Porous gold cluster film prepared from Au@BSA microspheres for electrochemical nonenzymatic glucose sensor

Accepted Manuscript Title: Porous gold cluster film prepared from Au@BSA microspheres for electrochemical nonenzymatic glucose sensor Author: Lei Han Shu Zhang Lihui Han Da-Peng Yang Chuantao Hou Aihua Liu PII: DOI: Reference:

S0013-4686(14)01277-8 http://dx.doi.org/doi:10.1016/j.electacta.2014.06.095 EA 22957

To appear in:

Electrochimica Acta

Received date: Revised date: Accepted date:

29-4-2014 17-6-2014 17-6-2014

Please cite this article as: L. Han, S. Zhang, L. Han, D.-P. Yang, C. Hou, A. Liu, Porous gold cluster film prepared from Au@BSA microspheres for electrochemical nonenzymatic glucose sensor, Electrochimica Acta (2014), http://dx.doi.org/10.1016/j.electacta.2014.06.095 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.

Porous gold cluster film prepared from Au@BSA microspheres for electrochemical nonenzymatic glucose sensor

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Lei Hana,b,1, Shu Zhanga,c,1, Lihui Hanc, Da-Peng Yanga, Chuantao Houa, and Aihua

Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology,

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Liua,b,∗

and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China

University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049,

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b

China

Key Laboratory of Marine Chemistry Theory and Technology of Ministry of

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c

Education, and College of Chemistry and Chemical Engineering, Ocean University of

author.

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∗Corresponding

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China, 238 Songling Road, Qingdao 266100, China

Tel: +86 532 80662758;

Fax: +86 532 80662778.

E-mail address: [email protected] (A. Liu).

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These two authors contributed equally to this work.

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ABSTRACT

To obtain hierarchically porous gold-cluster film as the direct electrochemical sensing

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interface for nonenzymatic glucose detection, a facile, cost-effective, environmentally friendly and bottom-up approach was developed by calcination of Au@BSA

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microspheres. The fabricated gold-cluster film was composed of the network structure interconnected with Au particles and the disordered 3D hierarchical pores. Due to

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large surface area, high electroconductivity and excellent electrocatalysis towards glucose oxidation, the as-prepared sensor showed high current response so that the

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glucose determination could be carried out at a less positive potential where common interferents (ascorbic acid, acetamidophenol and uric acid) were not oxidized.

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Importantly, 0.15 M chloride ions (Cl-) had no poisoning effect on the gold-cluster

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electrode. Meanwhile, the sensor also exhibited a rapid response (5 s), high sensitivity

(10.76 μA mM-1 cm-2), a low limit of detection (1 μM), a wide and useful linear range

(0.01−10 mM), good operational stability and storage stability. This work not only demonstrated an excellent direct electrochemical sensing interface for nonenzymatic detection of glucose, but also would extend to other noble metal films and metal oxide films as various electrodes and electrochemical interface.

Keywords: Au@BSA microsphere; hierarchically porous gold-cluster; direct electrochemical sensing; nonenzymatic glucose sensor

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1. Introduction

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Diabetes mellitus, known as a major public health problem [1], results in hyperglycemia, which can be diagnosed by monitoring blood glucose concentration

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and then compared with the normal glucose concentration (4.4–6.6 mM) [2]. To detect glucose concentration, various techniques were developed, such as optical

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sensor [3], colorimetry [4], the electrochemical sensors [5-8]. Among these, the electrochemical sensor has attracted increasing interests and has become a promising

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technology for its rapid response, good sensitivity, excellent selectivity, high

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reliability, low cost, portability and ease of use [2, 9, 10]. Since Clark and Lyons reported the first enzyme electrode in 1962, many efforts have been made to improve electrochemical glucose sensor [11]. So far, the glucose sensors have gone through three generations. The first-generation relied on detection of oxygen consumption or hydrogen peroxide formation, however, which is easily restricted by “oxygen deficit”. To overcome this problem, the second generation sensors have been developed based on mediators such as non-physiological electron acceptors and nanomaterial electrical connectors. Subsequently, the third-generation sensors were also made without mediators. Due to the direct electron transfer from glucose to the electrode, the lower operating potential can be applied, leading to a very high sensitivity and fast response [2]. The majority of third generation glucose sensors rely on immobilized enzymes for their good selectivity and high sensitivity [12]. But the bioactivity of the enzyme can be easily affected by the immobilization method and experimental conditions such as 3

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temperature, humidity, pH and toxic chemicals, resulting in poor stability and reproducibility [13-15]. On the other hand, using inorganic materials (noble metals and metal oxides), the non-enzymatic third-generation sensors overcome these drawbacks of enzyme sensors

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[16, 17]. Some recent efforts have been widely made for non-enzymatic sensors by immobilizing a variety of noble metal materials onto electrodes, such as platinum,

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gold, silver and their alloy [18-21]. However, such non-enzymatic sensors generally have many drawbacks. For example, they may be easily poisoned by adsorbed

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intermediates [18]. They also may be lack of selectivity due to the high operating voltage [22]. In addition, it should not be neglected that their preparation processes

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may be complex and tedious for large-scale production due to the multistep modification. Therefore, the once shaping preparation of selective and stable noble

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metal electrode without complicated modification is highly desirable. Due to the marvelous physical and chemical properties (particularly, good electrical

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conductivity and excellent catalytic activity of oxidation), the research of three-dimensional (3D) hierarchically porous gold materials with high surface area

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and low density has being one of the hottest topics in biology, catalysis and

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nanotechnology [23, 24]. Although the individual nanoparticles show unique shapeand size-dependent physicochemical properties [25], the superstructures, assembled from them, may give rise to some unexpected mechanical, electrical, catalytic and optical performances, which can be stimulated a variety of potential applications such as photoelectricity, catalysis, sensor and surface enhanced Raman scattering [26-28]. Inspired by biomineralization of bones and seashells in nature [29, 30], bovine

serum albumin (BSA), a commercially available protein, has being actively used to synthesize gold superstructures, such as BSA-Au nanoclusters [31]. In our previous work, we prepared the Au@BSA microspheres by using BSA as template [32]. For the preparation of gold film with high surface area and low density, a “bottom up” synthesis strategy by calcining Au@BSA microspheres was adopted in this work. Unlike the “top-down” dealloying method, the “bottom up” method is cost-effective, environmentally friendly and simple. The resultant gold-cluster film was composed of 4

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the network structure interconnected with gold particles and the disordered 3D hierarchical pores. Recently, we constructed the laccase/3D macroporous gold-cluster based biocathode, which exhibited an onset potential of 0.62 V vs. SCE to oxygen reduction and a high catalytic current of 0.61 mAcm-2 [33]. In this work, we report the

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use of the as-prepared gold-cluster film as the direct electrochemical sensing interface. Further, the amperometric detection of glucose using this nonenzymatic electrode at a

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low potential is demonstrated, which is of good sensitivity, excellent stability and

anti-interference performance. This work provides a useful idea of the once shaping

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preparation of noble metal or metal oxide film electrode for non-enzymatic direct

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electrochemical sensing interface by protein-templated micromaterials. 2. Materials and methods

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2.1. Chemicals and reagents

Chloroauric acid (HAuCl4), ascorbic acid (AA), acetamidophenol (AP) and uric

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acid (UA) were purchased from Sinopharm Chemical Reagent Co. Bovine serum

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albumin (BSA) was purchased from Xiamen Sanland Chemicals Company. Ultrapure water was prepared using a Millipore Milli-Q system and used throughout. Phosphate

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buffered saline (PBS, pH 7.4) was used as the supporting electrolyte. All other reagents were of the highest grade and all solutions were prepared with ultrapure water. The fluorine-doped tin oxide conducting glass (FTO) was purchased from Midwest Group Company (USA). 2.2. Synthesis of Au@BSA microspheres The Au@BSA microspheres were synthesized according to the previous report [32].

Briefly, 10 mL of HAuCl4 aqueous solution (10 mM) was dropwise added into the 10 mL of BSA aqueous solution (5 mg/mL) under vigorous stirring, at room temperature. After the stirring for 5 min, 50 mg of AA was fleetly added to the above mixture solution. After the reaction for 5 min, the obtained Au@BSA microspheres were centrifuged, washed with water and stored at 4 °C. 5

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2.3. Preparation of gold-cluster film electrode In the typical synthesis of preparation of the gold-cluster film electrode, 5 μL of Au@BSA aqueous solution was carefully dripped onto the FTO to make a circular

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drop. After drying at 60 °C, the modified FTO was directly calcined at 400 °C in air for 2 h and cooled down to the room temperature.

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2.3. Apparatus and methods

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The morphology of the as-prepared Au@BSA microspheres and gold-cluster film was observed by the field emission scanning electron microscopy (FE-SEM,

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HITACHI S-4800).

All electrochemical experiments were performed on a CHI660D electrochemical workstation (CH Instruments, Chenhua, Shanghai, China) in a conventional

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three-electrode system using a modified FTO as working electrode, a Pt wire as counter electrode and a saturated calomel electrode (SCE) as reference electrode. All

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potentials in this article were recorded versus SCE. All electrochemical measurements

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were carried out at room temperature (~25ºC). The PBS solution was deaerated with high purity nitrogen for 20 min previously and kept on feeding nitrogen through the

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entire experimental process. Amperometric detection was carried out by the continuous addition of glucose into the stirring NaOH solutions under the specified potentials. The effect of dilution on the final concentration has been taken into consideration.

3. Results and discussion

3.1. Characterization of Au@BSA microspheres and gold-cluster film Au@BSA microspheres were synthesized by bio-templated method [32]. When AA

was added to the mixture solution of HAuCl4 and BSA, a rapid colour change occurred from yellow, to colourless, to gray, and finally to black in a short time, implying the formation of Au microspheres. After the calcination of Au@BSA 6

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microspheres, gold-cluster film became golden, implying that the BSA and AA were removed and the gold-cluster film formed. Meanwhile, the circular gold-cluster film (~3 mm in diameter) was firmly immobilized on FTO (7.3 mm × 4.9 cm). The SEM image showed the good monodispersity and uniform morphology of Au@BSA

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microspheres with an average size of 500 nm in diameter (Fig. 1A). Subsequently,

Au@BSA microspheres were dripped onto FTO and heated at 400 °C in air to form

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gold-cluster film and the removal of BSA. The calcined Au-cluster film has uniformly interconntected network structure with the disordered worm-hole-like pores in a

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hierarchical scale (ranging from 50 nm to 2 μm) (Fig. 1B). The hierarchical porous structure of gold-cluster film provided large surface area and much active sites for

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electrocatalysis.

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3.2. Electrocatalytic behavior of gold-cluster film electrode

Prior to the study about the electrocatalytic behavior, the gold-cluster film-modified

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electrode was activated by cyclic voltammetry (CV) in 1.0 M H2SO4 solution. With the scan cycles increasing, the peak currents were enhanced and finally become stable

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in both the cathode peak and the anode peak, which indicates that the gold film was

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clean and almost inclusion-free. The CV curves of the as-prepared electrode in 1.0 M H2SO4 solution (curve a, Fig.2). The oxidation current starting at about 1.1 V was orginating from the formation of gold oxides and the reduction peak at about 0.85 V due to the reduction of the gold oxides. The smooth gold electrode (curve b, Fig.2) showed a similar result but lower current response, implying that gold-cluster film had larger surface area and activated. Subsequently, the electrocatalytic activities of gold-cluster film towards glucose

oxidation were detected to evaluate its potential application in glucose sensor. Fig. 3A depicts the CV profiles of gold-cluster film electrode in nitrogen-saturated 0.2 M NaOH solution in the absence and presence of 1 mM glucose. The bare FTO has not obvious current response in the absence and presence of 2 mM glucose. In the NaOH solution, there were a pair of redox peaks (purple curve), presumably resulting from the oxidation from gold to gold oxides (Au2O3) (peak-I, at 0.4 V) and the following 7

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reduction of gold oxides, respectively (peak-II, 0.05 V). The both peaks presumably involved a continuous redox reaction, which can be formulated as Au ↔ AuOH ↔ Au2O3 [34]. After the addition of glucose (1 mM) to the NaOH solution, three additional peaks obviously appeared in CV curve (blue curve). In the positive scan,

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the first increase in current (starting from about -0.6 V) could be due to the oxidation of glucose (glucose + AuOH → gluconolactone + Au), resulting in an oxidation

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peak-III at about -0.45 V. Subsequently, the further oxidation peak-IV of the gluconolactone arose at about 0.2 V [35]. The glucose oxidation was dependent on the

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amount of AuOH. At negative potentials, the formation of Au-OH was limited on the sensing interface, and glucose was incompletely oxidized to gluconolactone. With the

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increasing potential, the number of AuOH increased, and the further oxidation of gluconolactone take place. However, when the applied potential was larger than 0.3

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V, the formation of Au2O3 led to the decrease of AuOH, which suppresses the oxidation of glucose. Therefore, the current decreased and the similar peak to the

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blank NaOH solution (purple curve, Fig.3A) appeared. Due to the freshly generated Au reduced by glucose and gluconolactone on the surface of gold-cluster film, the similar peak was

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slightly higher than that in blank NaOH solution. In the negative scan, the reduction of

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Au2O3 provided enough AuOH (starting from about 0.1 V), which led to a greatly enhanced oxidation current in the presence of glucose (peak-V at about 0 V). These results suggested the excellent catalytic activities of the gold-cluster film to glucose oxidation in comparison with the blank NaOH solution. The catalytic activity of the gold-cluster film to the glucose was further supported by increasing glucose concentrations. The currents of all oxidation peaks (peak-I/III/IV/V) were increased as the glucose concentration increased (Fig. 3B), suggesting that the as-prepared electrode could be effectively used to determine glucose. 3.3. Non-enzymatic amperometric detection of glucose For electrochemical sensors, amperometric detection is usually applied because of its real-time monitoring and simple operation. For amperometric detection, it is important to select a suitable potential to obtain highly sensitive but selective current 8

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response for glucose oxidation. As mentioned above, all corresponding peak potentials can be used to measure the catalytic activity to glucose. However, the detection at low potential such as -0.45 V easily suffered from interference of oxygen, for which deoxygenization is needed, while the detection at potentials higher than

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+0.2 V easily interfered from common oxidizable interferents, such as AA, AP and

UA. Therefore, we selected -0.05−0.1 V as the candidate potentials for the

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amperometric detection of glucose and compared the corresponding current responses of successive and consistent addition of glucose using the sensor. The results

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demonstrated that the current responses at 0.1 V were most sensitive than those at other potentials (Fig. 4). Hence, +0.1 V was applied to the non-enzymatic

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amperometric detection of glucose (Fig. 5). After the successive addition of glucose, the current rapidly reached to ~96% of the steady-state values within less than 5 s,

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suggesting that the as-prepared electrode could respond rapidly to the change of glucose concentration. Furthermore, the current response increased linearly with the

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increasing concentration of glucose from 10 μM to 10 mM. The calibration curve was obtained by plotting the current with the varying glucose concentration (inset in Fig.

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5). The linear regression equation was y = 0.753x + 0.556 with the correlation

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coefficient (R) of 0.995. The limit of detection (LOD) was estimated to be about 2 μM glucose (S/N = 3), which was much lower than 50 μM for glucose sensors based on

PtRu modified multi-walled carbon nanotubes [36], and 0.1 mM for GOx-modified nanoporous PdCu and PtAg alloy [37]. The sensitivity for the gold-cluster film electrode was calculated to be 10.65 μA mM-1 cm-2, which was higher than 9.6 μA mM-1 cm-2, 1.06 μA mM-1cm-2 for the glucose electrochemical sensor based on

mesoporous platinum [18] and single-walled carbon nanohorns [38], respectively. 3.4 Selectivity of the sensor In human blood, the normal physiological level of glucose is 4.4–6.6 mM [2], and AA, AP and UA levels are 0.1 mM, 0.1 mM, 0.3 mM, respectively [39]. For blood glucose detection, AA, AP and UA were the common interfering substances. Therefore, it is very necessary to measure the effect of these interferent components 9

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on glucose detection. In doing so, the CVs of 5 mM glucose solutions were measured at scan rate of 50 mV/s. Then these interferents were separately added into fresh glucose solution (5 mM), CVs were recorded again. The anodic current (ia) at 0.1 V was measured for each CV and the relative ratios of ia for 5 mM glucose containing

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different interferents to ia for 5 mM glucose were calculated. As shown in Fig. 6A, the

relative ia ratios for glucose in the presence of AA, AP and UA were 103.6%, 101.8%

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and 101%, respectively. That is, the presence of interferents only arose less than 4%

increase in ia, suggesting that AA, AP and UA had negligible interferences on glucose

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detection at 0.1 V. Therefore, the as-prepared sensor has fair selectivity as a promising nonenzymatic sensor.

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It was reported that the chloride ions (Cl-) had a serious poisoning effect on some metal electrocatalysts such as Au, Pt and their alloys, inducing them to lose their

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activities toward the oxidation of glucose [40, 41]. In order to understand whether Clcould poison the electrocatalytic activity of the gold-cluster film, the CV response

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was examined in 0.2 M NaOH solution containing 5 mM glucose with (blue) and without (red) 0.15 M NaCl (Fig. 6B). Both CV curves overlap almost completely,

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implying that Cl- has no observable poisoning effects on gold-cluster film and the

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as-prepared sensor can be used for glucose detection in a high concentration of Cl-. This is an important advantage for the practical application of glucose sensor. 3.5 Stability and reproducibility of the sensor In order to evaluate the operational stability of the glucose sensor, a continuous

measurement of glucose by chronoamperometry at an applied potential of +0.1 V was performed. The current response drifted less than 5% over a period of 3 h (Fig. 7), demonstrating the good stability of the proposed sensor. This result also illustrated that the surface properties (such as surface area and electrochemical activity) of gold-cluster film had not obvious change under the continuous measurement of glucose. As a distinguished feature, non-enzymatic sensors have long term stability compared to enzymatic sensors. To evaluate the storage stability, the current response 10

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of gold-cluster film electrode to 2 mM glucose was tested over a period of 30 days of storage at room temperature. The electrode maintained about 98% of activity (data not shown) after 30 days, implying that the as-prepareed electrode can be used for a long time.

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To verify the reproducibility of the proposed glucose sensors, five gold-cluster film

electrodes were prepared with the same procedure. The relative standard deviation

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(RSD) of the response currents was 2.2% for 2 mM glucose measured at +0.1 V, which was calculated from the amperometric experiments for five gold-cluster film

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electrodes, indicating that the as-prepared sensor displayed the better reproducibility.

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4. Conclusions

In summary, the gold-cluster/FTO electrode was prepared by calcining Au@BSA

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microspheres on FTO. The resulted gold-cluster film was composed of the network structure interconnected with Au particles and the disordered 3D hierarchical pores. Due to large surface area, high electro-conductivity and excellent electrocatalysis, the

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gold-cluster/FTO electrode was used as a promising nonenzymatic sensor for

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detection of glucose at low potential, where it has not only high sensitivity but also excellent selectivity. In addition, the as-prepared electrode was stable and resistant

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against presence of excess of Cl-. The simple and cost-effective strategy for the

preparation of 3D hierarchical porous electrode will be promising for the large-scale production of direct electrochemical sensor. Meanwhile, this strategy may also extend to other noble metal films and noble metal oxide films for various potential applications including sensor, supercapacitor, biological fuel cell and solar cell. Acknowledgments

This work was financially supported by National Natural Science Foundation of China (Nos. 91227116 and 21275152) and the Hundred-Talent-Project (No. KSCX2-YW-BR-7), Chinese Academy of Sciences. References [1] J.D. Newman, A.P.F. Turner, Home blood glucose biosensors: a commercial perspective, Biosens. 11

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glucose sensors, Anal. Chem., 73 (2001) 1599.

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Figures and Figure captions:

Fig. 1. The SEM images of the as-prepared Au@BSA microspheres (A) and gold-cluster film (B).

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Fig. 2. CV curves of gold-cluster film electrode (a) and Au disk electrode (b) at

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different scan cycles in 1.0 M H2SO4 solution. Scan rate, 50 mV s-1.

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Fig. 3. (A) CV curves of bare FTO in N2-saturated 0.2 M NaOH solution in the

absence (orange) and presence (green) of 2 mM glucose, and gold-cluster film/FTO in N2-saturated 0.2 M NaOH solution in the absence (purple) and presence (blue) of 2 mM glucose. (B) CV curves of Au-cluster/FTO in N2-saturated 0.2 M NaOH solution

with 0 (yellow), 1 (green), 2 (blue) and 5 mM (purple) glucose. Scan rate, 50 mV s-1.

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Fig. 4. The i-t curves of gold-cluster film/FTO in stirring 0.2 M NaOH solution with successive addition of glucose (the final concentration from 10 μM to 18 mM) under

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M

different applied potentials of 0.1 V (a), 0.05 V (b), 0 V (c) and -0.05 V (d).

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Fig. 5. The typical i-t curve of gold-cluster film/FTO in stirring 0.2 M NaOH solution with successive addition of glucose (the final concentration from 0.01 to 28 mM) at

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applied potential of +0.1 V. Inset, the calibration curve for glucose.

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Fig. 6. (A) Relative ia ratio at 0.1V of gold-cluster film/FTO with the respective

addition of 5 mM glucose and 5 mM glucose mixed with AA (0.1 mM), AP (0.1 mM) and UA (0.3 mM) by CV measurements in 0.2 M NaOH solution. Relative ia ratio at

0.1V for 5 mM glucose was defined as 100%. Scan rate, 50 mV s-1. (B) CVs of

gold-cluster film/FTO in 0.2 M NaOH solution containing 5 mM glucose in the absence (red) and presence (blue) of 0.15 M NaCl. Scan rate, 50 mV s-1.

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Fig. 7. Operational stability of the sensor. Applied potential, +0.1 V.

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