Food Anal. Methods DOI 10.1007/s12161-013-9705-6
Optimisation of Glucose Biosensors Based on Sol–Gel Entrapment and Prussian Blue-Modified Screen-Printed Electrodes for Real Food Analysis Donatella Albanese & Adriana Sannini & Francesca Malvano & Roberto Pilloton & Marisa Di Matteo
Received: 22 April 2013 / Accepted: 20 August 2013 # Springer Science+Business Media New York 2013
Abstract In this study, we report the construction of amperometric screen-printed glucose biosensors for food analysis by using two procedures for Prussian Blue (PB) deposition and different membranes for enzymatic immobilisation. The comparison between the screen-printed electrodes modified with PB by electrochemical and chemical deposition showed higher analytical performance (detection limit of 1 μM, linear range from 0.5 to 500 μM and a sensitivity of 823 μA mM−1 cm−2) when the latter was employed. Then, the immobilisation of glucose oxidase (GOD) by silica sol–gel and polyvinyl alcohol (PVA) hydrogel was performed on electrochemically modified PB electrodes. The electrochemical response of two glucose biosensors was evaluated by flow injection analysis. Biosensors constructed by silica sol–gel entrapment showed a wider linear range (0.005–1 mM) and a detection limit (0.02 mM) that was 10-fold lower than using entrapped GOD in PVA. The selected glucose biosensor showed negligible interference from ascorbic acid when the Nafion membrane was used to cover the PB-modified electrode surface. Additionally, it exhibited an operating lifetime of 8 h under continuous glucose injections ranging from 0.01 to 2 mM. Finally, the biosensor was applied for specific determination of glucose in red and white wines, juices and dried fruit.
Keywords Glucose biosensor . Prussian Blue . Screen-printed electrodes . Food analysis D. Albanese (*) : A. Sannini : F. Malvano : M. Di Matteo Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, SA, Italy e-mail:
[email protected] R. Pilloton National Research Council, Institute of Photonic and Nanotechnology (CNR-IFN), Via Cineto Romano 42, 00156 Rome, Italy
Introduction The use of quick, reliable and cheap analytical devices for the detection of chemical compounds for quality control and realtime monitoring of food processing represents an important need in the food sector. Among the analytes that need to be monitored and measured in order to guarantee the quality of final food products and to verify that industrial processes are under control, a prominent role is assumed by glucose. The traditional methods for glucose determination, such as HPLC, spectrophotometry and titration, are often time-consuming, expensive and require expensive laboratory equipment and qualified personnel. The use of amperometric glucose biosensors represents a powerful alternative for the monitoring of glucose because of their specificity, high sensitivity, short response time, simple operation and rapidity of use. Biosensors are based on the intimate contact between a biorecognition element (nucleic acid, enzyme, antibody, receptor or tissue cell) and a transducer. Most glucose biosensors are based on electrochemical (amperometric) transducers and the immobilisation of the glucose oxidase (GOD) enzyme on the electrode surface. In this configuration, GOD catalyses the conversion of glucose and molecular oxygen (O2 dissolved in solution) to gluconic acid and hydrogen peroxide, which can be oxidised at the electrode surface when a specific potential is applied between the working and reference electrodes. The hydrogen peroxide decomposition causes the flow of electrons, which can be measured by means of an amperometric transducer according to the following reactions: glucose oxidase
D glucose þ O2 → D glucose acid þ H2 O2
anodic oxidation
H2 O2 → 2Hþ þ O2 þ 2e− E ¼ −680 mV:
Food Anal. Methods
One of the most important problems related to the use of glucose amperometric biosensors is due to the presence of some biologically active substances, such as polyphenols, flavonoids, carotenoids and vitamins, in real food matrices (i.e. grape must, fruit juices, etc.). These compounds can be oxidised on the surface of electrodes at the same potential as hydrogen peroxide and reduce the selectivity of the biosensors. To prevent interference problems, many reports have proposed the modification of electrodes by electrochemical mediators for the development of GOD amperometric biosensors (Karyakin 2001; Koncki 2002; de Tacconi et al. 2003; Chaubey and Malhorta 2002; Zen et al. 2003; Newman et al. 1995). In this field, the redox mediators Prussian Blue (PB) and ferric hexacyanoferrate are widely used in enzyme oxidase biosensor applications, allowing the mediated reduction of hydrogen peroxide at low applied potentials (Ricci and Palleschi 2005), according to the following reactions: h i h i Fe4 ðIII Þ FeðII Þ ðCN Þ6 þ 4e− þ 4Kþ →K4 Fe4 ðII Þ FeðII Þ ðCN Þ6 3
3
ð1Þ h i h i Fe4 ðII Þ FeðII Þ ðCN Þ6 þ 2H2 O2 →Fe4 ðIII Þ FeðII Þ ðCN Þ6 þ 40H− þ 4Kþ : 3
3
ð2Þ There are many reports on PB-modified glucose biosensors (De Mattos et al. 2003; Liang et al. 2008; Karyakin et al. 2002; Derwinska et al. 2003; Li et al. 2004), and most of which are constructed by glassy carbon or noble metal electrodes, such as Pt and Au. During the past several years, screen-printed carbon electrodes (SPCEs) have assumed a very important role in disposable biosensor construction. In fact, screen printing technology allows the production of electrodes at low costs with high reproducibility, versatility and miniaturisation, providing compact and portable analysis devices, while graphite materials offer a cheaper and simpler technological processing alternative (Dominguez Renedo et al. 2007). Some studies on the development of glucose biosensors by PBmodified SPCEs have been reported in the literature (De Mattos et al. 2003; Ricci et al. 2003; O'Halloran et al. 2001). The critical points in manufacturing PB screen-printed biosensors are the effectiveness of the redox mediator deposition on the electrodes surface (Ricci and Palleschi 2005) and the immobilisation procedure used for the bioactive membranes (Ricci et al. 2003; Tudorache and Bala 2007). These two factors influence the storage lifetime and operational stability of the biosensors and excluding them from the realistic employment in food process monitoring. In this study, the electrochemical and chemical deposition of PB on SPCEs was evaluated in order to obtain a suitable and stable redox mediator based biosensor. Moreover, different materials, such as silica sol–gel and polyvinyl alcohol (PVA) gel entrapping GOD on a PB-modified working electrode surface, were
investigated. Sol–gel immobilisation of biomolecules has attracted considerable interest because it provides an interesting method to prepare a three-dimensional network suitable for encapsulation of a variety of biomolecules and microbial cells (Habib et al. 2012). Moreover, sol–gel materials can be prepared under mild conditions and exhibit tunable porosity, high thermal stability, chemical inertness and negligible swelling in aqueous and non-aqueous solutions (Wang et al. 1998). PVA is a synthetic polymer with unusual properties through extremely mild freezing and thawing of its aqueous solution (Lozinsky 1998). During the freeze-thaw process, crosslinking among PVA chains by semi-permanent entanglement, molecular association or crystallisation can occur (Lee and Mooney 2001; Lozinsky and Plieva 1998). This threedimensional structure is an appropriate matrix for immobilisation of biomolecules for biosensor preparation (Tsai et al. 2007). Moreover, the large number of hydroxyl groups in PVA provides a biocompatible microenvironment for the enzymes (Kumar and D'Souza 2008; Imai et al. 1986). The aim of this work was to develop a suitable, stable, and low-cost amperometric glucose biosensor based on PB-modified SPCEs for food analysis and real-time monitoring of food processing by flow injection analysis. Two different PB deposition procedures and enzymatic entrapments, based on the use of silica sol–gel and PVA (hydrogel) cross-linking, were evaluated. The biosensors developed were characterised in terms of sensitivity, operational stability and reproducibility. Additionally, the influences of interfering substances were analysed. The potential use of the devices was demonstrated by comparing the biosensor with the HPLC method for the determination of glucose in wine and fruit juice samples.
Materials and Methods Reagents Glucose oxidase from Aspergillus niger X-S (100–250 U/mg solid), tetraethyl orthosilicate (TEOS), ferric chloride (FeCl3), potassium ferricyanide (K3Fe(CN)6), hydrolysed polyvinyl alcohol (Mw 13,000–23,000), Nafion®, potassium chloride (KCl), sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic monohydrate (NaH 2 PO 4 -H 2 O) and hydrochloric acid (HCl) were purchased from Sigma-Aldrich. Screen-Printed Carbon Electrode (SPCE) Construction Sensors based on a three-electrode (working/auxiliary/reference) layout were produced in three steps, as described in Albanese et al. (2011), by screen printing of different consecutive ink layers on transparent polyester films. Specifically, a first layer of a carbon/graphite ink (G-Went, UK) was deposed to define the conducting track and the working electrode. The
Food Anal. Methods
second one was a silver/silver chloride ink (Acheson Colloiden B.V., NL), used as a pseudo-reference electrode. The third layer consisted in an insulating ink (Acheson Colloiden B.V., NL), which was UV polymerisable. Between the first and the second screen printing step, the strips were cured at 80 °C for 25 min. The diameter of the working electrode was 2.8 mm. PB Deposition The deposition of PB was conducted on the screen-printed electrode surface after an electrochemical treatment (3 min) at 1.7 V versus Ag/AgCl in a 0.05 M buffer phosphate solution (0.1 M KCl, pH 6.8). Chemical deposition was carried out by dropping 5 μL of a 1:1 ratio solution of 0.1 M K3Fe(CN)6 in 10 mM HCl and 0.1 M FeCl3 in 10 mM HCl (Karyakin et al. 2002) onto the working electrode area. After 10 min, the PB excess was washed with a 10 mM HCl solution and placed for 1 h in a heater at 100 °C. Electrodeposition was performed by the cyclic voltammetry (CV) method (Guan et al. 2004). The SPCEs were dipped in 2 mM K3Fe(CN)6, 2 mM FeCl3, 0.1 M HCl and 0.1 M KCl, and then, a potential scan between 0 and 0.5 V at a scan rate of 50 mV/s was cycled 20 times. After deposition, the PB excess was rinsed with a solution of 10 mM HCl. The modified electrode was activated in 0.1 M KCl and 0.1 M HCl by 20 cycles between −0.1 and 0.4 V at a scan rate of 50 mV/s. It was then placed in a heater at 100 °C for 1 h. GOD Immobilisation Procedures GOD was immobilised on PB-modified SPCEs through PVA cross-linking or silica sol–gel entrapment. PVA Cross-linking First, 5 % (w /v) PVA in buffer solution (0.05 M phosphate and 0.1 M KCl, pH 6.8) was heated for 1.5 h at 85 °C with stirring periodically. Then, the solution was cooled at room temperature and frozen at −18 °C for at least 3 h. After thawing at 6 °C for 5 h, 18 μL of the PVA solution was mixed with 5 μL of GOD buffer solution (0.2 mg/μL). Then, 3 μL of PVA/GOD solution was dropped on the PB-modified working electrode surface and was stored overnight in a wet environment at 4 °C. Silica Sol–Gel Entrapment Silica sol–gel was prepared according to Zuo et al. (2008) with some modifications. The silica hybrid gel was prepared by mixing 1 mL TEOS and 0.5 mL ethanol in 10 mM HCl (15 % v/v). The mixture was sonicated for 1.5 h until a homogenous solution was obtained. Then, 20 μL of PVA (5 % (w /v)) was
added to 90 μL of the above TEOS sol. Then, 18 μL of TEOS/ PVA solution was mixed with 5 μL of GOD buffer solution (0.2 mg/μL), and 3 μL was dropped on the PB-modified working electrode surface. The formation of silica sol–gel membrane occurred at room temperature for the first 10 min and then overnight in a wet environment at 4 °C.
Measurements All the electrochemical experiments were carried out using the PalmSens potentiostat/galvanostat. The amperometric measurements were performed in a flow injection analysis (FIA) apparatus as described in Albanese et al. (2010). The biosensor was placed in an electrochemical flow cell, while a constant potential of 0.0 V versus Ag/AgCl was applied. Carrier solution (0.05 M phosphate buffer and 0.1 M KCl, pH 6.8) from a reservoir was pumped with a peristaltic pump (Miniplus 3, Gilson, France) at flow rate of 0.5 mL/min to the injection valve (sample injection valve; Omnifit) equipped with a 100 μL sample loop. Analysis of Food Samples Glucose biosensors were used for the determination of glucose in food samples. In order to verify the accuracy of the response, different kinds of food samples, such as red and white wine, fruit juices and dried fruits, were tested. We chose these samples on the basis of different glucose/interferent ratios. Dried grape (10 g) was weighed using an analytical balance (Mod. E50S/3 Gibertini, IT) and rehydratated in 40 mL phosphate buffer (pH 6.8) for about 30 min. Then, the solution was homogenised by Ultra-Turrax (T 25 Basic IKA, IT) for 3 min and then filled to a volume (100 mL) with buffer solution. Finally, the dispersion was stirred for 20 min at 30 °C and filtrated through a filter paper. Liquid food samples were properly diluted in phosphate buffer to a minimum of 20 times for white wine to a maximum of 250 times for pineapple juice. The dilution ratio applied for each sample analysed was calculated taking into consideration both the glucose levels in different food samples and the linear range of the biosensors. The results obtained using the glucose biosensors were compared with those obtained by HPLC (Hewlett Packard, Agilent). The HPLC system was equipped with a 4.6 mm× 250 mm steel carbohydrate cartridge column (Waters, USA). The mobile phase was acetonitrile/water solution (75:25), with the column at 30 °C and using a 1 mL/min flow rate. Peaks were detected by a refractive index detector (Hewlett Packard, Agilent), and concentrations were calculated with external standards.
Food Anal. Methods
Results and Discussion Investigation of PB Deposition on SPCEs A major drawback of the use of glucose oxidase amperometric biosensors is the high overpotential needed for hydrogen peroxide oxidation (approximately 0.7 V versus Ag/AgCl). At this potential, many electroactive substances (i.e. ascorbic acid, polyphenols, etc.), which are usually present in real samples, could also be oxidised and thus produce interfering signals. As already stated (Eqs. 1 and 2), PB is an electrochemical mediator that catalyses the reduction of hydrogen peroxide. These redox reactions at low potentials happen when a stable PB layer is present on the surface of the sensor. Figure 1 shows the voltammograms of the PB-modified SPCE obtained by electrochemical and chemical procedures. Both PB depositions showed the typical two pairs of redox peaks (cathodic and anodic peaks at 0.0 and 0.1 V, respectively), with a well-defined redox behaviour attributed to the reversible redox interconversion of PB into Prussian White. The latter is the reduced form that has a catalytic activity towards hydrogen peroxide reduction (Itaya et al. 1984). Chemical deposition of PB on SCPEs has been reported in the literature (Ricci et al. 2003; Rejeb et al. 2007), whereas the electrochemical deposition was used to modify carbon glass electrodes (Haghighi et al. 2004) or Pt/Au screen-printed electrodes (De Mattos et al. 2003). Because the performances of PB-modified electrodes are dependent on the method used for their preparation, we carried out a comparison between the two deposition procedures. The electrochemical characterisations of the two PB-modified SPCEs were evaluated by chrono-amperometry at 0.0 V versus Ag/AgCl injecting H2O2 at several concentrations into a flow injection apparatus. Even if the chemical procedure represents a fast method for PB deposition, the electrochemical performance parameters, such as sensitivity, limit of detection
(LOD) and linear range, recorded for these modified electrodes were lower than those obtained for electrochemical deposition (Table 1). Moreover, an acceptable reproducibility for both types of PB electrodes was obtained with a relative standard deviation percent (RSD%) (at 50 μM H2O2) of 5.22 and 2.97 for chemical and electrochemical PB deposition, respectively. Because electrochemical deposition of PB yielded the highest electrochemical performances, these modified electrodes were used for further immobilisation of the GOD in order to prepare a glucose biosensor with enhanced sensitivity. Characterisation of Analytic Performance of PB-Modified Screen-Printed Carbon GOD Biosensors The purpose of any immobilisation method is to retain a maximum activity of the biological component on the surface of the transducer. Moreover, other benefits that can be obtained from an efficient method of immobilisation are the prolonged use of the biosensor, working stability and the capability to be extended to an industrial scale. In order to obtain high performances for PB-modified screen-printed GOD biosensors, different immobilisation techniques by PVA cross-linking and sol–gel entrapment were investigated. Although numerous enzyme entrapment methods have been described for biosensor development (Mello and Kubota 2002), optimising an efficient protocol able to reach the proper analytic characteristics is a very important goal in the food industry. Results reflecting the main analytic performances related to the calibration of PVA and TEOS–PVA GOD biosensors are reported in Table 2. The immobilisation by a mix of TEOS and PVA was able to reach higher analytical parameters in terms of sensitivity, linear range and LOD. It is worth noting that the LOD for TEOS–PVA GOD biosensors was 10 times lower than GOD entrapped by PVA. The higher performances reached by TEOS–PVA GOD biosensors can be attributed to the 3D porous structure of the silica sol–gel matrix, which provides a biocompatible microenvironment able to maintain the activity of the enzyme and facilitate a Table 1 Analytical characteristics of PB-modified SPCE by chemical and electrochemical deposition
Linear range (μM) LOD (μM)a RSD%b (n =5) Sensitivity (μA mM−1 cm−2) R2 a
Fig. 1 Cyclic voltammograms of PB–SPCEs produced by a chemical and b electrochemical deposition
b
Chemical deposition
Electrochemical deposition
5–500 5 5.22 168.53 0.99
0.5–500 1 2.97 823.08 0.99
Defined as the glucose concentration that yields a signal-to-noise ratio of 3
Defined as the percent ratio between standard deviation and average [(SD/average)×100]
Food Anal. Methods Table 2 Analytical characteristics of GOD biosensors developed by PVA and TEOS–PVA on PB–SPCEs PVA
TEOS–PVA
Linear range (mM) LOD (mM)a Sensitivity (μA mM−1 cm−2) RSD%b (n =5)
0.01–1 0.2±0.14 1.6±0.21 4.44
0.005–1 0.02±0.017 2.06±0.35 5.09
R2
0.99
0.99
a
Defined as the glucose concentration that yields a signal-to-noise ratio of 3
b
Defined as the percent ratio between standard deviation and average [(SD/average)×100]
rapid mass transport of analytes and products (Li et al. 2007). Moreover, silica sol–gel showed negligible swelling in aqueous solution, increasing the stability of the immobilisation over time when the biosensor is in use (Zuo et al. 2008). The main disadvantage of enzyme immobilisation on the electrode surface through silica sol–gel matrix by manual dropping is the cracking phenomena during sol–gel transition, observed in our previous trials, that reduces the reproducibility of biosensors (data not shown). This drawback was solved by the addition of a small amount of PVA (0.9 %) to the TEOS solution, which reduced the rigidity of the TEOS sol–gel and prevented the cracking phenomena during the preparation of sol–gel silica membrane. A comparison of some analytical characteristics of previously reported GOD biosensors on PBmodified carbon screen-printed electrodes is reported in Table 3. Evaluation of the Selectivity (Interferences) in the Analysis of Real Food Samples Selectivity is an important feature that has to be evaluated when a glucose biosensor is used for food analysis. Food samples (i.e. grape must, wine and fruit juice) contain different
electrochemical interfering species, in particular, ascorbic acid and other sugars that can modify the current response due to glucose concentration. In order to evaluate the selectivity of the TEOS–PVA GOD biosensors and considering that in food samples, the ratio between glucose and interfering molecules changes depending of the type of foodstuff, different solutions containing glucose and ascorbic acid in several molar ratios ranging from 200 to 20 were evaluated. The range of molar ratios used in this study was chosen taking into consideration the amount of glucose and ascorbic acid existing in fruit juice (molar ratio of about 200) and in white wine (molar ratio of about 20). A large interference due to ascorbic acid was observed, with a decrease of up to 50 % of the glucose signal, when a solution containing an analyte/interferent molar ratio equal to 20 was injected. These results are in contrast to previous PB/ GOD biosensors reported by Ricci et al. (2003), O'Halloran et al. (2001) and Liang et al. (2008), which recorded irrelevant interferences due to ascorbic acid. However, our results were in accordance with the electrocatalytic activity of PB film and ferrocene derivative mediators in the oxidation of ascorbic acid described by Castro et al. (2001) and Pournaghi-Azar and Ojani (1995). As a matter of fact, our experiments showed an electrooxidation current when ascorbic acid (0.1 mM) was injected into the electrochemical cell with minimum (60 nA) and maximum (300 nA) peaks registered at −0.05 and 0.2 V, respectively (plot not shown). In order to lower the electroactivity of PB towards ascorbic acid, two aliquots (3 μL) of Nafion solution (1 %) were dropped on the PBmodified working electrode. Nafion is a negatively charged polyelectrolyte matrix that produces a repulsive force against negatively charged molecules. Its use as a thin membrane over the electrode surface can reduce the interference caused by electroactive (i.e. ascorbate ion) anions present in biological media (Pan and Arnold 1996). As shown in Fig. 2, the deposition of a Nafion coating remarkably reduced the oxidation current of ascorbic acid without influencing the electroactivity of PB toward hydrogen peroxide. On the basis of these results,
Table 3 Comparison of analytical characteristics of various GOD/PB-modified SPCEs reported in the literature Schematic sensor assembly
Sensitivity (μA mM−1 cm−2)
Linear range (μM)
LOD (μM)
Operational stability
Measurement assembly
References
PB/GOD/NAFION
3.21
Up to 3000
220
Batch
PB/GOD/GA/Nafion PB/GOD/PEI/GA PB/GOD/GA/Nafion
63 No data 54
2–100 Up to 5000 Up to 1000
1 1 20
PB/GOD/TEOS– PVA/NAFION
2.06
5–1000
20
The sensor lost 50 % of its stability after 4 h of continuous use No data No data 20 % decrease after 12 h of continuous glucose injection (0.5 mM) No decrease after 8 h of 55 injection of glucose (0.5 mM)
O'Halloran et al. (2001) Ricci et al. (2003) Lupu et al. (2004) Ricci and Palleschi (2005) This work
GA glutaraldehyde, PEI polyethylenimine
Batch Batch Micro-FIA FIA
Food Anal. Methods Table 5 Results for glucose determination in real samples by means of TEOS/PVA/PB GOD biosensors and reference method HPLC Sample
Biosensor (g/L)
HPLC method (g/L)
Red wine (Tavernello) White wine Pineapple juice Pear juice Dry grape (or raisins) (mg/g d.w.)
3.08±0.23 3.33±0.06 49.8±1.27 9.4±0.3 251.8+0.6
0.24±0.08 2.65±0.3 47±1.2 7.6±0.5 262.21±0.79
All measurements were repeated three times and reported as average ± standard deviation
Fig. 2 Amperometric response of H2O2 (0.05 mM) and ascorbic acid (AA; 0.05 mM) recorded with and without Nafion layers on PB-modified electrodes at 0.05 V
the selectivity of GOD/sol–gel biosensors produced by PB electrodes coated with two Nafion layers was evaluated and reported in Table 4. Because food samples exhibit a molar ratio of glucose to ascorbic acid ranging from 20 in wine to 200 in fruit juices, the results obtained highlight the capability of our biosensor for analysis of real samples. In order to evaluate biosensor reliability during the determination of glucose in real food samples, Table 5 shows the comparison between biosensors and HPLC data. A high correlation of the two sets of data was observed, while a greater difference was observed for red wine, probably due to the high concentration of polyphenols and/or anthocyanins combined with the low glucose concentration in this kind of matrix. Repeatability, Reproducibility, and Operational Stability of PB-Modified Screen-Printed Carbon GOD Biosensors
Operational stability is another important parameter that affects the potential of biosensors as analytical devices for routine analysis of real samples and monitoring of food process transformation. Operational stability is related to the retention of activity of an enzyme when in use. This is often the most quoted parameter in biosensor publications, and it relates to the operating lifetime and reusability of a device (Gibson 1999). To assess the applicability of glucose biosensors for the routine analysis of real samples and during the monitoring of food processes, the operational stability of PB-modified GOD biosensors was analysed by injecting 55 glucose samples ranging from 0.01 to 2 mM during an interval of 8 h. Through the comparison of signals corresponding to glucose samples at 100 μM, injected in FIA system in the beginning and at the end of the test period, no significant response decrease was observed. In order to evaluate the effect of a food matrix on the stability of the biosensor current responses, three consecutive glucose injections (0.1 mM) were measured and compared to those registered after the injection of food
The repeatability of the current response of GOD–SPCE (PB/ GOD/TEOS–PVA/NAFION) was evaluated using a 0.1 mM glucose solution with a RSD of 4.19 % for seven successive injections. The reproducibility of five different GOD–SPCEs had an RSD of 8.3 % for 0.1 mM glucose. Table 4 Effect of AA at different molar ratios on the response of PB– Nafion–GOD Glucose/ascorbic acid molar ratio
200 150 100 50 20
Decrease (%) of peak current Without Nafion
With Nafion
2.25 21.60 41.00 53.60 No data
0.00 0.00 0.00 0.00 3.20
Fig. 3 Influence of different food matrices on the current response of glucose (0.1 mM) measured by glucose oxidase biosensors (PB/GOD/ TEOS–PVA/NAFION). The data correspond to mean value of three consecutive injections for glucose and diluted real samples
Food Anal. Methods
samples (white wine, pineapple juice and dried grape). The results (Fig. 3) showed no significant differences between the first and the final current corresponding to 0.1 mM of glucose. After use, the biosensors were stored in phosphate buffer at 4 °C and characterised by calibration curves every day for 7 days. Under these conditions, the lifetime (t L50), defined as the storage time necessary for the sensitivity within the linear range to decrease by a factor of 50 %, was 4 days. The possible cause of the biosensor activity loss could be the inactivation/release of immobilised enzymes and/or the disintegration of the PB layer due to the high concentration of hydroxyl ions produced (Karyakin et al. 1999). Our previous study on the development of screen-printed TEOS/PVA GOD biosensors showed a t L50 longer than 3 months. Moreover, a rising decrease of signal corresponding to H2O2 (0.05 M) injected during the storage periods into PB/TEOS/PVA GOD biosensors was observed. These results are in agreement with the other studies (Itaya et al. 1984) that reported that the instability of biosensors based on PB depends on the presence of OH− generated by the redox reaction between Prussian White and H2O2 (see Eq. 2) that induces the cleavage of PB complexes due to the formation of ferric hydroxide.
Conclusion In this work, a study on the optimisation of amperometric screen-printed glucose biosensors to be applied to real food samples was reported using two procedures for PB deposition and different enzymatic immobilisation methods. Electrochemical deposition of PB on SPCEs showed the highest analytical performance towards hydrogen peroxide. The immobilisation of GOD by silica sol–gel onto a PBmodified electrode surface yielded a biosensor characterised by a lower detection limit, higher sensitivity and extended linear range in comparison to PVA entrapment. The wide linear range, low detection limit, high sensitivity, operational stability and low interference measurements obtained with the Nafion barrier for negatively charged interfering molecules (i.e. ascorbic acid) highlight the potential of the proposed biosensor as a highly capable analytical device for glucose monitoring in food processes. Moreover, the reduced interferences obtained with PB coupled with the Nafion barrier will be useful for new oxidase-based biosensors to be used in real food samples.
Conflict of Interest Donatella Albanese declares that she has no conflict of interest. Adriana Sannini declares that she has no conflict of interest. Francesca Malvano declares that she has no conflict of interest.
Roberto Pilloton declares that he has no conflict of interest. Marisa Di Matteo declares that she has no conflict of interest.
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This article does not contain any studies with human or animal subjects.
