Biosensor based on diamine oxidase/platinum nanoparticles/graphene/chitosan modified screen-printed carbon electrode for detection of histamine in fish
Abstract
This work describes the developing and optimisation studies of a novel amperometric biosensor for the detection and quantification of histamine in fish samples. The proposed biosensor is based on a carbon screen-printed electrode, modified with graphene and platinum nanoparticles, which detects the hydrogen peroxide produced by the reaction catalysed by the enzyme diamine oxidase immobilised onto the surface of the receptor element. The amperometric measurements have been accomplished in phosphate buffer solution at pH 7.4 applying an optimal low potential of 0.4 V. Novel biosensor exhibits high sensitivity (0.0631 A×M), low limit of detection (2.54×10-8 M) and a wide linear interval range from 0.1 to 300 M.
The analytical performance of this biosensor has been assessed for the detection of histamine in fish samples. An excellent correlation between results obtained with the developed biosensor and those obtained with the standard method (ELISA) for all fish samples has been achieved.
1. Introduction
Biogenic amines are organic compounds with low-molecular weight and basic character, with different chemical structures, formed mainly by the decarboxylation of amino acids (Suzzi and Torriani, 2015).
BAs are usually considered indicators of microbial decomposition in fish or shellfish (Al Bulushi, Poole, Deeth and Dykes, 2009; Apetrei and Apetrei, 2015a) even they are also present in different foods (Apetrei and Apetrei, 2013; Shalaby, 1996; Spano, Russo, Lonvaud-Funel, Lucas, Alexandre, Grandvalet, Coton, Coton, Barnavon, Bach, Rattray, Bunte, Magni, Ladero, Alvarez, Fernández, Lopez, de Palencia, Corbi, Trip, and Lolkema, 2010) and beverage (Preti, Antonelli, Bernacchia and Vinci, 2015; De Borba and Rohrer, 2007). BAs could be found in meat, fermented foods, cheese or wine (Suzzi and Torriani, 2015).
The principal biogenic amines associated with spoilage in food are histamine, putrescine, cadaverine and tyramine. However, histamine is one of the most biochemically active compounds from this class of compounds (Lieberman, 2011). Therefore, it is important to determine histamine level since it causes scombroid syndrome without changing the fish normal aspect and odour (Feng, Teuber and Gershwin, 2015). When ingested, this compound have an negative effect on the normal functions of heart, motor neurons, smooth muscle and gastric acid secretion (Feng, Teuber and Gershwin, 2015).
Histamine appears in fresh fish as a result of unsuitable refrigerated handling or preserving after being caught (Schmidt and Rodrick, 2005). Therefore, it is important to develop rapid, selective, sensitive and reduced cost methods for the detection and quantification of histamine as an alternative or complementary to the classical ones, which principally consist of the use of chromatographic techniques such as high performance liquid chromatography (Kim, Shin, Lee, Oh and Ban, 2011), cation-exchange chromatography (Cinquina, Calì, Longo, De Santis, Severoni and Abballe, 2004), gas chromatography (Pittertschatscher, Hochreiter, Thalhamer and Hammerl, 2002) or thin layer chromatography (Hua, Sato, Han, Tan, Yamaguchi and Nakano, 2011). Detection of BAs by HPLC methods require the use of a pre-column or post-column derivation stage to improve their detection in ultraviolet increasing the time of analysis and its cost. Other detection methods such as enzymatic determination (Landete, Ferrer and Pardo, 2004), spectrophotometric methods (Leng, Zhao, Yin and Ye, 2015), fluorometric methods (Staruszkiewicz, Waldron and Bond, 1977) or chemiluminometric methods (Toubanaki, Christopoulos, Ioannou and Flordellis, 2009) have also been carried out. For detection of BAs, biosensors could be attractive analytical tools offering rapid and short time analysis methods, adequate selectivity and sensitivity, low price (Kiviranda and Rinkena, 2011; Turner, 2013). Furthermore, the biosensors could be used for on-line, in-line or real time detection of such compounds (Di Fusco, Federico, Boffi, Macone, Favero and Mazzei, 2011; Son, Cho, Lim, Park, Hong, Ko and Park, 2015).
Graphene has recently appeared as an appealing material in a multitude of applications because of its excellent mechanical and electronic properties (Kuila, Bose, Khanra, Mishra, Kim and Lee, 2011; Song, Luo, Zhu, Li, Du and Lin, 2016). In the least years, graphene has been used as electrode material for development of electrochemical sensors and biosensors, battery, and fuel cell (Novoselov, Fal′ko, Colombo, Gellert, Schwab and Kim, 2012; Shao, Wang, Wu, Liu, Aksay and Lina, 2010; Apetrei and Apetrei, 2015b).
Noble metal nanoparticles, such as platinum or gold nanoparticles, exhibit electrocatalytic behaviour to hydrogen peroxide (H2O2) and were widely used for sensing applications (Li, Lu, Wu, Wang and Shi, 2013). It will be of great interest to prepare platinum nanoparticles – functionalized GPH nanocomposite, because such a functionalized GPH may generate synergy on electrocatalytic activity and consequently enhance the sensitivity of the biosensor.
In this work, we present a novel approach to develop a novel amperometric biosensor for the detection and quantification of histamine based on the immobilization of diamine oxidase into a platinum nanoparticles / graphene / chitosan modified carbon screen-printed electrode. To this purpose, experimental parameters have been optimized in order to found the optimal conditions that improve its electrochemical response characteristics. Finally, the novel biosensor has been tested in the quantification of histamine amount in different freshwater fish samples to assess its applicability, compare the biosensor obtained values with the ones obtained by using a reference method.
2. Experimental
2.1. Chemicals
Diamine oxidase (DAO, from porcine kidney, ≥0.05 unit×mg-1 solid, EC 1.4.3.6), histamine, chitosan, phosphate buffer solutions, potassium chloride, acetic acid, potassium tetrachloroplatinate(II), sulphuric acid, hydrogen peroxide (H2O2), L-lysine, L-tyrosine, L-histidine, L-tryptophan were all acquired from Sigma-Aldrich. Veratox® kit ELISA for histamine quantification was purchased from Neogen® Corporation. All solutions necessary in the experiments were prepared with ultrapure water (18.3 MΩ×cm, Milli-Q Simplicity® Water Purification System from Millipore Corporation).
2.2. Materials
Reduced graphene oxide (GPH) from Sigma-Aldrich was used for CSPE modification. Carbon screen-printed electrodes (4 mm in diameter) were purchased from Dropsens Ltd (Spain, http://www.dropsens.com/), model 110, further modified with graphene, chitosan, Pt nanoparticles and DAO.
2.3. Development of GPH/chitosan/CSPE
The suspension of GPH was prepared by dispersing 1 mg GPH in 1mL of 0.2% chitosan solution in acetic acid (pH 5). The mixture was sonicated 2 h to obtain a homogeneous dispersion. The GPH suspension was cast on the surface of a CSPE (diameter of 3mm) to prepare the GPH-modified carbon screen-printed electrode. Ten microliters of 1mg×mL-1 GPH suspension was cast on the CSPE and dried at room temperature.
2.4. Development of nPt/GPH/chitosan/CSPE
Platinum nanoparticles were electrochemically deposited on the GPH/chitosan/CSPE applying a constant potential of 0.2 V (referred to the standard hydrogen electrode) for a desired time in a solution containing 2×10-3 M H2PtCl4 (Potassium tetrachloroplatinate(II) + sulphuric acid). Solution was degassed with nitrogen before each experiment. A Pt gauze (1cm2) and Ag/AgCl (in saturated KCl) electrode were used as counter and reference electrodes, respectively, in the electrodeposition process. After the deposition, the electrode was thoroughly washed with water and kept to dry at room temperature (Minch and Es-Souni, 2011).
2.5. Development of DAO-nPt/GPH/chitosan/CSPE
DAO was dissolved in a 0.2% chitosan solution, and the DAO concentration was 10 mg×mL-1. Ten microliters of the solution were cast on the nPt/GPH/chitosan/CSPE. Right after casting, the DAO-modified screen printed electrode was stored into a refrigerator at 4 °C to dry for 12 h. Prior the experiment, the biosensors were stored at 4 °C until use in a closed box.
2.6. Apparatus
Cyclic voltammetric and amperometric measurements were performed on a Biologic SP 150 potentiostat/galvanostat (Bio-Logic Science Instruments SAS, France) connected to a personal computer and controlled by EC-Lab Express software. A three electrode cell of 25 mL capacity was employed with a modified CSPE as the working electrode, an Ag electrode as the pseudo-reference electrode, and carbon as the auxiliary electrode. The amperometric response of the DAO-nPt/GPH/chitosan/CSPE to the sequential addition of a desired amount of histamine was measured at +0.4V under continuous stirring in a 10 mL phosphate buffer solution 0.01M.
The pH of the buffer solutions was measured with a Inolab pH 7310 (WTW Instruments, Germany). A centrifuge Cencom II (JP Selecta, Spain) was used in real sample pre-treatment.
2.7. ELISA measurements
Histamine contents of real samples were analysed with a reference method to assess the viability of the biosensor. Quantification of histamine in fish samples was carried out using Neogen’s Veratox kit ELISA. This kit allows quantifying histamine in the range from 2.5 to 40 ppm. The assay is based on the competition of enzyme-labelled histamine (conjugate) with free histamine in the samples. After a washing stage, enzyme substrate was added, which reacts with the bounded conjugate producing a change of colour (from red to blue). After that, the test plate was read in a microwell reader at 620 nm to yield optical densities. Finally, obtained values were interpolated into the calibration plot founding the concentration in each sample.
2.8. Real samples
Different fish samples such as carp (Cyprinus carpio), tench (Tinca tinca), Prussian carp (Carassius gibelio), Pontic shad (Alosa Pontica), and Wels catfish (Silurus glanis) which were studied in order to test the developed biosensor, were acquired from a local fish market. The samples were analyzed immediately after purchasing and after 48 h. Prior to carry out the amperometric and ELISA determinations, samples were pre-treated to extract the biogenic amines. Therefore, fish samples were cleaned, eviscerated, washed and then cut in thick slices. Then, thick slices were blended to obtain a homogenous mixture. 5 g of this mixture plus 45 mL of ultrapure water were added to an extraction funnel and shacked during 10 min to improve the extraction process. After 5 min, this procedure was repeated two more times. Finally, the extract was centrifuged at 4000 rot/min for five minutes. The supernatant was recovered being fish samples then suitable for electrochemical analysis (Pérez, Bartrolí and Fàbregas, 2013).
3. Results and Discussion
3.1. Cyclic voltammetry
In the present study, the electrochemical response of the DAO-nPt/GPH/chitosan/CSPE biosensor was resulting from the anodic oxidation current of the enzymatic product hydrogen peroxide (H2O2). Components of sensitive layer and the scheme of the reactions that take place at the biosensor surface are shown in Figure 1.
Figure 1.
The enzymatic and electrochemical processes at the biosensor surface are presented in the following reaction scheme:
It is well-known that H2O2 could be detected by electrochemical oxidation processes at the solid electrodes (Al-Akraa, Mohammad, El-Deab and El-Anadouli, 2015). Usually, the oxidation or reduction take place at relatively high potentials that cause interference from other electroactive compounds present in samples. Noble metals nanoparticles, such as platinum or gold, were widely used as electrocatalysts for oxidation or reduction of H2O2 (Yu, Wu, Pan, Zhao, Wei and Lu, 2015). In order to study the electrocatalytic effect of the GPH/chitosan and Pt/GPH/chitosan films to H2O2, cyclic voltammograms were registered. The electrochemical responses of all electrodes do not present any electrochemical peaks in phosphate buffer solution of pH=7.4 in the absence of H2O2 (data not shown).
Figure 2 (A) shows the cyclic voltammograms obtained at the modified SPE with a nPt/GPH/chitosan nanocomposite film in a 10-2 M phosphate buffer solution of pH=7.4 containing 10-4 M H2O2 (curve a). In the same Figure are shown the cyclic voltammograms of GPH/chitosan/CSPE (curve b) and unmodified CSPE (curve c).
As shown in Figure 2, the oxidation and reduction currents of H2O2 at the CSPE are very small, and the oxidation of H2O2 starts at 0.55 V. On the other hand, the electrochemical currents obtained at the GPH/chitosan/CSPE and the nPt/GPH/chitosan/CSPE are much larger than that obtained at the CSPE. In addition, it can be observed that the oxidation of H2O2 started at a relative low potential, 0.25 V. Electrochemical process of the reduction of H2O2 started at a potential of 0.15 V at the modified electrodes. Furthermore, the reduction current increased as the potential decreased.
Figure 2.
The decrease of the redox potential of H2O2 is attributed to the electrocatalytic activity of the platinum nanoparticles and graphene. Moreover, it can be seen that the magnitude of the oxido-reduction current at these different electrodes increased in the subsequent order: nPt/GPH/chitosan/CSPE > GPH/chitosan/ CSPE > CSPE. From these results it was concluded that the nanocomposite film with both GPH and nPt demonstrated a synergistic effect on the electrocatalytic detection to H2O2. From all electrodes, the nPt/GPH/chitosan/CSPE revealed the highest electrocatalytic activity towards H2O2.
The influence of the Pt electrodeposition time on the electrocatalytic activity of nPt/GPH/chitosan/CSPE to H2O2 was studied. The electrocatalytic activity of the developed electrode was measured at +0.4 V in a solution containing 10-3 M H2O2. It was found that the signal (oxidation and reduction current) increased as the deposition time increased until it achieved the plateau at 300 s deposition. Then the electrochemical response started to decrease with more deposition time, which may be related to the reduction of electroactive surface area. This result could be related to large-sized particles deposited on the electrode surface when deposition time is larger than 300s. Therefore, a 300 s deposition time was selected for this work.
Figure 2 (B) shows the cyclic voltammogram of the DAO-nPt/GPH/chitosan/CSPE biosensor in a 10-2 M phosphate buffer solution of pH=7.4 containing 10-4 M histamine. The cyclic voltammogram of the DAO-nPt/GPH/chitosan/CSPE biosensor in 10-5 M of histamine showed an anodic peak associated to the electrochemical oxidation of the H2O2 at 0.60V and a cathodic peak at -0.15V associated with the reduction of the H2O2, respectively. Diamine oxidase catalyzes the oxidation of histamine. One from the reaction products is electrochemically detected at biosensor surface at appropriate potential, the detection principle of the biosensor developed in this study.
Influence of scan rate in the biosensor response was carried out by registering the cyclic voltammograms of the DAO-nPt/GPH/chitosan/CSPE biosensor at different scan rates, from 0.05 to 1.00 V×s-1 (data not shown).
The intensity of peak related to the electrochemical oxidation of H2O2 (anodic peak) increases linearly with the square root of the scan rate () suggesting a diffusion controlled processes according to the Randles-Sevcik equation (Apetrei, Alessio, Constantino, de Saja, Rodriguez-Mendez, Pavinatto, Fernandes, Zucolotto and Oliveira, 2011):
Ipa=2.687×105×n3/2×1/2×D1/2×A×C
where Ipa is the anodic peak current (Ampere), n is the number of electrons involved in the redox process (n=2), ν is the potential scan rate (V×s-1), D is the diffusion coefficient (cm2×s-1), A is the electrode surface area (cm2) and C is the concentration (mM). From the slope of Ipa vs. 1/2 plot, the electrode surface area, A, was calculated. Diffusion coefficient of H2O2 is 6×10-6 cm2×s-1 (Hall, Khudaish and Hart, 1998). The calculated electrode surface area was 1.2434±0.0121 cm2. The electroactive area of biosensor is much larger than the geometrical electrode surface area (0.1256 cm2). Therefore, the DAO-nPt/GPH/chitosan/CSPE biosensor presents fast electrochemical processes increasing sensitivity of the biosensor.
3.2. Optimization of the biosensor working conditions
In order to establish the optimal working potential for the determination of histamine, a study of the obtained electrochemical response when varying the applied potential in the electrochemical system was carried out. Presence of -COOH and -OH groups facilitate the immobilization of DAO on biosensor surface (nPt/GPH/chitosan) by means of electrostatic, hydrophobic, van der Waals, hydrogen bonding interactions, and combination of those (Kim and Herr, 2013). The sensitive layer is stable and cross-linking process is not necessary increasing the sensitivity of the biosensor (Pavinatto, Fernandes, Alessio, Constantino, de Saja, Zucolotto, Apetrei, Oliveira Jr and Rodriguez-Mendez, 2011).
As before mentioned, the obtained signal is due to the H2O2 produced in the biocatalytic reaction, which normally requires the application of high potentials. In the case of biosensor developed in this study, based on the enzymatic system together with the incorporation of GPH for increasing the electroactive surface of biosensor and facilitate the electron transfer and nPt as a catalyst for oxido-reduction of H2O2, it is allows reducing this potential to +0.4 V vs. Ag.
The optimization of applied potential was carried out by amperometry under constant stirring of solution containing histamine. The applied potential has the main effect over the biosensor response towards histamine contributing to the sensitivity and selectivity. Potential range analyzed was among 0.0V vs. Ag and 0.08V vs. Ag. The most intense response is obtained at +0.4 V vs. Ag. Therefore, this value was selected as the optimal applied potential because it showed the combination of the highest and the major stable current response to histamine (10-4 M), comparing with the signal obtained in phosphate buffer solution at the same working potential.
Given the importance of the pH in the DAO enzyme activity, a study of the effects of the pH of the phosphate buffer solution was also carried out. Its influence in the signal towards a 10-4 M histamine solution at different pH values (from 6.4 to 8.4) was investigated.
As shown in Figure 3 (a), it was found that there was a increase in the biosensor response as the pH increased, until achieving its maximum at pH 7.4. After that, from 7.4 to 8.4 there was a decrease in its response. Therefore, taking into account this dependence profile, pH 7.4 was chosen as the optimal one to perform further electrochemical measurements.
Figure 3.
In order to establish the optimal quantity of DAO on surface of nPt/GPH/chitosan/CSPE different biosensors were prepared increasing the quantity of DAO immobilized. Figure 3 (b) shows the variation of biosensor response in function of the amount of DAO immobilized. The peak current increases as the DAO quantity is increased. For higher quantity than 9 U of DAO a slight decrease of the biosensor response is observed. This behaviour point out that at low quantities, DAO increase the rate of the enzymatic process. High quantities of DAO might cause diffusion limitation. Therefore, 9 U of DAO was chosen to prepare the biosensor, because no significant variation on the biosensor response between 9 U and 11 U of DAO was observed.
3.3. Characteristics of the biosensor
Histamine biosensor was further characterized with regard to its linear interval range, sensitivity and detection limit, storage and operational stability and its reproducibility.
Amperometric technique was employed for the evaluation of the diamine oxidase / platinum nanoparticles / graphene – based biosensor for histamine detection. Figure 4 (a) shows the typical amperometric response of DAO/nPt/GPH/chitosan/CSPE to successive additions of histamine with an applied potential of +0.4 V. As can be seen from Figure 4 (a), the electrochemical response increased as the histamine concentration increased.
Figure 4.
Figure 4 (b) shows the plot of the anodic current vs. the histamine concentration. It can be observed from this figure that this biosensor shows a wide linear range from 0.1 M to 300 M histamine. Biosensor's linear equation is I = 0.0631×c + 1.6633 with a coefficient of determination of 0.9962. The detection limit of the histamine biosensor is 2.54×10-8 M based on 3× / m equation (where – standard deviation of the blank sample; m – slope of the calibration plot). Such a sensitivity of the biosensor (0.0631 A×M, R2=0.9962) could be related to a large surface area of the electrode, a fast electron transfer facilitated by GPH, and an electrocatalytic synergism of platinum nanoparticles and graphene. Inside of the nanocomposite sensitive layer, DAO can contact both GPH and platinum nanoparticles, which facilitates the rapid electron exchange with a relatively small barrier between the enzyme and the electrode. The histamine biosensor based on DAO/nPt/GPH achieved a better detection limit in comparison with the DAO/graphite with peroxidase and Os mediator biosensor (Bóka et al., 2012) (a detection limit of 5 M), DAO / hydrogel film of photo-2-hydroxyethyl methacrylate /carbon paste SPE biosensor (Keow, AbuBakar, Salleh, Heng, Wagiran and Bean, 2007) (a detection limit of 0.65 ppm), DAO-HRP /C- SPE biosensor (Alonso-Lomillo, Domínguez-Renedo, Matos and Arcos-Martínez, 2010) (a detection limit of 0.40 M), and DAO / Pt / Copolymerization (GA-membrane) biosensor (Bouvrette, Male, Luong and Gibbs, 1997) (a detection limit of 25 M).
From the calibration data, the Hill coefficient was calculated from the plot of log[I/(Imax-I)] vs. log [S] (the logarithm of histamine concentration). A Hill coefficient of 1.02 0.02 was calculated demonstrating that the kinetics of the enzymatic reaction of histamine fitted into a Michaelis–Menten type kinetics (Apetrei et al., 2011).
The apparent Michaelis–Menten constant ( ) and maximum current response (Imax) was calculated from Lineweaver-Burk equation (Uematsu and Katano, 2013):
where [S] is the concentration of histamine, I is the response current and Imax is the steady-state current.
The Imax is 22.4 A and = 120.6 M. The is lower but comparable to that obtained using histamine as the substrate (Bouvrette, Male, Luong and Gibbs, 1997; Frébort, Skoupá and Peč, 2000). These results explain the high sensitivity of the developed biosensor.
3.4. Biosensor stability studies
Relative standard deviation of the biosensor towards 10-4 M histamine was 5.5% for five different electrodes, indicative of the good reproducibility of the biosensor fabrication.
Storage stability of the developed biosensor was studied. For this, two sensors were prepared in the same conditions and their sensitivity toward 10-4 M histamine was evaluated during a month. The amperometric response of the first biosensor was studied by triplicate in different days during a month. The electrochemical response towards histamine of the second biosensor was evaluated just after its preparation and the last day of the study. During this time, biosensors were stored at 4 °C in a closed plastic box when not in use. Results obtained showing excellent and comparable storage stability in both cases, since the decrease was only 10.2 and 12.6%, respectively.
On the other hand, repeatability of the biosensor response after 5 consecutive calibrations using the same biosensor toward 10-4 M histamine was studied. A high reproducibility was obtained with RSD value of 2.6%.
3.5. Study of interferences
The study of interfering compounds is an essential step to evaluate the response of the biosensor prior to the analysis of real samples to demonstrate its applicability. For this purpose, biosensor response towards principal amino acids involved in the biosynthesis of biogenic amines such as L-hystidine, L-tyrosine, L-lysine and L-tryptophan. The effect of these compounds was examined by comparison of the amperometric response obtained for a 10-5 M and a 5×10-6 M histamine standard solution vs. the amperometric response obtained for solutions of the same concentration of those amino acids. Obtained results reveal that these amino acids have a reduced influence in the histamine quantification. Only lysine produces an amperometric response at these concentrations levels and optimal measurement conditions, representing only a 3.4% of the histamine response when compared.
3.6. Real samples analysis: application in fish samples
Different types of fish samples were analysed with the developed biosensor. The purpose of this diversity of samples was to evaluate if the biosensor was able to quantify the amount of histamine in different fish species with good reliability.
The quantification of histamine was carried out with the biosensors. Histamine quantification was performed by means of direct interpolation in the histamine calibration plot. The standard addition method was also used in order to study the influence of matrix effect. Under the optimum established conditions, amperometric measurements were carried out in an electrochemical cell containing 20 mL of phosphate buffer solution at pH 7.4 and applying a potential of 0.4 V vs. Ag. All amperometric measurements were done in triplicate.
As can be observed in Table 1, there are relative small differences between interpolation and standard addition methods, concluding that the matrix effect was not significant in histamine detection.
The evolution of histamine content in fish samples stored at 4 °C was studied initially and after 48h for all fish samples. Obtained results are shown in Figure 5.
Figure 5.
For all samples there was a clear increasing of histamine content, demonstrating an increment of toxicity as the time of storage increases. For this reason, histamine concentration levels could be used as an indicator of fish quality or fish freshness. Control of quality and freshness is especially important to prevent scombroid syndrome, which results from eating spoiled fish.
As an additional proof of the proposed methods, a Student’s paired samples t-test was performed between the results obtained with the amperometric biosensor and the ones obtained with the ELISA kit. In the case of measurements by interpolation and standard addition, obtained experimental t values was 0.4678, while the critical tabulated t value was 2.2621 (95% confidence level and 9 degrees of freedom). Therefore, there are no significant differences between the concentrations found by interpolation and standard addition method. On the other hand, experimental t values were 0.1678 and 0.7263, respectively, when were compared interpolation method vs. ELISA and standard addition method vs. ELISA. Experimental t values are lower than the critical t value in both cases (2.2621 at 95% confidence level and 9 degrees of freedom). It was concluded that there are no significant differences between the concentrations found with the amperometric method and the ELISA kit method.
4. Conclusions
We present a novel nanobiocomposite film consisting of DAO/nPt/GPH/chitosan for histamine biosensing. The biosensor exhibits excellent sensitivity (0.0631 A×M) with a detection limit of 2.54×10-8 M histamine. Such a sensitivity is related to the synergy of GPH and Pt nanoparticles on the electrocatalytic detection of H2O2. The histamine biosensor has good amperometric responses for the reason that exists a large surface area and a fast electron transfer facilitated by Pt nanoparticles and graphene. The biosensor also has good reproducibility and long-term stability. The interfering signals from L-hystidine, L-tyrosine, L-lysine and L-tryptophan are negligible compared with the response to histamine. Fish samples were tested with the histamine biosensor and good and correlate results were obtained by interpolation method and standard addition method. Excellent correlations between the histamine amounts obtained with developed biosensor and ELISA method were found. This work shows that the hybrid DAO/nPt/GPH/chitosan nanocomposite-based biosensor is a reliable method for the quantification of histamine in fish samples such as carp, tench, Prussian carp, Pontic shad and Wels catfish.
Figure captions
Figure 1. Scheme of the sensitive layer and the reactions that take place at the biosensor surface.
Figure 2. (A) Cyclic voltammograms obtained at (a) CSPE; (b) GPH/chitosan/CSPE; (c) nPt/GPH/chitosan nanocomposite film in a 10-2 M phosphate buffer solution of pH=7.4 containing 10-4 M H2O2.
(B) Cyclic voltammogram of the DAO-nPt/GPH/chitosan/CSPE biosensor in a 10-2 M phosphate buffer solution of pH=7.4 containing 10-4 M histamine.
Figure 3. (a) Influence of the pH in the biosensor response towards a 10-4 M histamine solution in phosphate a 10-2 M phosphate buffer solutions. Measurements were carried out at +0.4 V in triplicate (RSD=2.24 %).
(b) Variation of biosensor response in function of the amount of DAO immobilized. Measurements were carried out at +0.4 V in triplicate (RSD=4.46 %) in 10-4 M histamine solution in phosphate a 10-2 M phosphate buffer solution of pH=7.4.
Figure 4. (a) Amperometric response of DAO/nPt/GPH/chitosan/CSPE to sequential addition of a series of histamine concentrations from 0.1 M to 100 M at +0.4 V.
(b) The calibration curve.
Figure 5. Evolution of total histamine content in fish samples. Samples were stored at 4 °C. Values of histamine concentration are the average of all quantification methods.