Control 14 (2003) 7-12 .

AC

[8] P.F. Swann, The toxicology of nitrate, nitrite and N-Nitroso compounds, J. Sci. Fd. Agric, 26 (1975) 1761-1770. [9] Official Journal of the European Communities, No. L229, 15 July 1980 (EPA). [10] Guide lines for Drinking-Water Quality, Vol. 1, 3 rd ed., World Health Organization, Geneva 2004.

18

ACCEPTED MANUSCRIPT [11] W.L. Daniel, M.S. Han, J.S. Lee, C.A. Mirkin, Colorimetric nitrite and nitrate detection with gold nanoparticle probes and kinetic end points, J. Am. Chem. Soc. 131 (2009) 6362-6363. [12] V.V. Kumar, S.P. Anthony, Highly selective silver nanoparticles based label free colorimetric

T

sensor for nitrite anions, Anal. Chim. Acta 842 (2014) 57-62.

of p-aminothiophenol

to

p,p’-dimercaptoazobenzene on

CR

catalytic conversion

IP

[13] X. Liu, L. Tang, R. Niessner, Y. Ying, C. Haisch, Nitrite-triggered surface plasmon-assisted gold

nanoparticle: surface-enhanced Raman scattering investigation and potential for nitrite

US

detection, Anal. Chem. 87(2014) 499-506.

AN

[14]. J.M. Doyle, M.L. Miller, B.R. McCord, D.A. McCollam, G.W. Mushrush, A multicomponent mobile phase for ion chromatography applied to the separation of anions from the residue of

M

low explosives, Anal. Chem. 72 (2000) 2302-2307.

ED

[15] M.D. Croitoru, Nitrite and nitrate can be accurately measured in samples of vegetal and animal origin using an HPLC-UV/Vis technique, J. Chromatogr. B 911 (2012) 154-161.

PT

[16] M.C.B. Alonso, R. Prego, Determination of silicate, simultaneously with other nutrients

CE

(nitrite, nitrate and phosphate) in river waters by capillary electrophoresis, Anal. Chim. Acta 416 (2000) 21-27.

AC

[17] A.F. Lagalante, P.W. Greenbacker, Flow injection analysis of imidacloprid in natural waters and agricultural matrixes by photochemical dissociation, chemical reduction and nitric oxide chemiluminescence detection, Anal. Chim. Acta 590 (2007) 151-158. [18] J. Li, Q. Li, C. Lu, L. Zhao, Determination of nitrite in tap waters based on fluoro surfactantcapped gold nanoparticles-enhanced chemiluminescence from carbonate and peroxynitrous acid, Analyst 136 (2011) 2379-2384. 19

ACCEPTED MANUSCRIPT [19] H.Wang, P. Chen, F. Wen, Y. Zhu, Y. Zhang, Flower-like Fe2O3@MoS2 nanocomposite decorated GCE for the determination of nitrite. Sens. Actuators B 220 (2015) 935-945. [20] M. Liu, L. Wang, Y. Meng, Q. Chen, H. Li, Y. Zhang, S. Yao, 4-Ferrocenylethyne phenylamine functionalized Graphene oxide modified electrode for sensitive nitrite sensing,

T

Electrochim. Acta 116 (2014) 504-511.

IP

[21] N. Wang, X. Cao, X. Cai, Y. Xu, L. Guo, Porous cuprite films: Facile solution deposition

CR

and their application for nitrite sensing, Analyst 135 (2010) 2106-2110. [22] R. Sardar, A.M. Funston, P. Mulvany, R.W. Murray, Gold nanoparticles: past, present and

US

future, Langmuir 25 (2009) 13840-13851.

AN

[23] M.C. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantumsize-related properties and applications toward biology, catalysis and nanotechnology, Chem.

M

Rev. 104 (2004) 293-346.

ED

[24] P. Wang, Z. Mai, Z. Dai, Y. Li, X. Zou, Construction of Au nanoparticles on choline chloride

(2009) 3242-3247.

PT

modified glassy carbon electrode for sensitive detection of nitrite, Biosens. Bioelectron. 24

CE

[25] T.S. Liu, T.F. Kang, L.P. Lu, Y. Zhang, S.Y. Cheng, Au-Fe(III) nanoparticle modified glassy carbon electrode for electrochemical nitrite sensor. J. Electroanal. Chem. 632 (2009)

AC

197-200.

[26] P. Muthukumar, S.A. John, Gold nanoparticles decorated on cobalt porphyrin modified GCE for the sensitive determination of nitrite ion, J. Colloid. Interface Sci. 421 (2014) 78-84. [27] B.X. Guoa, F. Wub, Y. Nia, S. Kokotc, Synthesizing a nano-composite of BSA-capped Au nanoclusters/graphitic carbon nitride nanosheets as a new fluorescent probe for dopamine detection, Anal. Chim. Acta. 942 (2016) 112-120. 20

ACCEPTED MANUSCRIPT [28] M.S. Mathew, A. Baksi, T. Pradeep, K. Joseph, Choline-induced selective fluorescence quenching of acetylcholinesterase conjugated Au@BSA clusters, Biosens. Bioelectron. 81 (2016) 68-74. [29] B. Zhang, X. Wang, F. Liu, Y. Cheng, D. Shi, Effective reduction of nonspecific binding by

T

surface engineering of quantum dots with bovine serum albumin for cell-targeted imaging.

IP

Langmuir 2 (2012) 16605-16613.

CR

[30] I. Gallardo, J. Pinson, N. Vila, Attachment of amines to carbon and metallic surfaces, J. Phys. Chem. B 110 (2006) 19521-19529

R.A. Singh, A.M. Raichur, N. Chandrasekaran, A. Mukherjee, Studies on interaction of

US

[31]

AN

colloidal Ag nanoparticles with bovine serum albumin, Colloids Surf. B Biointerfaces 76 (2010) 32-37.

J. Mariam, P.M. Dongre, D.C. Kothari, Study of interaction of silver nanoparticles with

M

[32]

ED

bovine serum albumin using fluorescence spectroscopy, J. Fluoresc, 21 (2011) 2193–2199. [33] R. Tantra, J. Tompkins, P. Quincey, Characterization of the de-agglomeration effects of

CE

75 (2010) 275-281.

PT

bovine serum albumin on nanoparticles in aqueous suspension, Colloids Surf. B: Biointerfaces

[34] T. Okamoto, I. Yamaguchi, Optical absorption study of the surface plasmon resonance in gold

AC

nanoparticles immobilized onto a gold substrate by self-assembly technique, J. Phys. Chem. B 107 (2003) 10321-10324. [35] J.F. Moulder, W.F. Stickle, P.E. Sobol, K.D. Bomben, Handbook of X-Ray Photo-electron Spectroscopy, Perkin-Elmer Corp., MN, 1992.

21

ACCEPTED MANUSCRIPT [36] X. Gao, W. Wei, L. Yang, M. Guo, Carbon nanotubes/poly(1,2‐diaminobenzene) nanoporous composite film electrode prepared by multipulse potentiostatic electropolymerisation and its application to determination of trace heavy metal ions, Electroanalysis, 18 (2006) 485-492. [37] B. Piela, P.K. Wrona, Oxidation of nitrites on solid electrodes; Determination of the reaction

T

mechanism on the pure electrode surface, J. Electrochem. Soc, 149 (2002) E55.

IP

[38] D. M. Stankovic, E. Mehmeti, J. Zava, K. Kalcher, Determination of nitrite in tap water: A

CR

comparative study between cerium, titanium and selenium dioxide doped reduced graphene

US

oxide modified glassy carbon electrodes. Sens. Actuators B 236 (2016) 311-317. [39] S. Yang, X. Zeng, X. Liu, W. Wei, S. Luo, Y. Liu, Y. Liu, Electrocatalytic reduction and

AN

sensitive determination of nitrite at nano-copper coated multi-walled carbon nanotubes modified glassy carbon electrode, J. Electroanal. Chem. 639 (2010) 181-186.

M

[40] F. Liang, M. Jia, J. Hu, Pt-implanted indium tin oxide electrodes and their amperometric sensor

ED

applications for nitrite and hydrogen peroxide, Electrochim. Acta 75 (2012) 414-419. [41] S. Radhakrishnan, K. Krishnamoorthy, C. Sekar, J. Wilson, S.J. Kim, A highly sensitive

PT

electrochemical sensor for nitrite detection based on Fe2O3 nanoparticles decorated reduced

CE

graphene oxide nanosheets. Appl. Catal. B 148-49 (2014) 22-28. [42] B.S. He, D.D. Yan, One-pot preparation of wavy graphene/Au composites and their application

AC

for highly sensitive detection of nitrite, Anal. Methods. 10 (2018) 3654-3659. [43] J. Losada, M.P.G. Armada, E. Garcia, C.M. Casado, B. Alonso, Electrochemical preparation of gold nanoparticles on ferrocenyl-dendrimer film modified electrodes and their application for the electrocatalytic oxidation and amperometric detection of nitrite, J. Electroanal. Chem. 788 (2017) 14-22.

22

ACCEPTED MANUSCRIPT [44] K. Promsuwan, P. Thavarungkul, P. Kanatharana, W. Limbut, Flow injection amperometric nitrite sensor based on silver microcubics-poly(acrylic acid)/poly (vinyl alcohol) modified screen printed carbon electrode, Electrochim. Acta 232 (2017) 5357-369. [45] N. Jaiswal, I. Tiwari, C.W. Foster, C.E. Banks, Highly sensitive amperometric sensing of

T

nitrite utilizing bulk-modified MnO2 decorated graphene oxide nanocomposite screen-printed

IP

electrodes, Electrochim. Acta 227 (2017) 255-266.

CR

[46] S. Kesavan, D.R. Kumar, M.L. Baynosa, J.J. Shim, Potentiodyanmic formation of diaminobenzene films on an electrochemically reduced graphene oxide surface: Determination

US

of nitrite in water samples, Mater. Sci. Eng. C, 85 (2018) 97-106.

AN

[47] Y. Zhang, F. Wen, J. Tan, C. Jiang, M. Zhu, Y. Chen, H. Wang, Highly efficient electrocatalytic oxidation of nitrite by electrodeposition of Au nanoparticles on molybdenum

AC

CE

PT

ED

M

sulfide and multi-walled carbon nanotube, J. Electroanal. Chem. 786 (2017) 43-49.

23

ACCEPTED MANUSCRIPT Highlights

 BSA-AuNPs were fabricated on glassy carbon electrode by direct attachment  Modified electrode was exploited for the determination of nitrite

IP

T

 Electrochemical oxidation current enhanced by 3-fold towards oxidation of nitrite

CR

 Limit of detection was found to be 2 nM in the present modified electrode

AC

CE

PT

ED

M

AN

US

 The practical application was demonstrated by determining nitrite ion in tap water samples

24

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7