Chitosan cryogel with embedded gold nanoparticles decorated multiwalled carbon nanotubes modified electrode for highly sensitive flow based non-enzymatic glucose sensor

Accepted Manuscript Title: Chitosan Cryogel with Embedded Gold Nanoparticles Decorated Multiwalled Carbon Nanotubes Modified Electrode for Highly Sensitive Flow Based Non-Enzymatic Glucose Sensor Authors: Tawatchai Kangkamano, Apon Numnuam, Warakorn Limbut, Proespichaya Kanatharana, Panote Thavarungkul PII: DOI: Reference:

S0925-4005(17)30332-5 http://dx.doi.org/doi:10.1016/j.snb.2017.02.105 SNB 21836

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

31-10-2016 24-1-2017 16-2-2017

Please cite this article as: Tawatchai Kangkamano, Apon Numnuam, Warakorn Limbut, Proespichaya Kanatharana, Panote Thavarungkul, Chitosan Cryogel with Embedded Gold Nanoparticles Decorated Multiwalled Carbon Nanotubes Modified Electrode for Highly Sensitive Flow Based Non-Enzymatic Glucose Sensor, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.02.105 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.

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Chitosan Cryogel with Embedded Gold Nanoparticles Decorated Multiwalled Carbon Nanotubes Modified Electrode for Highly Sensitive Flow Based Non-Enzymatic Glucose Sensor

Tawatchai Kangkamano a, b, c, Apon Numnuama, b, c, Warakorn Limbuta, b, d, Proespichaya Kanatharanaa, b, c and Panote Thavarungkula, b, e*

a

Trace Analysis and Biosensor Research Center, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand b

Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla

University, Hat Yai, Songkhla 90112, Thailand c

Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand d

Department of Applied Science, Faculty of Science, Prince of Songkla University, Hat Yai,

Songkhla 90112, Thailand e

Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla

90112, Thailand * Corresponding author at: Department of Physics, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Tel.: +66 74 288753; fax: +66 74 558849. E-mail address: [email protected] (P. Thavarungkul).

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Highlights

 A novel cryogel of AuNPs-MWCNTs-CS non-enzymatic sensor was developed.  The sensor can detect glucose within a short time, highly sensitive and selective.  LOD of the sensor was sufficient low for a 10,000 times of samples dilution.  The sensor showed a good stability with 525 analysis for one modified electrode.  The sensor would be useful for glucose and other oxidizable analytes detection.

Abstract This work describes the combined electrocatalytic and synergistic properties of gold nanoparticles (AuNPs) decorated multiwalled carbon nanotubes (MWCNTs) and the large surface area of chitosan (CS) cryogel for the fabrication of a highly sensitive and stable electrochemical non-enzymatic sensor. MWCNTs were pre-mixed with citrate ions that serve as the substrate for gold deposition along their chains. The AuNPs-MWCNTs nanocomposite and chitosan cryogel were easily modified on a gold electrode in only 1 hour by freezing and thawing of the mixture after casting it on the electrode surface. Glucose, as a model analyte, was measured by the developed electrode in a flow-injection amperometric system. A fast response time and excellent sensitivity were obtained. The sensor exhibited a linear range between 0.001 and 1.0 mM with a low detection limit (0.5 M) and a high operational stability (525 injections). There were no effects from the common interferences found in physiological levels in blood samples. After measuring glucose in human blood plasma using this sensor, the results were in good agreement (P  0.05) with those obtained from the standard spectrophotometrically-measured hexokinase method employed clinically. These good performances made this sensor a potential alternative tool for the detection of glucose and other oxidizable analytes.

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Keywords: flow based non-enzymatic glucose sensor; gold nanoparticles decorated multiwalled carbon nanotubes; chitosan; nanocomposite; cryogel

1. Introduction Non-enzymatic electrochemical sensors have received a lot of attention due to their simplicity and reproducibility in various types of biological sample detection [1-4]. However, in some cases chemisorbed intermediates can lead to poor operational stability and the lack of recognition units also contributes to interferences by other electro-active species [5]. Thus, enhancing electrocatalysis and avoiding electro-active interferences are crucial in the development of non-enzymatic electrochemical sensors. One way of reaching this goal is by developing an electrode platform with materials and strategies that improve the detection of an analyte of interest. Research in electrode modification has focused on the use of nanomaterials, especially transition metal nanoparticles [6, 7] and their alloys [8, 9] and oxides [10, 11]: predominant among them, gold nanoparticles (AuNPs) [1, 12, 13]. Compared to other metals, they have greater response, better biocompatibility and more potential for negative oxidation of analytes in neutral and alkaline solution [14]. To heighten their performance, AuNPs have been developed with other molecules or nanoscaled materials into nanocomposites that combine their unique properties [15]. Examples are AuNPs-carbon nanotubes (AuNPs-CNTs) [16], AuNPs-graphene (AuNPs-Gr) [17], AuNPs-polymer [18, 19] and AuNPs-biomolecules [20]. These nanocomposites exhibit synergic effects that produce distinctly higher performance than AuNPs alone. Among these nanocomposites, multiwalled carbon nanotubes (MWCNTs) [15, 16], with properties such as a unique tubular structure, large specific surface, modifiable sidewalls, high strength, extremely high conductivity and

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biocompatibility, composited with AuNPs have attracted considerable attention due to their simple preparation and potential applications [15, 21]. This kind of nanocomposite helps to enhance the rate of electron transfer and increase catalytic ability [22]. Moreover, MWCNTs can be used as a substrate for gold deposition, showing an effective dispersion along their chains [15]: a key factor in reducing electrode fouling [23]. Another important factor in the modification of nanomaterials onto the electrode is the use of an effective supporting material that prevents separation from the electrode surface. An interesting supporting platform is a macroporous chitosan (CS) cryogel, which can be very easily made by freezing and thawing chitosan. This cryogel can stabilize the entrapped nanomaterials and its large surface area to volume ratio can help increase the amount of active sensor material [24, 25]. Therefore, as a supporting matrix, chitosan cryogel has certain advantages. The objective of the present work is to combine the excellent electrocatalytic and synergistic properties of AuNPs decorated MWCNTs, that have been entrapped in the cryogel, with the large surface area of the porous chitosan cryogel for the fabrication of a flow-based electrochemical non-enzymatic sensor. Glucose was selected as the model analyte due to its importance in fields such as medical diagnostics, the food industry, and process control [26]. This is the first report of the use of chitosan cryogel as a supporting matrix for AuNPs-MWCNTs composite modified onto an electrode to be incorporated into a flow-based non-enzymatic sensor system. Affecting parameters were optimized to obtain the highest sensitivity of the sensor. The sensor was evaluated for its analytical performance, used to test glucose in real blood plasma samples, and the obtained results statistically compared to those obtained by a conventional analytical method. 2. Materials and methods 2.1 Materials

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Hydrogen tetrachloroaurate hydrate (III) (HAuCl4, with a purity of 99.9%) and chitosan from shrimp shells (low viscosity, <200 mPa.s, 1% in acetic acid at 20 ◦C) were from Sigma-Aldrich (St. Louis, USA); D-(+)-glucose anhydrous (≥98.0%), dopamine (≥98.0%), uric acid (≥98.0%) and glutaraldehyde (25% solution) from Fluka (Buchs, Switzerland); ascorbic acid from Alfa Aesar (Tianjin, China); sodium hydroxide (97.0%) from RCl Labscan (Bangkok, Thailand); and sodium chloride from Merck (Damstadt, Germany). The MWCNTs (purity ≥95%) had an average diameter of 60-100 nm and a length of 2-5 m and were purchased from Shenzhen Nano-Technologies Port Co., Ltd. (Shenzhen, China). They were functionalized using a 3:1 (v/v) concentrated H2SO4 and HNO3 solution to form carboxylated MWCNTs, filtered and left in a vacuum desiccator before use. All other chemicals used were analytical grade. 2.2 Preparation of AuNPs-MWCNTs nanocomposite The nanocomposite of AuNPs-MWCNTs was prepared following the method described by Zhang et al. with a slight modification [15]. In brief, 1.5 mg of the carboxylated MWCNTs were added in 2.0 mL of 1.0% (w/v) sodium citrate solution and ultra-sonicated for 5 min at room temperature until they were completely dispersed within the solution, their surfaces coated with sodium citrate [15]. This suspension was transferred to a beaker containing 25.0 mL ultrapure distilled water and heated to the boiling point while stirring. Then 250 L of 1.0% (v/v) HAuCl4 was rapidly added, mixed thoroughly for 5 min and maintained at boiling point for another 5 min until the color of the solution changed to watermelon red. During this process the heat-treated MWCNTs adsorb citric acid onto their side walls via electrostatic interaction, which facilitates the in-situ reduction of the Au3+ on the nanotubes to Au0 nanoparticles [27]. The morphology of the synthesized nanocomposite was observed by transmission electron microscopy (TEM, JEM-2010, JEOL, Japan).

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2.3 Immobilization of AuNPs-MWCNTs-CS cryogel on gold electrode A gold rod, 3.0 mm in diameter and of 99.99% purity, was polished using successively finer alumina slurries of particles of 5, 1, and 0.3 m. It was then rinsed with distilled water and electrochemically cleaned in 0.5 M sulfuric acid for 25 cycles (0.1-1.5 V, scan rate 100 mV s-1). Fig. 1 shows the preparation of AuNPs-MWCNTs-CS nanocomposite cryogel layer on this gold electrode surface. A composite mixture combining 50.0 L of AuNPs-MWCNTs nanocomposite solution (Section 2.2) with 50.0 L of 2.0 % (w/v) chitosan solution in 1.0 % (v/v) acetic acid (a suitable concentration for the formation of a cryogel from a preliminary study) was ultra-sonicated for 5 min, and 5.0 L of 5.0% (v/v) glutaraldehyde, the cross-linking reagent, was then added. A 4.0 L aliquot of this mixture was dropped onto the surface of the gold electrode, which was immediately stored at -20 °C for 1 h for cryogelation. It was thawed at 4 °C for 10 min and rinsed with distilled water. For comparison, we also prepared cryogel modified Au electrodes of different compositions,: CS/Au, AuNPs-CS/Au, MWCNTs-CS/Au; and a non-cryogel modified electrode of AuNPsMWCNTs-CS/Au prepared without the cryogelation process. The surface morphologies of the cryogel and non-cryogel electrodes were observed using a scanning electron microscope (SEM, Quanta 400, FEI, Japan). While not in use, the sensor was simply kept in distilled water at room temperature. Fig. 1 here 2.4 Electrochemical characterization of the modified electrodes Electrochemical characterization of the modified electrodes was performed in a batch system using an Autolab Type III (Metrohm Autolab B.V., KM Utrecht, The Netherlands) controlled by GPES 4.9 software (General Purpose Electrochemical System). The threeelectrode system comprised a modified gold working electrode, a silver/silver chloride

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reference electrode and a platinum wire counter electrode. The interface properties of different surface-modified electrodes (Section 2.3) were characterized by cyclic voltammetry from - 0.30 to 0.70 V at a scan rate of 100 mV s-1 in a 10 mM [Fe(CN)6]4-/3- with 0.10 M KCl solution. The responses of different modified electrodes to glucose oxidation were investigated by cyclic voltammetry from - 0.40 to 0.70 V at a scan rate of 100 mV s-1 in 0.050 M of NaOH. 2.5 Amperometric glucose detection The amperometric detection of glucose was performed in a flow injection system. The AuNPs-MWCNTs-CS cryogel modified electrode, a custom built silver/silver chloride electrode (3.0 M KCl) and a platinum wire were inserted into a custom built flow cell (10 μL) (Fig. 1) and were connected to the Autolab. Standard glucose solutions were prepared in 0.050 M NaOH (the running electrolyte) and injected through a six-port injection valve (Valco Instrument, USA). A peristaltic pump (Miniplus 3, Gilson, France) provided a constant flow of glucose to the flow cell for detection. The working potential was applied and the peak current related to glucose concentration was recorded. 2.6 Optimization of operational conditions We optimized the conditions affecting the flow-injection amperometric detection by varying the applied potential, concentration of the alkaline electrolyte, flow rate, and sample volume. A series of standard glucose solutions (six replicates for each concentration) were tested and the optimized conditions were considered those that provided a balance between a short analysis time and a high sensitivity (slope of the calibration curve). 2.7 Interference study The developed sensor would be tested with blood plasma samples, thus, its selectivity to glucose was investigated with possible common blood interfering species under the

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optimal conditions. Ascorbic acid (AA), uric acid (UA) and dopamine (DA) were tested as the oxidized species that can cause false positive signals. Chloride ions (Cl-) were also investigated as a species that can cause a serious interference on the gold electrode by forming complex intermediates [5, 26, 28]. They were tested at physiological concentrations, and at 100 times higher. Since the matrix effect for blood samples is usually relieved by 20100 fold sample dilutions [24, 29, 30], the interference study was performed by mixing the normal physiological concentration of 5.0 mM glucose [31] with different concentrations of interferences and analyzed after a 100 times dilution using 0.050 M NaOH and compared with that from glucose alone. 2.8 Operational and storage stabilities Operational stability was assessed by repeatedly injecting 0.050 mM glucose (100 times dilution of glucose concentration found in normal human plasma; 5 mM [31]) into the flow system under the optimal conditions. The material’s storage stability was also evaluated by fabricating a number of cryogel electrodes which were kept in a closed container containing distilled water at room temperature. We tested them after 1, 3, 5, 7, 9, 13, 15, 30, 60 and 90 days by using them one by one to measure a series of standard glucose solutions from 0.10-0.50 mM. 2.9 Real sample analysis Blood plasma samples obtained from Songklanagarind Hospital, Hat Yai, Thailand were spiked with a series of glucose concentrations (before dilution). Slopes obtained from the standard curve of standard glucose solutions and that of plasma spiked with glucose were compared by two-way ANOVA to account for any matrix effects. Samples of a suitable dilution factor with no matrix interference (Section 3.6) were then measured by the developed

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sensor. The results obtained from the non-enzymatic glucose sensor and those from the standard clinical hexokinase method were compared using the Wilcoxon signed rank test.

3. Results and discussion 3.1 AuNPs-MWCNTs nanocomposite and surface morphology of the modified electrode The TEM image of the nanocomposite (Fig. 2A) shows a dense loading of AuNPs decorated on the MWCNTs with very little aggregation of AuNPs. The average diameter of the AuNPs was 81 nm (n=349) (Supplementary data Fig. S1). Fig. 2B shows the SEM image of the surface without any porous structure of the non-cryogel nanocomposite. During the freezing of the cryogel, the solvent turned into ice, and, when thawed, the ice melted leaving behind a porous interconnected matrix with pore diameters ranging from 20-140 m (Fig. 2C). The embedded MWCNTs can be observed as the coated protruding surface (Fig. 2D). This porous chitosan cryogel incoporates the AuNPs-MWCNTs composite, providing a large conductive surface area for glucose oxidation, hence an increased sensitivity. Fig. 2 here 3.2 Electrochemical behaviors of the modified electrode Electrochemical characterization of the different surface modification was observed from the cyclic voltammograms. The bare Au electrode showed well-defined oxidation and reduction peaks of [Fe(CN)6]4-/3-. After modification with CS cryogel (CS cryogel/Au), there was a marked decrease in the peak current attributed to the non-conductive properties of CS. When either AuNPs (2.94 nmol of gold, 2.61  1010 particles of 13 nm, see Supplementary

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data for the calculation) or MWCNTs (0.30 g, see Supplementary data) was added to the cryogel (AuNPs-CS cryogel/Au and MWCNTs-CS cryogel/Au), there was an increase in the peak current compared to the cryogel alone, due to the good conductivity of AuNPs and MWCNTs. However, the peak currents of both materials were still less than the bare Au electrode due to the blocking behavior of CS. But when the AuNPs-MWCNTs-CS cryogel/Au was fabricated with the same amount of gold and MWCNTs as the AuNPs (2.94 nmol of gold, 1.12  1011 particles of 8 nm) and MWCNTs (0.30 g), the peak current was more than double (2.2 and) that of the AuNPs-CS cryogel/Au electrode and almost double (1.6 and 1.8 times) that of the bare Au and MWCNTs-CS/Au electrodes, respectively (Supplementary data Fig. S2). These results indicated that a better conductivity of AuNPs combined with MWCNTs can effectively increase the response. When the AuNPsMWCNTs-CS composite was modified onto the electrode without the freezing and the thawing process (AuNPs-MWCNTs-CS non-cryogel/Au), the peak current was much lower. This was because the flat layer of the non-cryogel provided smaller surface area of the modified electrode led to a lower rate of electron transfer in the voltammetric system than the porous cryogel that allowed the analyte to easy diffuse through. 3.3 Glucose oxidation at AuNPs-MWCNTs-CS cryogel modified electrode The electrocatalytic reactivity of the different surface-modified electrodes toward 4.0 mM glucose was first tested by cyclic voltammetry in 0.050 NaOH which showed two oxidation peaks (Fig. 3A) at about - 0.10V (peak a) and 0.25V (peak b). These were similar to those reported earlier where peak a corresponded to the direct electrochemical oxidation of glucose (dehydrogenation) to the adsorbed intermediates on the AuNPs-MWCNTs surface. Peak b is attributed to the continuous oxidation of dehydrogenated glucose to gluconolactone by the adsorbed OH species on the AuNPs before reacting with hydroxide ions in the solution to form gluconate [28, 32, 33]. The activity of glucose oxidation on the Au surface, especially

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via the influence of the adsorbed OH- anions and the gold oxide surface monolayer formation, depended on the surface area and structure [33-35]. Thus, the exposure of more AuNPs, on the MWCNTs embedded in the large surface area of the pore walls of the cryogel modified electrode, could help enhance catalytic activity for the electrochemical oxidation of glucose. After 0.40 V, Au was further oxidized to form gold oxide on the surface (peak c). On the reverse scan, the reduction of gold oxide back to gold occurred (peak d). As soon as the gold oxides were reduced, they provided the fresh surface of AuNPs for the direct reoxidation of glucose by which another oxidation peak appeared at approximately 0.0 V (peak e) [20, 28]. Compared to other modified electrodes this AuNPs-MWCNTs-CS cryogel/Au provided the highest responses while the CS cryogel modified Au electrode (CS cryogel/Au) exhibited the lowest, likely because of its insulating properties. The AuNPs-CS cryogel/Au showed a higher catalytic reactivity toward glucose than that obtained from MWCNTs-CS cryogel/Au. This was due to the better catalytic property of AuNPs toward glucose compared with MWCNTs. In the case of the AuNPs-MWCNTs-CS non-cryogel modified electrode, because of the smaller surface area of the non-cryogel film, its peak current was much less than that of the cryogel form (Supplementary data Fig. S3). Fig. 3 here These electrodes were then used to test a series of concentrations of glucose (0.10, 0.20, 0.30, 0.40, 0.50 mM) by measuring the height of the oxidation peak (peak b Fig. 3A). As expected the AuNPs-MWCNTs-CS cryogel/Au provided the best sensitivity (slope of the calibration plot) in response to glucose (Fig. 3B), which corresponded to the results from cyclic voltammetry (Supplementary data Fig. S2 and S3). To evaluate the kinetics of the glucose oxidation on the AuNPs-MWCNTs-CS cryogel modified electrode, we recorded cyclic voltammograms in a 0.050 M NaOH solution containing 4.0 mM glucose, at different scan rates. The anodic peak currents (peak b Fig. 3A)

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were found to be directly proportional to the square root of scan rate (Supplementary data Fig. S4), indicating that the electrochemical kinetics was a diffusion-controlled process [36]. The result is in agreement with that reported by previous works [37, 38].

3.4 Optimization of the flow injection system Further amperometric glucose measurements by the AuNPs-MWCNTs-CS cryogel modified Au electrodes were performed in a flow injection system. The effects of the various flow based measurement conditions were investigated. The initial operating conditions were; 350 l sample volume, flow rate 500 Lmin-1 and NaOH 0.050 M. The obtained optimal condition in each parameter was then used for further experiments. 3.4.1 Glucose oxidation potential An ideal applied potential would be one at which a high current response of glucose oxidation could be achieved while those from the interferences are negligible. A series of potentials (0.00, 0.10, 0.15, 0.20, 0.25 and 0.30 V) covering the oxidation peak (Fig. 3A) was applied for the amperometric detection of glucose (0.10, 0.20, 0.30, 0.40 and 0.50 mM). The results indicated that the sensitivity increased with the applied potential from 0.00 to 0.20 V then gradually decreased, which corresponded well with the CV (Supplementary data Fig. S5). Therefore, 0.20 V was used for further experiments. 3.4.2 The concentration of NaOH For glucose oxidation on Au, the current response also depends on the NaOH concentration since OH- neutralizes the protons generated from the dehydrogenation steps of the reaction. Increasing the solution alkalinity would increase the rate of the formation of a more easily oxidized glucose intermediate. In addition, the amount of the catalytic oxide, AuOH, present, is dependent on the concentration of OH- on the electrode surface [39].

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Therefore, we tested NaOH concentrations of 0.005, 0.050, 0.100, 0.150, 0.200 and 0.300 M (i.e., pH 11.70, 12.70, 13.00, 13.18, 13.30 and 13.48, respectively) and the sensitivity of glucose detection increased with concentration up to 0.050 M and decreased at higher concentrations (Supplementary data Fig. S6). With the increased NaOH concentration, the amount of the catalytic AuOH generated by the chemisorbtion of hydroxide ions was increased. It could facilitate the adsorption of glucose to AuNPs [32, 39, 40]. The reduced response at higher NaOH concentrations was likely due to the transformation of Au to the water-soluble ion (HAuO32-) [28]. As a result, 0.050 M NaOH was chosen for further glucose detection. 3.4.3 Flow rate and sample volume For a flow-based sensor, flow rate and sample volume are two main factors in the system’s performance. Different flow rates (400, 500, and 600 μL min-1) and sample volumes (200, 250, 350, and 500 μL) were studied simultaneously. From the results, higher current responses were achieved with a larger sample volume and a lower flow rate due to a longer contact time. On the other hand, a faster flow rate and a smaller sample volume could speed up the analysis: however, a lower sensitivity was obtained. To strike a balance between high current response and short analysis time, a 500 L min-1 flow rate and a 250 L sample volume were chosen for further glucose detection (Supplementary data Fig. S7). In summary, the optimum conditions for the flow injection amperometric enzymelessglucose sensor were: applied potential 0.20 V, 0.050 M NaOH of alkaline electrolyte solution, flow rate 500 μL min-1, and sample volume 250 μL. Under the optimal conditions, the analysis time for glucose was about 2.5 min for each injection. 3.5 Performance of the AuNPs-MWCNTS-CS cryogel modified electrode 3.5.1 Linearity and limit of detection

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Under the optimal conditions, the relationship between oxidation current and glucose concentration (0.001-20.0 M) in a flow based amperometric system (Fig. 1) is shown in Fig. 4A together with an example of the amperometric current signal for different concentrations of glucose in Fig.4B. A linear range was found between 1.0M and 1.0 mM (Fig.4C). The sensitivity, calculated against the electrode surface area, was 27.7 A mM-1 cm-2. At higher concentrations (Fig.4A) the response increased more slowly. One possible origin might be the transition from a monolayer adsorption state of analyte on electrode surface into a more complicated multilayer adsorption state [41]. Another interesting possibility is due to the different activity of the electrode surface with low and high concentration of glucose. In the lower concentrations, due to a high number of active sites (in relation to the total number of the analyte molecules), the responses increased more rapidly with concentrations. While in the higher concentration, due to decreasing active sites (mainly at the surface of the electrode), the responses increased more slowly [42]. For the detection limit, it was found to be 0.5 M (3/S where  = the standard deviation of blank and S= the slope of calibration curve). This value was more than sufficient for the direct detection of glucose in diluted human plasma samples, since the normal range of glucose in human blood is 4.1-5.6 mM [31], it means that almost 10,000 times of samples dilution is possible and this would help reduce the effect from sample matrix. Fig. 4 here 3.5.2 Selectivity of the modified electrode To evaluate the selectivity of the electrode, we prepared analytes of common interferences found in blood samples. These interferences were AA, UA, DA and Cl- and they were mixed with 5.0 mM glucose at concentrations of 0.100 mM, 0.50 mM, 12.0 nM, and 100 mM, respectively. These concentrations are at the higher values in the normal physiological range [31]. They were diluted 100 times before analysis with the developed

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electrode. It was found that at these levels there were no interferences of the current response of 5.0 mM glucose. At 100 times higher concentrations, the responses obtained from AA, UA and DA were also not significantly different from the response obtained from glucose alone (Supplementary data Fig. S8). The selectivity of the developed sensor is likely due to the synergistic effect of AuNPs-MWCNTs nanocomposite which provided better electron transfer efficiency between glucose and the working electrode and increased the catalytically active surface of the AuNPs-MWCNTs nanocomposite [43]. This enabled glucose to be oxidized at a potential of 0.20 V, lower than the oxidation potential of the co-existing interferences (AA, UA and DA) that normally found to be 0.30-0.40 V on gold electrode [44]. Besides, the use of alkaline media provided a restricted environment where interference from molecules such as uric acid and ascorbic acid is minimized [45]. In addition, with the low LOD (0.5 M) of this sensor, samples can be easily dilute many times to a much lower concentration of the interfering compounds, thus, reducing the effect of interferences. As for chloride ions that are the main inhibitor in the electro-oxidation of glucose on Au electrodes because they can be strongly chemisorbed [28], at a concentration higher than 5 times the normal level (500 mM), some interference started to show on the current response of 5 mM glucose. However, this should not affect glucose measurement in real blood plasma samples because the critical physiological concentration level of chloride ion, usually found only in hyperchloremic patients, is around 153 mM [46]. Thus, this sensor was highly selective for the quantification for glucose in blood samples. 3.5.3 Repeatability and reproducibility Repeatability refers to the agreement between successive measurements of the same analyte, whereas reproducibility describes the closeness of agreement between signals obtained using the same method under different conditions (using different fabricated electrode) [47]. Fifteen measurements for each of the three concentrations of glucose

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covering the lower linear range (Section 3.5.1), 0.01, 0.50 and 1.0 mM, produced RSDs of 0.31%, 0.33% and 0.92%, respectively. These are well within the acceptance limits, which are 11%, 7.3% and 5.3%, respectively [48]. For electrodes fabricated on different days, reproducibility was tested by measuring a series of glucose concentrations (0.10, 0.20, 0.30, 0.40 and 0.50 mM). The sensitivities of the six electrodes were 2.37±0.02, 2.44±0.09, 2.37±0.07, 2.38±0.05, 2.38±0.10 and 2.42±0.05 μA mM-1 (Supplementary data Fig. S9). The relative standard deviation of the average sensitivity was calculated to be only 1.2% indicating a good reproducibility in the fabrication of the developed glucose sensors. 3.5.4 Stability of the fabricated non-enzymatic sensor To assess the operational stability of the developed electrode toward glucose sensing, the modified electrodes were investigated by continuously measuring the glucose response from repeated injections of 0.050 mM glucose under optimal operational conditions. The modified electrode exhibited excellent operational stability up to 525 injections with an average response relative to the first response of 98.9±3.3% (Supplementary data Fig. S10A). After 525 injections, the relative response gradually decreased to below 90%. The loss of electrode stability was confirmed by CV after scanning the used electrode in 0.050 M NaOH: the oxidation peak of gold to gold oxide (0.4 V) and the reduction peak of gold oxide to gold (0.2 V) both decreased (Supplementary data Fig. S10B). This might be because the cryogel matrix on the electrode became swollen, leading to a loss of sensing surface area, which especially affected the exposure of AuNPs to the analyte. From the results, it could be said that the electrode has a good operational stability for glucose detection. To assess the electrodes’ stability during storage, we kept the fabricated electrodes in distilled water at normal room temperature and tested their sensitivities after 1, 3, 5, 9, 11, 13, 15, 30, 60 and 90 days. The relative sensitivity retained, up to 60 days, was more than 90 %

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(average 3.500.06 A mM-1) of the initial test after one day: after 90 days it was less than 90% (Supplementary data Fig. S10C). The excellent operational and storage stability obtained were most likely due to the the porosity of the chitosan supporting material and the strength of the MWCNTs that helped to increase both the chemical and mechanical stability of the sensing layer decorated with AuNPs. The results corresponded well to previous reports [49, 50] indicating that the CSMWCNT composite could affect a significant improvement in the mechanical properties and strength compared to pure chitosan. The analytical performance of the AuNPs-MWCNTs-CS cryogel modified Au electrode as a glucose sensor was compared with other AuNPs, MWCNTs and chitosan modified electrodes, as shown in Table 1. The developed electrode exhibited a relatively low limit of detection (0.0005 mM or 0.5 M) that was better than that in most cited reports (Table 1). Although the obtained LOD was slightly higher than LOD reported in some works [6, 51, 52], it was, nonetheless, more than sufficient for the direct detection of glucose, even with a 10,000 times diluted human plasma sample (Section 3.5.1). The developed sensor showed a linear range that covered more concentrations than sensors assessed in most reports. In the case of stability, this fabricated sensor provided better performance than all cited works. These good performances indicated that the AuNPs-MWCNTs-CS cryogel modified electrode is a powerful enzymeless sensor for the direct detection of glucose. This is most likely because of the large surface area of the cryogel porous structure that can help increase the amount of entrapped sensing materials and the accessibility of glucose solution to the electrode. The large amount and good distribution of AuNPs on the electrode, helped by the carboxylated MWCNTs, provided a good electron transfer and excellent electrocatalytic properties of the AuNPs-MWCNTs nanocomposite. The latter property not only enhanced sensitivity but also reduced interference of sensor through the lower measured potential.

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When considering the processes of electrode fabrication, the presented cryogel based glucose sensor was easily prepared in a short time, only 1 hour, by freezing and thawing of the composite mixture after casting on the electrode surface. In addition, a single electrode that retains good stability of response over approximately 525 uses makes it very convenient in real sample analysis, especially for a large number of samples. The storage stability of up to 60 days at room temperature also makes handling much easier. It can be concluded that the combined properties of a large surface area together with the enhancement of electron transfer of the sensing part, coupled with an on-line system led to a highly sensitive and stable flow based glucose sensor. This would be valuable as an alternative non-enzymatic based electrochemical method for glucose detection and a model procedure for adapting in other kinds of analyte detection. 3.6 Analysis of blood plasma samples The developed electrode was used to measure glucose in blood plasma samples. To study the matrix effect, the calibration plot obtained from standard glucose solutions of 0.01, 0.10, 0.20, 0.30, 0.40 and 0.50 mM were compared to that of the spiked samples. Spiked samples were diluted with electrolyte to obtain a 100 and 200 times dilution and were analyzed under optimized conditions. At a 100 times of dilution, at the same concentration, the response peak gradually decreased from the initial one. This was likely due to the fouling liked behavior caused by the non specific adsorption of molecules in the real sample onto the electrode surface. This effect was not observed at the 200 times of dilution. The slope of the spiked samples with 200 times dilution was further compared with that of standard glucose by two-way ANOVA. No significant difference (P  0.05) was obtained, thus, there was no matrix effect at this dilution. For real sample analysis, human plasma samples were diluted 200 times before being analyzed by the developed sensor. The glucose concentration was calculated from the calibration equation of the glucose standard curve using the obtained

19

responses, and the results of twenty samples are shown in Fig. 5. Using the Wilcoxon SignedRank test, we compared the glucose concentrations obtained from the developed sensor to the results from the standard hexokinase-spectrophotometric method in clinical use: the two sets of results were in good agreement (P  0.05). Fig. 5 here 4. Conclusions A flow based non-enzymatic glucose sensor based on AuNPs-MWCNTs-CS nanocomposite cryogel was developed. The fabrication method of the nanocomposite cryogel electrode is simple. The macroporous structure of chitosan cryogel provided a large surface area for the entrapped composite of AuNPs decorated MWCNTs and facilitated the analyte’s access to the sensing material. The developed sensing tool was used to detect the current obtained from glucose oxidation. Under optimal conditions, the sensor exhibited excellent performances. Moreover, common interfering species naturally present in blood plasma at the physiological level had no effects. No obvious change in response was observed after keeping the electrode for 2 months in distilled water at room temperature, indicating an excellent stability. Moreover, compared to commercial glucometer electrode that is used for a one time measurement, the developed sensor can be used for a large number of samples. Since one electrode can be used several times. It is also an enzymeless and this can help reduce the cost. The good electrocatalytic ability, selectivity, stability and easy preparation make this electrochemical sensor a potential alternative tool for the detection of glucose. It could also be applied to other analytes.

Acknowledgements

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This work was supported by the Thailand Research Fund (TRF) grant no. BRG 5680003. We would also like to thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Trace Analysis and Biosensor Research Center (TAB-RC) at Prince of Songkla University of Thailand. The financial support for Tawatchai Kangkamano from The National Science and Technology Development Agency (NSTDA), Ministry of Science and Technology of Thailand and Thaksin University of Thailand are also gratefully acknowledged. Thanks also to Mr Thomas Duncan Coyn, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand for assistance with the English.

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Biographies Tawatchai Kangkamano obtained his B.Sc. in Chemistry (2004) from Thaksin University, Thailand and his M.Sc. in Analytical Chemistry (2008) from Prince of Songkla University, Thailand. He is a faculty member at the Department of Chemistry, Faculty of Science, Thaksin University and currently doing his Ph.D. in Chemistry in the group of Trace Analysis and Biosensor Research Center, Faculty of Science, Prince of Songkla University, Thailand. His research interest focuses on the development of chemical sensor and biosensor based on nanomaterials modified electrode for trace analysis. Apon Numnuam has a Ph.D. in Chemistry obtained in 2008 at Prince of Songkla University, Thailand. He is an Assistant Professor at the Department of Chemistry, Prince of Songkla University, Thailand. He is also a member of the Trace Analysis and Biosensor Research Center, Prince of Songkla University. His research interests include electrochemistry, biosensors and chemical sensors. Proespichaya Kanatharana has a Ph.D. in Analytical Chemistry from Villanova University, USA. She is an Associate Professor at the Department of Chemistry, Prince of Songkla University, Thailand. She is also the Director of the Trace Analysis and Biosensor Research Center, Prince of Songkla University. Her research interests include trace analysis, chemical

28

sensors, biosensors, synthesis and development of nanomaterials and techniques, analytical methods development, application and techniques for flux measurement (greenhouse gases) from ecosystem. Warakorn Limbut has a Ph.D. in Chemistry obtained in 2007 at Prince of Songkla University. He is an Assistant Professor at the Department of Applied Science, Prince of Songkla University, Thailand. He is also a member of the Trace Analysis and Biosensor Research Center, Prince of Songkla University. His research interests include biosensors, chemical sensors and electroanalytical chemistry. Panote Thavarungkul has a D. Phil. in Physics (Biophysics) from University of Waikato, New Zealand. She is an Associate Professor at the Department of Physics, Prince of Songkla University, Thailand. She is also a member of the Trace Analysis and Biosensor Research Center, Prince of Songkla University. Her research interests include biosensors and chemical sensors for medical, environmental and industrial applications.

29

Figure legends

Fig. 1 The preparation of AuNPs-MWCNTs-CS cryogel modified gold electrode and its use as a working electrode in a flow injection system

30

Fig. 2 (A) TEM image of AuNPs-MWCNTs nanocomposite; SEM images of (B) AuNPsMWCNTs-CS non-cryogel and (C) the top view of AuNPs-MWCNTs-CS cryogel modified layer at x80 magnification showing the porous structure of cryogel and (D) the inside pore view at x20,000 magnification showing the surface of the chitosan cryogel with coated protrusions of the embedded MWCNTs

31

Fig. 3 (A) Cyclic voltammogram obtained from AuNPs-MWCNTs-CS cryogel modified Au electrode in 0.050 M NaOH; (B) The sensitivities of different modified Au electrodes for 0.10-0.50 mM glucose. The potential sweep was performed from -0.4-0.7 V with a 100 mVs-1 scan rate

32

Fig. 4 (A) The relationship between current response and concentration (0.001-20.0 mM) of the fabricated non-enzymatic glucose sensor; (B) an example of the amperometric current response for different concentrations of glucose in flow-injection analysis and (C) the linear regression of the lower range (0.001-1.0 mM)

33

Fig. 5 Comparison of the analytical results using the fabricated sensor and the standard hospital hexokinase method for glucose measurements in human plasma sample

34

Table 1 A comparison of the performance of the fabricated non-enzymatic glucose sensor and other reported sensors (both non-enzymatic and enzymatic sensors). Modification types

Applied potential (V)

LOD (mM)

Linearity (mM) Stability

References

0.0005

0.001-1.0

This work

Non-enzymatic based sensors AuNPs-MWCNTs-CS cryogel/AuE 0.20

90%, 525 inj 98%, 60 d

AuNPs-CS/GCE

0.00

0.37

0.4-10.7

93%, 14 d

[20]

AuNPs/GCE

0.24

0.05

0.1-25.0

NR %, 28 d

[1]

AuNPs-MWCNT-IL/GCE*

-

0.002

0.005-0.12

NR

[43]

AuNPs-Gr-MWCNTs/GCE

0.10

0.0015

0.005-1.0

NR

[53]

AuNPs-MWCNTs-CR/AuE

0.00

0.0005

0.005-37.0

NR

[54]

AuNP/GA/APBA /GCE

OCP

0.025

0.5-50

96.6%, 10 d

[55]

CuNPs/Gr/GCE

0.50

0.0005

up to 4.5

88%, 21 d

[56]

NiNPs/RGO/GCE

0.50

0.0001

0.002-2.1

NR

[6]

CuO NSs/CC

0.55

0.001

0.001-1.0

95.3%, 20 d

[10]

CuNPs-MWCNTs/GCE

0.65

0.00021

0.0007-3.5

80%, 35 d

[52]

0.010

0.010-30

90%, 400 inj

[24]

Enzymatic based sensors GOD/CS-BSA-Fc/MWCNTs/AuE 0.175

98.8%, 30 d GOD/Fc/MWCNTs/GCE

0.35

0.003

0.0012-3.8

91%, 28 d

[57]

GOD/LBL-AuNPs/CS/PtE

0.6

0.007

0.5-16

90%, 30 d

[58]

GOD-Gr-AuNPs-CS/AuE

-0.20

0.18

2.0-10

96.4%, 30 d

[59]

35 GOD/Gr-PEI-AuNPs/AuE

-0.38

0.00032

0.001-0.100

88%, 10 d

[51]

GOD-PoPD/AuNP-ATP-

0.60

0.015

0.05- 8.85

85%, 18 d

[60]

MWCNTs/GCE NPs, nanoparticles; NSs, nanosheets; MWCNTs, multiwalled carbon nanotubes; CS, chitosan, AuE, gold electrode; GCE, glassy carbon electrode; PtE, platinum electrode; CC, carbon clothes electrode; CR, Congo red; Cu, copper; IL, ionic liquid; Gr, graphene; RGO, reduced graphene oxide; GA, glutaraldehyde; APBA, poly (2-aminophenyl boronic acid); CuO, copper oxide; BSA, bovine serum albumin; Fc, ferrocence; GOD, glucose oxidase; PEI, polyethyleneimine; PoPD, poly o-phenylenediamine; ATP, 4aminothiophenol; LBL, layer-by-layer; OCP, open circuit potential; NR, not reported; d, days; inj, injections. *Voltammetric technique was used.