gold nanoparticle encapsulated-tubular tio 2 nanocluster as a scaffold...

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Article

A gold nanoparticle encapsulated-tubular TiO2 nanoclusteras a scaffold for development of thiolated enzyme biosensors

Xiaoqiang Liu, Jiamei Zhang, Shanhu Liu, Qing-You Zhang, Xiuhua Liu, and Danny K.Y. WongAnal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac303420a • Publication Date (Web): 12 Apr 2013

Downloaded from http://pubs.acs.org on April 16, 2013

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A GOLD NANOPARTICLE ENCAPSULATED-TUBULAR TIO2 NANOCLUSTER

AS A SCAFFOLD FOR DEVELOPMENT OF

THIOLATED ENZYME BIOSENSORS

Xiaoqiang Liu∗, Jiamei Zhang, Shanhu Liu, Qingyou Zhang, Xiuhua Liu, and

Danny K.Y.Wong#••

Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical

Engineering, Henan University, Kaifeng, Henan 475004, P. R. China

# Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW

2109, Australia

* Corresponding author: Tel: +86-378-2825854; Fax: +86-378-2825854; E-mail: [email protected].

••

Corresponding author: Tel: +61-2-9850-8300; Fax: +61-2-9850-8313; E-mail: [email protected].

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Abstract

In this work, a highly sensitive and stable sensing scaffold consisting of gold nanoparticle-

encapsulated TiO2 nanotubes, the hydrophilic ionic liquid, 1-decyl-3-methylimidazolium

bromide, and Nafion was developed for the fabrication of electrochemical enzyme biosensors.

A significant aspect of our work is the application of 12-phosphotungstic acid as both a highly

localised photoactive reducing agent to deposit well-dispersed gold nanoparticles on TiO2

nanotubes and an electron mediator to accelerate the electron transfer between an enzyme and

the electrode. After characterising the nanocomposite component of the scaffold by Fourier

transform infrared spectroscopy, X-ray diffraction and transmission electron microscopy,

thiolated horseradish peroxidase (as a model enzyme) was immobilised on the scaffold and the

biosensor was applied to the detection of H2O2. The direct electron transfer between the

enzyme and the electrode was promoted by the excellent biocompatibility and conductivity of

the scaffold. In addition, a thiolated enzyme has significantly improved the stability and direct

electron transfer of horseradish peroxidase on the biosensor, which could be ascribed to the

strong affinity between the sulfhydryl group on the enzyme and gold nanoparticles on the

biosensor surface. Cyclic voltammetry, chronoamperometry and square wave voltammetry

were used to study the electrochemistry and analytical performance of the biosensor. A

dynamic range from 65 to 1600 µM, a limit of detection of 5 µM and a sensitivity of (18.1±

0.43)×10-3 µA µM-1 H2O2 were obtained. The sensing scaffold based on the nanocomposite was

demonstrated to be effective and promising in developing enzyme biosensors.

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INTRODUCTION

Redox enzyme modified electrodes have attracted increasing attention due to their

potential applications to the development of biosensors, bioelectronics and biofuel cells.1,2

In

these applications, satisfactory electrical communication between the active sites of enzymes

and the electrodes is critical to achieve an optimal performance.3 In this respect, the material

and method used to immobilise the redox enzyme were considered among the most important

factors affecting the direct electron transfer between the enzyme and electrode.4, 5

Owing to

good biocompatibility, large surface area and strong ability to facilitate the electron transfer

kinetics for redox reactions, TiO2 nanomaterials were often applied as a matrix for

immobilising enzymes to the development of electrochemical and photoelectrochemical

biosensors.3, 6

More recently, noble metals, particularly Au nanoparticles, were used to modify

TiO2 nanomaterials for use in constructing enzyme biosensors.7, 8

For example, Zhu et al.

fabricated a photoelectrochemical biosensor based on gold nanoparticle-modified TiO2

nanoneedles, where gold nanoparticles were deposited by incubating the TiO2 nanoneedles in a

gold nanoparticle suspension for 12-15 h.7 The as-prepared biosensor exhibited a 4-fold

increase in sensitivity for detection of H2O2 under visible light illumination, compared to that

without the corresponding illumination. Similarly, a photoelectrochemical sensor based on a

gold nanoparticle-modified nanostructured anatase electrode was constructed by incubating

sintered TiO2 anatase nanoparticle films in a gold nanoparticle dispersion for 20 min.9 This has

resulted in more than 10% increase in photocurrent in 0.05 M NaOH. In contrast, Wang et al.

used a one-step method to reduce gold nano-seeds on TiO2 nanoparticles to construct a

horseradish peroxidase (HRP) biosensor.10

The biosensor not only exhibited a response within

3 s and a low limit of detection of 5.9 µM H2O2, but also retained 90% of its initial response

after a 2-week storage. In their work, the Au nanoparticles provided a favourable

microenvironment for biomolecules,11

and they also acted as an electron transfer bridge for

direct electrical contact between redox proteins and electrode support.12

Unfortunately, there

are several limitations associated with gold nanoparticle deposition methods hitherto reported,

which may affect the performance of gold nanoparticle-TiO2 nanomaterial based enzyme

biosensors. For example, the incubation method often resulted in weak binding between gold

nanoparticles and TiO2, causing the gold nanoparticles to easily detach from TiO2 during the

detection process.7 In addition, the chemical reduction method also yielded gold nanoparticles

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that tended to agglomerate and they were shown to be non-uniformly dispersed in the TiO2

nanomaterial, all of which will negatively affect the repeatability and reproducibility of

biosensors constructed.13

Furthermore, both the incubation method and chemical reduction

method produce a mixture of free gold nanoparticles and gold nanoparticle modified-TiO2,

which is undesirable for practical applications.

Alternatively, noble metal (Au, Pt) nanoparticles can be attached to semiconductor

nanoparticles using polyoxometalates (e.g. PW12

O40

3- , SiW12

O40

4- and W10

O32

4- ) as a linker.14

The

significance of this technique is that the noble metal nanoparticles were found to be well

dispersed and they strongly adhered to the semiconductor nanoparticle surface without any free

noble metal nanoparticle contamination.14,15

Moreover, polyoxometalates were shown to

exhibit photocatalytic capability, thermal stability and electrical communication ability.14

In

particular, polyoxometalates have demonstrated ability to undergo reversible multi-electron

redox processes without decomposition, making them excellent electrochemical catalysts in the

detection of nitrite, H2O2 and iodate.16,17

All of above features will benefit the development of

electrochemical and photoelectrochemical biosensors.

In comparison to TiO2 nanoparticle-based biosensors, TiO2 nanotube-based biosensors

have exhibited dramatically improved performance due to their relatively higher specific

surface area, better biocompatibility and higher conductivity of the special tube-like geometry.8

For example, the special hollow structure of TiO2 nanotubes, compared to the bulkier

nanoparticle spheres, will potentially facilitate a stable immobilisation of small biological

molecules on the inner wall of the nanotubes, which may enhance the performance of

nanocomposite-based biosensors.

In this work, we will initially use the polyoxometalate anion of 12-phosphotungstic acid

(PTA; PW12

O40

3- ) as a linker to attach gold nanoparticles to TiO2 nanotubes to construct a

scaffold for the development of an electrochemical enzyme biosensor using HRP as a model

enzyme. In this scaffold, photochemically reduced PTA acts not only as a localised reducing

agent to deposit gold nanoparticles on TiO2 nanotube surface, but also as an electron mediator

to improve the enzyme catalytic reaction. To further increase the stability of the biosensor and

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to enhance direct electron transfer at the biosensor interface, it is necessary to strengthen the

contact between HRP and gold nanoparticle-PTA modified TiO2 nanotubes. One strategy to

achieve this is by introducing a sulfhydryl group to HRP using a dithio crosslinker so that it can

readily bind to the gold nanoparticles. In this way, the strong interaction between thiolated

HRP and gold nanoparticles is expected to enhance the stability of HRP in this scaffold.

Previously, the amine groups on Protein G and antibodies were successfully modified in a

similar manner to facilitate an orientation-controlled immobilisation on gold electrodes for the

construction of electrochemical immunosensors with improved performance.18,19

Finally, the

ionomer, Nafion, and the hydrophilic ionic liquid, 1-decyl-3-methylimidazolium bromide

([Demim]Br), were applied to bind the thiolated HRP | gold nanoparticle-PTA-TiO2 nanotube

scaffold to the electrode surface. Specifically, [Demim]Br was used because it offers excellent

conductivity, biocompatibility, wide electrochemical windows and high chemical stability.20,21

The electrocatalytic response to the reduction of H2O2 by HRP at the thiolated HRP | gold

nanoparticle-PTA-TiO2 nanotube | [Demim]Br-Nafion biosensor (hereafter referred to as a

thiolated HRP biosensor) was then studied by cyclic voltammetry, chonoamperometry and

square wave voltammetry.

EXPERIMENTAL SECTION

Chemicals and Materials. TiO2 nanotubes were prepared using a procedure reported

prviously.22

Briefly, hydroxyl functionalised TiO2 nanotubes were synthesised by reacting

polycrystalline TiO2 with NaOH solution at 110°C for 20 h in a in a high pressure Teflon

reactor. HRP, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), dithiothreitol (DTT),

propan-2-ol, bicinchoninic acid (BCA) QuantiPro Protein Assay Kit, HAuCl4·3H2O and

dimethylsulfoxide (DMSO) were purchased from Sigma–Aldrich, USA. PTA was obtained

from J&K Scientific Ltd., Beijing, China. [Demim]Br was purchased from Shanghai Chengjie

Chemical Co., Ltd., China. Zeba™ spin desalting columns were purchased from Thermo

Fisher Scientific Inc. All solutions were prepared in Milli-Q ultrapure water.

Instrumentation. Electrochemical measurements were performed using a CHI630

electrochemical workstation (CH Instruments, Shanghai, China) with a conventional three-

electrode system consisting of a 3-mm diameter glassy carbon working electrode, a platinum

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wire counter electrode and a Ag|AgCl (3.0 KCl) reference electrode. All electrodes were

purchased from Gaoshiruilian Co. Ltd., Wuhan, China. During electrochemical measurements,

the working solutions were deoxygenated with N2 gas for 15 min and then a nitrogen

atmosphere was kept over the solutions until the measurements were completed. Philips

tubular ultraviolet low-pressure 16-W mercury lamps were purchased from Philips Lighting

B.V., The Netherlands. Morphological characterisation of the nanocomposites was

investigated by Fourier transform infrared spectroscopy (FT-IR; Nicolet 170, USA), X-ray

diffraction (XRD; X-PertPro, Netherland) with Cu Kα radiation (λ = 1.5406 nm) and field

emission transmission electron microscopy (FETEM; Tecnai G2 20, FEI Co. Ltd., USA). The

bioactivity of thiolated HRP was determined using uv–vis spectrophotometry (UV-1750

spectrophotometer, SHIMADZU, Japan) and FT-IR.

Preparation of Nanocomposites. Firstly, TiO2 nanotubes (20 mg) was dispersed in 20 mL

PTA aqueous solution (10 mM) and left overnight under mechanical stirring. The products

were then centrifuged and washed with deionised water to facilitate the removal of

uncoordinated PTA molecules. Next, 4 mL of PTA–modified TiO2 nanotubes (4 mg mL–1

)

solution was mixed with 1 mL of propan-2-ol in a quartz cell, purging with N2 gas for 15 min.

The mixed solution was photoexcited under four UV lamps (16 W, λex = at 253 nm) for 4 h to

reduce PTA on the TiO2 nanotubes before 5 mL of 1 mM HAuCl4 solution was added. After

the colour of the mixture solution had changed from dark blue to light purple, the mixture was

allowed to mature for 2 h. Finally, the suspension was centrifuged to obtain the gold

nanoparticle-PTA-TiO2 nanocomposite.

Thiolation of HRP. ZebaTM

desalt spin columns used in the enzyme thiolation process contain

a high-performance resin which offers exceptional purification, desalting and buffer-exchange

for protein samples, and they therefore do not require any chromatographic system and

cumbersome preparation or equilibration procedures. The spin-column method also eliminates

delay caused by samples emerging at the end of a column. Compared with our previous

enzyme thiolation method,19,23

the whole thiolation time has been reduced from approximately

1 day to half day and the enzyme recovery has been increased from 44% to approximately 65%

using the spin column method. The specific procedure was described as follows. Firstly, 1 mg

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SPDP was dissolved in 160 µL of DMSO, and then 12.5 µL of the 20 mM SPDP solution and 1

mg HRP was dissolved in 500 µL of phosphate buffer saline containing 1 mM EDTA (PBS-

EDTA), and this was allowed to react for 30–60 min at room temperature. Two desalt spin

columns were centrifuged at 2500 rpm for 5 min to remove storage solution, and acetate buffer

was exchanged 2-3 times to equilibrate two desalting columns, then the SPDP-modified HRP

was centrifuged to remove reaction by-products and excess non-reacted SPDP. Next, 200 µL

DTT solution was added to 500 µL SPDP-modified HRP solution, followed by a 30-min

incubation to produce thiolated HRP. Finally, PBS-EDTA solution was employed to

equilibrate desalting columns and thiolated HRP was desalted using the equilibrated columns to

remove the excess DTT. The recovery of thiolated HRP was determined by estimating the

quantity of residual protein content in the final solution using a BCA-based QuantiPro Protein

Assay Kit. This detection procedure was similar to that reported previously,18,19

except that

thiolated HRP was used in place of Protein G.

Preparation of a thiolated HRP Biosensor. Initially, a glassy carbon electrode was

successively polished on a polishing cloth with 0.3 µm and 0.05 µm alumina powder and rinsed

with deionised water and ethanol. The polished electrode was then allowed to dry at room

temperature. The mixture of thiolated HRP, gold nanoparticle-PTA-TiO2, [Demim]Br and

Nafion was kept agitated at 2°C for 4 h in a full temperature oscillation incubator before being

applied to the electrode surface. Specifically, 300 µL thiolated HRP aqueous solution (1.5 mg

µL-1

) and 50 µL Nafion were added in [Demim]Br and gold nanoparticle-PTA-TiO2 composite

(1:2, w/w). Finally, 4 µL of the suspension was applied to the glassy carbon electrode and

dried at 4°C for 2 h to obtain an HRP biosensor.

RESULTS AND DISCUSSION

Briefly, an electrochemical HRP biosensor was developed in this work by initially

immobilising on a glassy carbon electrode a scaffold consisting of gold nanoparticle-modified

TiO2 nanotubes using PTA as a linker. Thiolated HRP was then allowed to interact with the

gold nanoparticles before the scaffold was anchored on an electrode using Nafion and

[Demim]Br. One novel aspect of this work lies in the application of PTA to synthesise the gold

nanoparticle-TiO2 composite such that the gold nanoparticles are uniformly dispersed,

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minimising the aggregation of thiolated HRP that was subsequently immobilised on the

composite. PTA was also exploited as an electron mediator in the direct electron transfer of

HRP at the electrode. In the following sections, we applied a range of techniques to

characterise the individual components of the biosensor scaffold, before the thiolated HRP

biosensor was used to analytically detect H2O2.

FT-IR, XRD and FETEM Characterisation of Nanocomposites.

We have initially used FT-IR to examine whether PTA was successfully attached to TiO2.

Figure 1A shows the FT-IR spectra of TiO2 alone, PTA alone and PTA-TiO2. As expected, the

FT-IR spectrum of TiO2 nanotubes alone (trace a) did not exhibit any significant features above

800 cm-1

.14

In the FT-IR spectrum of PTA alone (trace b), there are four characteristics bands

at 1079, 983, 893, and 800 cm-1

attributable to the four bonds linking the tungsten atoms by

oxygen atoms with a phosphorus atom at the centre of a tetrahedron (P－Oa, W－Od, W－Ob－

W and W－Oc－W) in PTA.24,25

Then, the FT-IR spectrum of PTA-TiO2 (trace c) shows a

minor shift of the four PTA characteristic bands relative to those of PTA alone in trace b. We

attributed this to the effect on the vibrational modes of the oxygen atoms in PTA after being

attached to TiO2 nanotubes. Accordingly, we shall use this to infer successful adsorption of

PTA on TiO2 nanotubes by mechanical stirring in our work.

Next, XRD was used to characterise the crystal structure of TiO2 nanotubes, PTA-TiO2

nanotubes, and gold nanoparticle-PTA-TiO2 nanocomposite. The results are shown in Figure

1B. As reported previously, the diffraction peak at 2θ angle of 9° in trace a is a notable feature

of the TiO2 nanotubes successfully synthesised using a hydrothermal method.26

Moreover, the

peaks at 25.0°, 38.1°and 48.5° correspond to the spacing of (101), (004) and (200) of the

anatase (tetragonal) phase of the TiO2 nanotubes.6 However, the binding of PTA to TiO2

nanotubes did not result in any significant change in the XRD pattern (trace b) relative to that

of TiO2 nanotubes alone. This might be due to the presence of only a very thin PTA coating on

the TiO2 nanotube surface.14

Similar to a previous report,27

the peaks at 38.3°, 44.6°, 64.8°

and 77.8° in trace c can be assigned to (110), (200), (220), (311) reflection of gold

nanoparticles, which clearly indicate that gold nanoparticles were successfully deposited on

TiO2 nanotubes.

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Finally, the morphology of TiO2 nanotubes and gold nanoparticle-PTA-TiO2

nanocomposite synthesised in our laboratory was characterised using FETEM and the results

are shown in Figure 2A and 2B, respectively. In Figure 2A, the TiO2 nanotubes show a

relatively uniform geometrical dimension, with a mean diameter of approximately 5 nm and

length of a few hundred nm. The FETEM image of a gold nanoparticle-PTA-TiO2

nanocomposite shown in Figure 2B depicts relatively well dispersed gold nanoparticles

(denoted by dark spheres) in the TiO2 nanotube matrix with no observable free gold

nanoparticles. This also demonstrated the successful immobilisation of gold nanoparticles on

TiO2 nanotubes using PTA as a linker. The average particle size of gold nanoparticles on TiO2

nanotubes is approximately 15 nm and this is in good agreement with 15.4 nm evaluated using

the Scherrer equation28,29

based on the XRD data obtained above,

d =0.9×λ

kα

B(2θ)

cos θmax

where λkα is the wavelength of X-rays used (0.154056 nm), B(2θ) is the width of the peak at half

height (with the peak height evaluated from the baseline), and θmax is the Bragg angle at the

peak. In Figure 2B, the bird-nest like TiO2 nanotube matrix will offer a biocompatible

microenvironment for encapsulating the enzymes and sustaining their bioactivity. The high

specific area of TiO2 nanotube will also accommodate a substantial quantity of gold

nanoparticles and thereby promoting the binding of high load of thiolated HRP.

UV–Vis and FT-IR spectroscopic analysis of sulfhydryl modified HRP. Uv–vis

spectrometry was previously employed to investigate the native structure of HRP.30,31

The

Soret band shift in a uv–vis spectrum will aid in determining whether denaturation in heme

protein has occurred after modifying the microenvironment around the heme group.32

Figure

3A shows the respective uv-vis spectra of HRP, sulfhydryl-modified HRP and sulfhydryl-

modified HRP in the gold nanoparticle-PTA-TiO2 nanotube | [Demim]Br scaffold. In these

spectra, the Soret band absorption for HRP (trace a) and sulfhydryl-modified HRP (trace b) is

located at nearly the same wavelength (~403 nm), suggesting that the heme structure of HRP

was not significantly altered after the thiolation process. There was also no obvious shift in the

Soret band after HRP was mixed with the gold nanoparticle-PTA-TiO2 nanotube composite and

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[Demim]Br (trace c), indicating that the composite materials provided biocompatibility that

allowed HRP to remain in the native heme structure and to maintain its bioactivity.

FT-IR spectroscopy was also conducted to provide additional information for the secondary

structure of HRP. Previously, the amide I band at 1600–1700 cm−1

was attributed to the C=O

stretching vibration of the peptide bond in the backbone of HRP, while the amide II band

between 1500 and 1600 cm−1

was assigned to the combination of N–H bending and C–N

stretching vibration.33,34

Denaturation or any changes in the structure of an enzyme molecule is

expected to lead to diminished intensity of these bands or even their disappearance.35

As

supported by the results in Figure 3B, there was no significant difference in the amide I and

amide II bands between the FT-IR spectrum of HRP before thiolation (trace a) and that of

thiolated HRP (trace b). Furthermore, even after being incorporated in the nanocomposite film,

the amide I and amide II bands of HRP molecules exhibited minor changes in their position and

intensity (trace c). All of above results demonstrated that the native secondary structure of

HRP was retained after being thiolated and entrapped in the composite material. As the

bioactivity of HRP is strongly related to its heme group and secondary structure, its bioactivity

has not been significantly affected based on non-detectable changes in the FT-IR features.

Electrochemical characterisation of thiolated HRP biosensors. Figure 4A shows the cyclic

voltammograms at different modified electrodes in N2-saturated PBS at 50 mV s−1

. Initially,

the cyclic voltammogram (trace a) obtained at the PTA-TiO2 nanotube | [Demim]Br | Nafion-

coated electrode did not show any obvious redox peaks. Notably, PTA-modified TiO2

nanotubes were obtained in this work by incubating the two components together, followed by

copious rinsing, that only a relatively small quantity of PTA would be present on the TiO2

nanotubes. Therefore, PTA was not expected to yield any observable signals in the cyclic

voltammogram. After immobilising HRP on the gold nanoparticle-PTA-TiO2 nanocomposite,

a pair of redox peaks at 0.126 V and 0.010 V (peak separation of ~116 mV) arising from direct

electron transfer of HRP in the matrix was observed in the cyclic voltammogram (trace b).

Here, both the gold nanoparticles and PTA acted as electron transfer bridges to accelerate the

electron transfer between the enzyme and the electrode, while gold nanoparticles and TiO2

nanotubes provided a biocompatible microenvironment for HRP. Next, a thiolated HRP,

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instead of HRP, was immobilised on the nanocomposite and the cyclic voltammogram obtained

is shown in trace c. The reduction-to-oxidation peak current ratio was decreased from 2.98

(trace b) to 1.40 (trace c) and a corresponding decrease in the peak separation from 116 mV to

72 mV was also observed, indicating an improved reversibility of the direct electron transfer of

HRP at the thiolated HRP biosensor. In this work, the thiolation of HRP was achieved by

modifying the enzyme with a dithio crosslinker, SPDP. The sulfhydryl groups would facilitate

strong binding of HRP to the gold nanoparticle surface. After being stably anchored on the

electrode, direct electron transfer of HRP takes place more readily, giving rise to a reduction-

to-oxidation peak current ratio that is closer to unity and a smaller peak separation that are

indicative of a more electrochemically reversible reaction.

Next, the thiolated HRP biosensor was placed in 0.05 M PBS (pH 7.0) containing 0.1,

0.2 and 0.3 mM H2O2, respectively, before square wave voltammetry was performed and the

results obtained are shown in Figure 4B. The thiolated HRP exhibited a strong catalytic effect

towards H2O2, which can be used as additional evidence that the bioactivity and catalytic ability

of HRP was sustained after being thiolated and immobilised on the nanocomposite.

Accordingly, the proportional increase of catalytic current as a function of H2O2 concentration

demonstrated that the thiolated HRP biosensor could be applied to the determination of H2O2.

Current-Time Relationship for the H2O2 Measurement. The chronoamperometric responses

of (a) a HRP | TiO2 nanotube | [Demim]Br | Nafion biosensor, (b) a HRP | gold nanoparticle-

PTA-TiO2 nanotube | [Demim]Br | Nafion biosensor and (c) a thiolated HRP | gold

nanoparticle-PTA-TiO2 nanotube | [Demim]Br | Nafion biosensor to the successive additions of

H2O2 into continuously stirred 0.1 M PBS solution (pH 7.0) at -0.4 V are shown in Figure 5A.

As observed in trace (a), the current increased sluggishly after injection of H2O2, indicating the

slow response of the biosensor in the absence of PTA and gold nanoparticles. On the contrary,

the chronoamperometric responses of both biosensor (b) and (c) were enhanced dramatically

once H2O2 was injected, demonstrating that both PTA and gold nanoparticles aided in

significantly reducing the response time of the biosensors. Based on the slopes of the

calibration plots, the corresponding sensitivity for the three designs of sensors was estimated to

be (a) (1.72 ± 0.11)×10-3

µA µM-1

, (b) (10.7 ± 0.13)×10-3

µA µM-1

and (c) (18.1 ± 0.43)×10-3

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µA µM-1

respectively. Notably, biosensor (b) has displayed a larger than 500% enhancement in

sensitivity over biosensor (a), which can be explained by the presence of gold nanoparticles and

PTA in the scaffold of biosensor (b). However, based on our previous results,13

gold

nanoparticles alone were unlikely to yield such a dramatic increase in sensitivity of HRP

biosensors. This led us to consider PTA as another contributing factor affecting the

chronoamperometric response of the HRP biosensors. Previously, polyoxometalates including

PTA were reported to have directly catalysed the electrochemical reduction of H2O2 in strongly

acidic solutions.16,17

However, direct electrochemical catalysis by PTA was unlikely in our

work as H2O2 reduction occurred in a neutral phosphate buffer throughout our experiments.

Instead, we speculate that PTA played the role of an electron mediator, for example,

hydroquinone, in the redox reaction of HRP.8 In such a mechanism, Fe(III) within the heme

group of HRP was initially reduced by the reduced form of PTA yielding Fe(II) and the

oxidised form of PTA at -0.4 V.

HRP(Fe(III)) + PTA(Red) →HRP(Fe(II)) + PTA(Ox)

HRP(Fe(II)) was then oxidised back to HRP(Fe(III)) by H2O2. Finally, PTA(Ox) was reduced

back to PT(Red), which further enhanced the reduction current.

HRP(Fe(II)) + H2O2 → HRP(Fe(III)) + H2O

PTA(Ox) → PTA(Red)

The analytical performance of the thiolated HRP biosensor and several other HRP

biosensor designs are listed Table 1. Compared to the other HRP biosensors, the thiolated

HRP biosensor exhibited a superior sensitivity and a comparable limit of detection. Both

characteristics of the thiolated HRP biosensor may be ascribed to the following features. In the

gold nanoparticle-PTA-TiO2 nanotube | [Demim]Br | Nafion scaffold, the porous structure of

TiO2 nanotube-cluster provides a biocompatible matrix, exposes the gold nanoparticles to the

adsorbed enzyme and aids in electrolyte accessibility. Gold nanoparticles and [Demim]Br are

also biocompatible materials that would have facilitated electron transfer between enzyme and

the underlying electrodes.19,31

In addition, the uniform distribution of gold nanoparticles on

TiO2 nanotubes and high specific surface area of both gold nanoparticles and TiO2 nanotubes

have increased the load of enzyme molecules and decreased their aggregation in the scaffold.

Furthermore, the strong binding between sulfhydryl of HRP and gold nanoparticles has

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minimised the leakage of HRP and improved the electron transfer between enzyme and

electrode, which is also beneficial to the sensitivity of HRP biosensors. Finally, PTA acted as

an electron mediator to enhance the sensitivity and to significantly reduce the response time of

the thiolated HRP biosensor.

Stability study of the biosensors. Cyclic voltammetry of the thiolated HRP biosensor in 0.05

M PBS was conducted to evaluate its stability. For comparison, cyclic voltammetry of a non-

thiolated HRP biosensor was also conducted. More specifically, 20 repeated cyclic

voltammetric scans were conducted at the respective electrode at 50 mV s-1

and the results are

shown in Figure 5B. The HRP redox currents at the thiolated HRP biosensor exhibited a 12%

decrease after 20 continuous scans (trace a), while that at the non-thiolated HRP biosensor

decreased by ~26% (trace b), indicating a higher stability of the former biosensor than the

latter. Here, the strong interaction between sulfhydryl of HRP and gold nanoparticles has

minimised the leakage of the enzyme molecules from the biosensor. In addition, the uniformly

deposited, rather than agglomerated, gold nanoparticles would have aided in maintaining the

dispersion and therefore the bioactivity of HRP molecules. In summary, all these advantages of

the gold nanoparticle-PTA-TiO2 nanotube | [Demim]Br | Nafion scaffold contributed to the

improved stability and prolonged life span of the thiolated HRP biosensor.

CONCLUSIONS

We presented in this work a scaffold consisting of gold nanoparticle-PTA-TiO2 nanotube,

[Demim]Br and Nafion for the development of an electrochemical enzyme biosensor. In this

scaffold, PTA was employed as both a UV-switchable reducing agent to immobilise gold

nanoparticles on the TiO2 nanotube surface and as an electron mediator to accelerate enzyme

catalytic reaction. The FT-IR, XRD and FETEM results demonstrated that gold nanoparticles

were well dispersed in the TiO2 nanotube matrix and no observable free metal nanoparticles

were produced. In our work, the model enzyme, HRP, was modified with sulfhydryl using a

spin-column method to evaluate the performance of the scaffold. Uv-vis spectrometry, FT-IR

and voltammetry provided evidence for sustained bioactivity of thiolated HRP immobilised on

the scaffold. Finally, the chroamperometric and cyclic voltammetric results demonstrated that

high sensitivity and stability were achievable using the described scaffold on an

electrochemical enzyme biosensor.

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ACKNOWLEDGMENT

This work was financially supported by the China Postdoctoral Science Foundation funded

project (No. 2012M511569） and National Natural Science Foundation of China (No.

20875022, No.21105021).

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FIGURE CAPTIONS

Figure 1 (A) FT-IR spectra of (a) TiO2 nanotubes alone, (b) PTA alone and (c) PTA-TiO2

nanocomposite; (B) XRD patterns of (a) TiO2 nanotubes, (b) PTA-TiO2 nanotubes,

and (c) gold nanoparticle-PTA-TiO2 nanocomposite.

Figure 2 FETEM image of (A) TiO2 nanotubes; (B) gold nanoparticle-encapsulated TiO2

nanotubes.

Figure 3 (A) Uv–vis absorption spectra and (B) FT-IR spectra of (a) HRP, (b) sulfhydryl

modified HRP and (c) sulfhydryl modified HRP in gold nanoparticle-PTA-TiO2

nanotube | [Demim]Br composite.

Figure 4 (A) Cyclic voltammograms at (a) a PTA-TiO2 nanotube | [Demim]Br | Nafion-

coated electrode, (b) a HRP-gold nanoparticle-PTA-TiO2 nanotube | [Demim]Br |

Nafion-coated electrode, (c) a thiolated HRP-gold nanoparticle-PTA-TiO2 nanotube

| [Demim]Br | Nafion-coated electrode in N2-saturated PBS at 50 mV s−1

; (B)

square wave voltammograms of a thiolated HRP-gold nanoparticle-PTA-TiO2

nanotube | [Demim]Br | Nafion-coated electrode in 0.05 M PBS (pH 7.0) in the

presence of 0.1, 0.2 and 0.3 mM H2O2 (from trace a to c) at 50 mV s

-1.

Figure 5 (A) Chronoamperometric response of (a) a HRP | TiO2 nanotube | [Demim]Br |

Nafion electrode, (b) HRP-gold nanoparticle-PTA-TiO2 nanotube | [Demim]Br |

Nafion-coated electrode, and (c) a thiolated HRP-gold nanoparticle-PTA-TiO2

nanotube | [Demim]Br | Nafion-coated electrode to the successive additions of

H2O2 into continuously stirred 0.1 M N2-saturated PBS solution (pH 7.0) at -0.4 V.

The inset shows an enlarged portion of trace (c). (B) Twenty successive cyclic

voltammetric scans of (a) a thiolated HRP biosensor and (b) a non-thiolated HRP

biosensor in 0.05 M PBS (pH 7.0) at a scan rate of 50 mV s-1

.

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REFERENCES

(1) Wang, Y.; Shao, Y. Y.; Matson, D. W.; Li, J. H.; Lin, Y. H. ACS Nano 2010, 4, 1790-1798.

(2) Zafar, M. N.; Tasca, F.; Gorton, L.; Patridge, E. V.; Ferry, J. G.; Noll, G. Anal. Chem. 2009,

81, 4082-4088.

(3) Si, P.; Ding, S. J.; Yuan, J.; Lou, X. W.; Kim, D.-H. ACS Nano 2011, 5, 7617-7626.

(4) Zhang, L.; Geng, W. C.; Qiao, S. Z.; Zheng, H. J.; Lu, G. Q.; Yan, Z. F. ACS Appl. Mater.

Interfaces 2010, 2, 2767-2772.

(5) Holland, J. T.; Lau, C.; Brozik, S.; Atanassov, P.; Banta, S. J. Am. Chem. Soc. 2011, 133,

19262-19265.

(6) Han, X.; Zhu, Y. H.; Yang, X. L.; Li, C. Z. J. Alloys Compd. 2010, 500, 247-251.

(7) Zhu, A. W.; Luo, Y. P.; Tian, Y. Anal. Chem. 2009, 81, 7243-7247.

(8) Liu, X. Q.; Feng, H. Q.; Zhang, J. M.; Zhao, R. X.; Liu, X. H.; Wong, D. K. Y. Biosens.

Bioelectron. 2012, 32, 188-194.

(9) Lana-Villarreal, T.; Gomez, R. Electrochem. Commun. 2005, 7, 1218-1224.

(10) Wang, Y.; Ma, X. L.; Wen, Y.; Xing, Y. Y.; Zhang, Z. R.; Yang, H. F. Biosens. Bioelectron.

2010, 25, 2442-2446.

(11) Jia, J. B.; Wang, B. Q.; Wu, A. G.; Cheng, G. J.; Li, Z.; Dong, S. J. Anal. Chem. 2002, 74,

2217-2223.

(12) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877-1881.

(13) Liu, X. Q.; Feng, H. Q.; Zhao, R. X.; Wang, Y. B.; Liu, X. H. Biosens. Bioelectron. 2012, 31,

101-104.

(14) Pearson, A.; Bhargava, S. K.; Bansal, V. Langmuir 2011, 27, 9245-9252.

(15) Mandal, S.; Selvakannan, P. R.; Pasricha, R.; Sastry, M. J. Am. Chem. Soc. 2003, 125,

8440-8441.

(16) Ma, H.; Gu, Y.; Zhang, Z.; Pang, H.; Li, S.; Kang, L. Electrochim. Acta 2011, 56, 7428-

7432.

(17) Han, Z.; Zhao, Y.; Peng, J.; Liu, Q.; Wang, E. Electrochim. Acta 2005, 51, 218-224.

(18) Fowler, J. M.; Stuart, M. C.; Wong, D. K. Y. Anal. Chem. 2006, 79, 350-354.

(19) Liu, X. Q.; Wong, D. K. Y. Talanta 2009, 77, 1437-1443.

(20) Anderson, J. L.; Armstrong, D. W.; Wei, G.-T. Anal. Chem. 2006, 78, 2892-2902.

(21) Musameh, M.; Wang, J. Anal. Chim. Acta 2008, 606, 45-49.

Page 16 of 24



123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

17

(22) Yang, J. J.; Jin, Z. S.; Wang, X. D.; Li, W.; Zhang, J. W.; Zhang, S. L.; Guo, X. Y.; Zhang, Z. J.

Dalton Trans. 2003, 3898-3901.

(23) Liu, X. Q.; Duckworth, P. A.; Wong, D. K. Y. Biosens. Bioelectron. 2010, 25, 1467-1473.

(24) Sanyal, A.; Mandal, S.; Sastry, M. Adv. Funct. Mater. 2005, 15, 273-280.

(25) Kumbar, S. M.; Halligudi, S. B. Catal. Commun. 2007, 8, 800-806.

(26) Wang, Y.; Feng, C. X.; Jin, Z. S.; Zhang, J. W.; Yang, J. J.; Zhang, S. L. J. Mol. Catal. A:

Chem. 2006, 260, 1-3.

(27) Paramasivam, I.; Macak, J. M.; Schmuki, P. Electrochem. Commun. 2008, 10, 71-75.

(28) Park, K.-W.; Choi, J.-H.; Kwon, B.-K.; Lee, S.-A.; Sung, Y.-E.; Ha, H.-Y.; Hong, S.-A.; Kim,

H.; Wieckowski, A. J. Phys. Chem. B 2002, 106, 1869-1877.

(29) Yang, H.; Coutanceau, C.; Leger, J.-M.; Alonso-Vante, N.; Claude, L. J. Electroanal. Chem.

2005, 576, 305-313.

(30) Sun, X. M.; Zhang, Y.; Shen, H. B.; Jia, N. Q. Electrochim. Acta 2010, 56, 700-705.

(31) Zhu, Z. H.; Li, X.; Wang, Y.; Zeng, Y.; Sun, W.; Huang, X. T. Anal. Chim. Acta 2010, 670,

51-56.

(32) Arnstein, H. R. V.; Neuberger, A. Biochem J. 1953, 55, 259-271.

(33) Sun, X.; Zhang, Y.; Shen, H.; Jia, N. Electrochim. Acta 2010, 56, 700-705.

(34) Zhu, Z.; Li, X.; Wang, Y.; Zeng, Y.; Sun, W.; Huang, X. Anal. Chim. Acta 2010, 670, 51-

56.

(35) Song, Y. P.; Petty, M. C.; Yarwood, J.; Feast, W. J.; Tsibouklis, J.; Mukherjee, S. Langmuir

1992, 8, 257-261.

(36) Xiang, C.; Zou, Y.; Sun, L.-X.; Xu, F. Sens. Actuators, B 2009, 136, 158-162.

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Table 1 Analytical characteristics of several HRP biosensors in H2O2 detection. 1

2

HRP biosensors Sensitivity × 10

3 /

µA µM-1

Dynamic range /

µM

Limit of detection /

µM References

Thiolated HRP | gold nanoparticle -PTA-TiO2

nantube | [Demim]Br biosensor 18.1 65 – 1600 5 This work

HRP | gold nanoparticle -PTA-TiO2 nantube |

[Demim]Br biosensor

10.7 90 – 1400 6.5 This work

Nafion | HRP–gold nanoseed–TiO2 | glassy carbon

electrode biosensor

0.23 41 – 630 5.9 10

HRP | halloysite nanotube | chitosan | glassy carbon

electrode biosensor 12.3 2 – 75 0.7

30

HRP | flowerlike ZnO nanoparticle | gold

nanoparticle | Nafion | glassy carbon electrode

biosensor

6.36 15 – 1100 9 36

3

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Figure 1 4

5

6 7

8

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Figure 2 9

10

11

12

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Figure 3 13

14

15 16

17

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Figure 4 18

19

20 21

22

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Figure 5 23

24

25

26

27

28

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For TOC only 29

30

SPDP/DTT SH HRP HRP ZebaTM columns

PTA, N2/15 min,

UV 4 h,

HAuCl4, Mature

4 h

Tubular TiO2

nanocluster

Gold nanoparticle-

Tubular TiO2

nanocluster

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gold nanoparticle encapsulated-tubular tio 2 nanocluster as a scaffold...

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