Rapid Determination of Sucrose in Fruit Juices : A New Sensitive Carbon Nanotube Paste Osmium-Polymer Mediated Biosensor

The aim of the present work was the development of a novel amperometric biosensor for rapid detection of sucrose in fruit juices samples. Two enzymes, invertase and fructose dehydrogenase (FDH), were immobilized onto a single-walled carbon nanotube paste (SWCNTP) electrode by wiring with a highly original osmium-polymer hydrogel. A second biosensor, for fructose only, was constructed containing inactive invertase and used for signal subtraction. The biosensor exhibits a detection limit for sucrose of 2 M, linearity up to 5 mM, good sensitivity of 1.98 A cm mM, good reproducibility (RSD = 2.5%), fast response time (8s) and a stability of 4 months if kept under wet conditions at 4 °C. The biosensor was successively tested for specific detection of sucrose and fructose in several commercial fruit juice samples and the results were compared with those obtained with a commercial spectrophotometric enzymatic kit.


Introduction
Sucrose is an important monosaccharide contained in many foodstaffs and sweet drinks.The determination of sucrose is therefore extremely important for food and beverage industries where quality control is essential for both consumer protection and for food industries.Various conventional analytical methods are largely reported in literature for the determination of sucrose, such as gas-chromatography, UV-spectrophotometry, titration and electrophoresis (AOAC, 1995;Beutler, 1984).However, all these methods require expensive instrumentation, long time of analysis, well trained operators and sometimes also complicated sample pretreatments.It is therefore of extreme importance to develop a fast, selective and affordable method usable by food industries.Biosensors can represent a unique alternative: they are low cost devices with very good sensitivity and selectivity which can be easily used "in situ" without long or difficult sample pretreatments (Tran & Cahn, 1993;Eggins, 2002;Wagner & Guilbault, 1994).
We propose a more convenient method which used only two enzymes, invertase and fructose dehydrogenase, immobilized onto a single-walled carbon nanotube paste (SWCNTP) electrode properly modified with a highly original osmium-polymer hydrogel.
Invertase enzyme (E.C. 3.2.1.26)catalyzes the hydrolysis of sucrose to glucose and fructose.The coupling of the two enzymes can be therefore succesfully utilized for the construction of the sucrose biosensor (Clanek, Vydano, & Braunovà, 2000).
The aim of the present work is to develop a novel biosensor for sucrose determination by using a CNTP electrode modified with an osmium redox polymer.The osmium redox polymer showed excellent electron shuttling properties and for this reason it was successfully used for the electrical wiring of cells and enzymes (Timur, Haghighi, Tkac, Pazarlioglu, Telefoncu, & Gorton, 2006a;Timur, Yigzae, & Gorton, 2006b;Antiochia, Vinci & Gorton, 2013) and for biosensor and biofuel cells development (Antiochia & Gorton, 2007;Heller, 1992;Heller & Feldman, 2008).
The osmium redox polymer in the present paper was used for a dual function: redox mediator to shuttle the electrons between the immobilized enzyme and the electrode and support for direct wiring of both invertase and FDH into the paste by utilizing a cross-linking agent, poly(ethylene glycol) diglycidyl ether (PEDGE).
The proposed biosensor was finally tested for sucrose analysis in real fruit juice samples and the results obtained were validated by comparing the results with a standard spectrophotometric kit.

Preparation of the SWCNTP Electrode Modified With Os-polymer
The SWCNTP electrodes were prepared by hand-mixing in a mortar SWCNTs and mineral oil at a 60:40% ratio (w/w) (Valentini et al., 2003) and packed into a cavity (3 mm diameter).A copper wire connected to the paste allowed the electrical contact.
The CNTP electrode was modified with Os-polymer by depositing 10 L of a solution (10 mg/mL) of the Os-polymer and 1 L of an aqueous solution (2.5 mg/mL) of the cross-linker agent PEDGE (Antiochia et al., 2007) onto the CNTP electrode surface.The electrodes were then left to dry overnight at room temperature.

Preparation of the Sucrose Biosensor
Invertase and FDH were directly wired into the Os-polymer hydrogel by mixing 10 L of the Os-polymer solution (10 mg/ml), 1 L of an aqueous solution of PEDGE (2.5 mg/mL), 10 L of a solution containing 5 units of FDH enzyme and 10 L of a solution containing 5 units of invertase enzyme.Then 10 L of this solution was deposited onto the CNTP electrode surface and allowed to dry overnight (Antiochia et al., 2007).Figure 1 shows the wiring of invertase and FDH through the Os-polymer redox hydrogel onto the SWCNTP electrode.

Results
The operat

Sucrose is
The reduce 3).The en (3) on 2).action n. this is valid for sucrose concentration values lower than the Michaelis-Menten constant.The oxygen independent biosensor (Davidson & Jones, 1991) is based on a very simple mechanism due to the fact that FDH enzyme contains FAD as cofactor and does not require the adding of the most common used NAD + required by most other dehydrogenase enzymes.Figure 2 shows the stucture of the osmium mediator.(osmium redox polymer)

Electrochemical Properties of Invertase/FDH/Os-polymer/CNTP Electrode
An electrochemical study of a similar Os-polymer CNTP modified electrode was already reported in our previous work (Antiochia et al., 2007).Figure 3 shows cyclic voltammograms recorded at a scan rate of 10 mV/s in the absence and in the presence (curves a and b, respectively) of invertase and FDH enzymes.All voltammograms clearly showed the typical behavior of surface-bound species with small peak separations thus demonstrating that the presence of invertase and FDH enzymes does not change the electrochemical behavior of the modified electrode.From the voltammetric curve it has been calculated a formal redox potential of the Os-polymer of about +180 mV vs. Ag|AgCl, value which is in perfect agreement with the value previously reported in literature (Timur et al., 2006a;Timur et al., 2006b;Antiochia et al., 2007).Therefore the working potential of the biosensor was choosen at +200 mV for further experiments. .Experimental conditions: applied potential: 0.2 V (vs.Ag/AgCl); Os-polymer: 10 L of a 10 mg/mL solution; PEDGE: 1 L of a 2.5 mg/mL solution; invertase: 5U; FDH: 5U; =10 mV/s; 0.1 M phosphate buffer pH 7.0; electrode diameter: 3 mm

Sucrose Biosensor
Different amounts of the two enzymes were tested in order to optimize the sucrose-Os-polymer CNTP biosensor.
Figure 4 shows the sucrose calibration graphs derived from the amperometric study by using an Os-polymer CNTP electrode with invertase and FDH directly wired to the Os-polymer hydrogel.The effect of enzyme amounts on the biosensor current response was studied in the range 2-10 U for both enzymes.The three curves showed the same trend: a linear part at the beginning and a deviation from linearity when higher sucrose concentrations are employed.This behaviour can be ascribed to a possible saturation of the enzyme active site.The current values gradually increase at increasing invertase and FDH amount loaded for relatively low amount used (from 2 to 5 U, as reported in curves a and b, Figure 4) but slightly decrease at higher enzyme amounts employed (more than 5 U, curve c, Figure 4).This result can be explained by taking into account the fact that the enzyme amount is a rate limiting factor at quite low enzyme amounts employed but at higher enzyme values used other factors started to limit the rate of the reaction.An amount of 5 U of invertase and 5 U of FDH showed the higher current response (curve b, Figure 4) and therefore 5 U of FDH and 5 U of inveratse were used to prepare the biosensor in further experiments.The low amount of both enzymes employed and the relatively high current response can be understood by the fact that the enzymes are now wired through the Os-polymer onto the surface of the CNTP electrode.The enzyme is immobilized into the hydrogel without large structural changes and without any significative loss of activity and at the same time, due to the high surface area of SWCNTs, a major exposure of the enzyme's catalytic sites is possible and also a large loading of both enzymes with a consequent enhancement of the amperometric signal.
The linear part of the sucrose calibration curve registered by the optimized biosensor is shown in the inset of Figure 4.A a good linear range is shown from 0.1 to 5 mM with the following equation y = 0.14x + 1.03x10 -2 (r=0.9969,n=6), where y indicates the current (A) and x the sucrose concentration (mM).At higher sucrose concentrations the curve approaches a saturation value and starts to become nonlinear.The biosensor showed a good sensitivity of 1.98 µA cm -2 mM and a detection limit of about 2 µM, calculated with the following expression: 3S.D.a/b, with S.D.a the absolute standard deviation of the intercept and b the slope of the calibration curve.
The response time of the biosensor was quite short, resulting of about 8 s.

Dependance of Biosensor Response on pH and Temperature
In order to study the effect of different solution pHs on the sucrose biosensor response two different buffers were used and the pH ranges between 4 and 5.5 and between 6 and 7.5 in 0.1 M acetate buffer and 0.1 M phosphate buffer, respectively, were investigated.The biosensor maximum current response is registered in acetate buffer at pH 5.0, as clearly shown in Figure 5, and for this reason this pH was choosen for further experiments.At pHs higher than 6 a progressive decrease of the current response is observed until a loss of response of about 80% is registered when a pH value higher than 7 is used.This result is comparable with that reported in literature for FDH modified electrodes (Trivedi et al., 2009;Antiochia et al., 2004b) and for the optimum pH of the free enzymes, which resulted to be of 4.5 for both invertase and FDH (Ameyama, Shinagawa, Matsushita, & Adachi, 1981;Schiweck, Clarke, & Pollach, 2007).
Figure 5. Dependance on the maximum biosensor response at a function of different pHs.
[sucrose] = 5 × 10 -4 M; buffers employed: 0.1 M acetate buffer, 4<pH<5.5;0.1 M phosphate buffer, 6<pH<7.5.RSD % values are between 3.0 and 4.0% (n=5).Other conditions as in Figure 4 We also studied the effect of the temperature of the buffer solution on the biosensor current response.The study was carried out in the range 10-40 °C and the results reported in Figure 6.It is easy to observe that there is an initial increase of the biosensor relative response with increasing temperatures up to a maximum value at about 30 °C and successively a marked decrease.At higher temperautures there is in fact a possible thermal inactivation of FDH, although the optimum temperature reported in literauture for free invertase enzyme is 60 °C (Schiweck, Clarke, & Pollach, 2007).The temperature choosen for further experiments was 25 °C, at which only a 5% decrease of the maximum current response was obtained, for simplicity of operation being closer to room temperature.

.4 Reproducibility of the Sucrose Biosensor
The reproducibility of the sucrose biosensor was studied by using 5 different sensors.It resulted to be very good, with an RSD of 2.5% for a 0.5 mM sucrose solution for n=5, where n is the number of biosensors utilized in the test.

Lifetime of the Sucrose Bisensor
The storage stability and lifetime of the sucrose biosensor were also investigated.The current responses were consecutively measured for a 1 mM sucrose solution every day for a period of 10 days.The sucrose biosensor was carefully stored at 4 °C when not in use under dry and wet storage conditions and the results are shown in Figure 7.The biosensor retains about 80% of its initial activity after 1 day of 8 h/day operation and only 15% after 10 days is kept under dry storage conditions.Otherwise under wet storage conditions, the biosensor keeps almost 100% of its original response after 3 days of 8 h/day operation and almost 85% after 10 days.Under wet conditions the study was extended up to 4 months and the biosensor resulted to be able to retain 80% of original response even up to such a long period.These excellent stability results obtained for the proposed biosensor are superior to most of the results reported in literature for amperometric biosensors for sucrose (Damar et al., 2011).A possible explanation of this extraordinary result is the fact that under wet conditions the biosensor is kept with cotton wet with buffer solution in a closed bottle thus allowing the maintenance of a higher water content in the immobilized hydrogel layer.
Figure 7. Lifetime of the sucrose biosensor stored at 4 °C under dry conditions (curve a) and wet conditions (curve b).RSD% values between 2.8 and 3.5% (n=5).Other conditions as in Figure 4 3

.6 Selectivity of the Biosensor
Interferences such as ascorbic acid, ethanol and other mono-and disaccharide compounds present in food such as glucose, lactose, galactose and maltose were investigated by adding equal amounts of the specific interferent and sucrose.As shown in Table 1, no significant influence was noted for each interferent specie studied.Only for ascorbic acid was reported a slight interference of about 7%.
As for fructose, the sucrose biosensor is based on FDH and therefore reacts also towards fructose present in solution (Table 1).For this reason, all measurements of sucrose in real samples must be coupled with a measurement of the fructose concentration in the same sample by using the same sensor with inactive invertase and subtracting this current response from the total response of the sucrose biosensor.

Application on Food Analysis
The developed invertase-FDH sucrose biosensor was utilized for the analysis of foodstuffs like commercial fruit juices.The sucrose content was calculated by using the calibration graph method.Each sample was analysed in five replicates.From a comparison of the results obtained with the proposed biosensor and those obtained using a commercial spectrophotometric enzymatic assay kit, it was possible to observe that there is a very good agreement between the results obtained with the two methods (Table 2).More precisly, the results obtained with the reference spectrophotometric kit show slightly higher values.It may be interesting to note that this can be explained by the fact that the kit is based on multienzymatic cascade reactions which can cause interferences with other compounds, in particular with other sugars.

Conclusion
In this work the osmium redox polymer was successfully used for the modification of a CNTP electrode.The osmium redox polymer resulted to be able to shuttle the electrons between the immobilized enzyme and the single-walled CNTP electrode and was used also as a support for direct wiring of both invertase and FDH enzymes into the paste by using PEDGE as cross-linking agent.The modified electrode was used to develop a sucrose biosensor based on the immobilization of invertase and FDH.The biosensor assembly is simple, quick and not expensive.The sucrose biosensor showed the following analytical parameters: a good linear range, low detection limit (2 M), good sensitivity, high reproducibility, good stability and fast response time.Moreover, its response was not influenced by possible interfering species which can be present in food samples.
For these reasons this novel, cheap and "in situ" usable sucrose biosensor will form the basis for the development of an ideal commercial device for applications in food quality and safety control.

Table 2 .
Results for sucrose determination in real samples using the invertase-FDH biosensor and the reference enzymatic kit All measurements were repeated six times and reported as average  RSD (n=6). a