Screen-printed biosensor modified with carbon black nanoparticles for ...

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Microchim Acta (2015) 182:643–651 DOI 10.1007/s00604-014-1370-y

ORIGINAL PAPER

Screen-printed biosensor modified with carbon black nanoparticles for the determination of paraoxon based on the inhibition of butyrylcholinesterase Fabiana Arduini & Matteo Forchielli & Aziz Amine & Daniela Neagu & Ilaria Cacciotti & Francesca Nanni & Danila Moscone & Giuseppe Palleschi

Received: 18 June 2014 / Accepted: 16 September 2014 / Published online: 2 October 2014 # Springer-Verlag Wien 2014

Abstract We have developed a screen-printed electrochemical electrode (SPE) for paraoxon based on its inhibitory effect on the enzyme butyrylcholinesterase (BChE). The electrode was first modified by drop casting with a dispersion of carbon black nanoparticles (CBNPs) in a dimethylformamide-water mixture, and BChE was then immobilized on the surface by cross-linking. The resulting biosensor was exposed to standard solutions of paraoxon, and the enzymatic hydrolysis of butyrylthiocholine over time was determined measuring the enzymatic product thiocholine at a working voltage of + 300 mV. The enzyme inhibition is linearly related to the concentration of paraoxon up to 30 μg L−1, and the detection limit is 5 μg L−1. The biosensor is stable for up to 78 days of storage at room temperature under dry conditions. It was applied to determined paraoxon in spiked waste water samples. The results underpin the potential of the use of CBNPs in electrochemical biosensors and also demonstrate that they represent a viable alternative to other carbon nanomaterials F. Arduini (*) : M. Forchielli : D. Neagu : D. Moscone : G. Palleschi Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy e-mail: [email protected] A. Amine Faculté de Sciences et Techniques Laboratoire Génie des Procédés et Environnement, Université Hassan II-Mohammedia, B.P. 146 Mohammadia, Morocco I. Cacciotti Università degli Studi di Roma “Niccolò Cusano”, UdR INSTM, Via Don Carlo Gnocchi 3, 00166 Rome, Italy F. Nanni Dipartimento di Ingegneria dell’Impresa, Università di Roma Tor Vergata, UdR INSTM Roma-Tor Vergata, Via del Politecnico 1, 00133 Rome, Italy

such as carbon nanotubes or graphene, and with the advantage of being very affordable. Keywords Organophosphate . Butyrylcholinesterase . Screen-printed electrode . Carbon black nanoparticles . Inhibition

Introduction Recently in the frame of an European project, a list of emerging substances (Norman list) was drawn up, and the organophosphorus insecticides such as parathion methyl, parathion ethyl are included in Norman list [http://www.normannetwork.net Accessed 16 June 2014]. These insecticides, in fact, are the most used due to their high insecticidal activity and relatively low persistence in the environment, thus their detection is an important issue in analytical chemistry. The detection of organophosphorus insecticides is generally carried out using Gas or Liquid Chromatography, which are highly sensitive and selective techniques, but require skilled personnel, laboratory set-up, and expensive instrumentation [1, 2]. An alternative analytical system is the use of biosensors, which are cost-effective, miniaturized and friendly to use. Taking into consideration that the acetylcholinesterase (AChE) is inhibited by organophosphorus insecticides [3], this enzyme was properly used as biocomponent in the biosensor development; in fact, measuring the AChE activity before and after the biosensor exposure to environmental samples, it is possible to quantify the amount of organophosphorus insecticides present in the sample [4, 5]. The biosensors which offer the best guarantee for analytical applications, in terms of sensibility, reproducibility, and selectivity, often turn out to be the electrochemical ones based on disposable screen-printed electrodes produced by thick film

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technology [6]. In this overall scenario, an electrochemical amperometric biosensor for organophosphates can be a bienzymatic one that uses acetylcholine as substrate. The two enzymes are AChE, which hydrolyses the acetylcholine to choline and acetic acid, and Choline Oxidase (ChOx) that oxidises the choline to betaine with the production of H2O2. The use of ChOx is necessary in the case of amperometric biosensors because the enzymatic products of the AChE reaction are not electroactive, and the enzymatic activity can be detected by means of the O2 decrease quantification using a Clark’s electrode [7] or by the increase of H2O2 [8]. The alternative approach mainly used in the last years, was the monoenzymatic amperometric biosensor, in which a synthetic substrate is used; in fact, acetylthiocholine was adopted instead of the natural substrate acetylcholine. The enzymatic reaction hydrolyses the acetylthiocholine to acetic acid and thiocholine, and then the latter, being electrochemically active, can be quantified. In order to reduce the applied potential and fouling problems during thiocholine detection, two approaches can be followed: (i) the use of redox mediators such as cobalt phthalocyanine (CoPc) [9], Prussian Blue [10], t e t r a c y a n o q u i n o d i m e t h a n e ( T C N Q ) [ 11 ] , c o b a l t hexacyanoferrate [12], potassium ferricyanide [13] or (ii) the use of nanomaterials such as carbon nanotubes [14]. Recently our research group demonstrated the suitability of CBNPs as useful nanomaterial to modify SPEs in order to increase their electrochemical performances [15–18]; the improvement using CBNPs was also confirmed by the Compton group that highlighted “the potential improvement involved in the (largely unexplored) direct application of nano-carbon in electrode surface modification” [19]. In this work, we investigated for the first time the suitability of CBNPs-SPE as platform to immobilize the butyrylcholinesterase (BChE) in order to develop a biosensor based on BChE inhibition for organophosphate detection.

F. Arduini et al.

carried out in the same cell with a PC-controlled Autolab. A sinusoidal voltage perturbation of 10 mV amplitude was applied over the frequency range 10 kHz to 0.01 Hz, with 10 measurement points per frequency decade. For the fitting of the data obtained by EIS, Z-views software (Scribner Associates, Inc.; www.scribner.com) was used.

Reagents Commercial CB N220 of industrial standard grade was obtained from Cabot Corporation (Ravenna, Italy, www.cabotcorp.com). As reported by the manufacturer, the nanoparticles of CB N220 had a diameter comprised between 19 and 29 nm with a surface area of 124 m2 ·g−1 as measured by BET method (N2 absorption). Butyrylcholinesterase (BChE) from equine serum, bovine serum albumin (BSA), Sbutyrylthiocholine chloride, 5,5′-dithio-bis 2-nitrobenzoic acid (DTNB), glutaraldehyde and paraoxon (paraoxon-ethyl), Nafion (perfluorinated ion-exchange resin, 5 %v/v solution in lower alcohols/water) were purchased from Sigma Aldrich Company (St. Louis, USA, www.sigmaaldrich.com).

Preparation of SPE SPE was produced with a 245 DEK (Weymouth, UK; www. dek.com) screen-printing machine. Graphite based ink (Electrodag 423 SS) from Acheson (Milan, Italy; www. achesonindustries.com) was used to print the working and counter electrodes. Silver ink (Electrodag 477 SS) was used to print the reference electrode. As insulating ink Carboflex 25.101S was used. The substrate was a flexible polyester film (Autostat HT5) obtained from Autotype Italia (Milan, Italy). The electrodes were home produced in foils of 48. The diameter of the working electrode was 0.3 cm resulting in a geometric area of 0.07 cm2.

Experimental Carbon black nanoparticle dispersion Apparatus Cyclic voltammetry (CV) measurements were performed using an Autolab electrochemical system (Eco Chemie, Utrecht, The Netherlands; www.ecochemie.nl) equipped with PGSTAT-12 and GPES software (Eco Chemie, Utrecht, The Netherlands). Amperometric measurements were carried out using a VA 641 amperometric detector (Metrohm, Herisau, Switzerland), connected to a X-t recorder (L250E, Linseis, Selb, Germany; www.linseis.com). Micrographs of CBNPs-SPE and biosensor based CBNPsSPE were acquired by means of a field emission gun scanning electron microscopy (FEG-SEM, Leo Supra 35). Electrochemical impedance spectroscopy (EIS) measurements were

The dispersion of CBNPs was prepared by adding 20 mg of CBNPs powder to 20 mL of solvent (a mixture dimethylformamide (DMF): water (1:1)), and sonicated for 60 min at 59 KHz.

Preparation of CBNPs-SPE The SPE was modified with CBNPs via drop casting, pipetting a small volume (6 μL) of the dispersion onto the SPE working electrode surface in three steps of 2 μL each. After that, the solvent is allowed to volatilize at RT, and a CBNPs “film” is left on the electrode surface.

Biosensor based on butyrylcholinesterase inhibition and for paraoxon determination

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Thiocholine determination

Paraoxon determination

Thiocholine was enzymatically produced by BChE using butyrylthiocholine as substrate (because thiocholine is not commercially available). For this purpose, 1 mL of 1 M butyrylthiocholine solution was prepared in phosphate buffer 0.1 M (pH=8), and 100 units of BChE were added to this solution. After 1 h, the concentration of thiocholine produced by BChE was estimated spectrophotometrically by Ellman’s method. For this purpose, 900 μL of phosphate buffer solution (0.1 M, pH=8), 100 μL of 0.1 M DTNB, and 5 μL thiocholine solution (diluted 1:100 in water) were put in a spectrophotometric cells. The absorbance was measured, and the real concentration was evaluated by using the Lambert–Beer law with the known molar extinction coefficient of TNB (ε= 13,600 M−1 cm−1) [20]. After 2 h, the butyrylthiocholine hydrolysis was completed, and 1 mL solution of 1 M thiocholine was obtained. The solution was stable for 1 day at 4 °C.

The inhibitory effect of paraoxon on BChE biosensor was evaluated by determining the decrease in the current obtained for the oxidation of thiocholine that was produced by the enzyme. To do this, the response toward the substrate was analyzed as described above, after the BChE biosensor was incubated in the insecticide solution for a certain period (incubation time) and then rinsed three times with distilled water. After that, the response toward the substrate was registered and the degree of inhibition was calculated as a relative decay of the biosensor response (Equation 1). I% ¼ ½ðI0 −Ii Þ=I0   100

ð1Þ

where Io and Ii represent the biosensor response before and after the incubation procedure, respectively Sample collection and measurement

Preparation of BChE biosensor In the case of cross-linking method, two steps were adopted. 2 μL of a glutaraldehyde solution 0.25 %v/v (diluted in water) were applied with a syringe exclusively on the working electrode. Then, 2 μL of a mixture of BSA, enzyme and Nafion were placed onto the working electrode. The mixture was obtained by mixing 25 μL of BSA (3 %w/v prepared in water), 25 μL of Nafion (0.1 %v/v diluted in water) and 25 μL of enzyme stock solution (see scheme 1).

Two samples of waste water were supplied by Tover Italia, Rome (paint industry) and collected in different days (i.e. samples A and C). One sample of waste water was supplied by BASF The Chemical Company (Rome, Italy) (i.e. sample B). All the samples were tested before and after spiking with paraoxon. The samples supplied by Tover Italia were filtered before the analysis using Acrodisk syringe filter 37 mm and glass membrane 1 μm filter. All samples were diluted 1:2 (v/v) with phosphate buffer 0.1 M+KCl 0.2 M for successive electrochemical analyses.

Butyrylthiocholine determination Butyrylthiocholine analyses were performed using an amperometric “drop” procedure in phosphate buffer solution (0.05 M+0.1 M KCl, pH 7.4) with an applied potential of + 300 mV vs Ag/AgCl. In details, a drop (50 μL) of buffer containing different amounts of butyrylthiocholine was placed onto the BChE biosensor in such a way that the working, counter and reference electrodes were covered. After applying the potential, the signal was continuously recorded and the current value at the steady state was detected.

Scheme 1 Schematic preparation of the BChE-CBNPs-SPE biosensor and its typical response

Results and discussion Electrochemical properties of CBNPs towards thiocholine In a previous paper, we demonstrated the ability of CBNPs using commercial available SPE modified with CBNPs dispersion (1 mg · mL − 1 prepared in acetonitrile) to electrocatalyze the oxidation of several thiols such as cysteine, cysteamine, glutathione and thiocholine [16]. The high sensitivity reached for thiol detection was used to measure mercury ions. In this paper we have tested CBNPs-SPE prepared using the dispersion of CBNPs in DMF: H2O 1:1 (v/v) with thiocholine, the cholinesterase enzymatic product. The cyclic voltammograms are shown in Fig. 1. We have observed an increase of the oxidation current and a decrease of the oxidative potential; a broad peak was in fact observed at +300 mV, while in the case of the bare SPE it was placed at +700 mV. Thus, also in this case, we have demonstrated the ability of CBNPs to electrocatalyze the thiocholine detection, and these

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i(A)

6e-6 4e-6 2e-6 0 0,0 0,2 0,4 0,6 0,8

1,0

E vs Ag/AgCl (V) Fig. 1 CVs in phosphate buffer 0.05 M+KCl 0.1 M, pH=7.4, in absence of 1 mM thiocholine using bare (continuous lines) and CBNPs-SPE (dotted lines) and in presence of 1 mM thiocholine using bare (dashed lines) and CBNPs-SPE (dashed-dotted lines)

electrocatalytic properties were also observed when varying the CBNPs-SPE preparation procedure using different SPEs and dispersions, demonstrating the robustness of the sensor production. The valuable electrochemical properties obtained using CBNPs toward thiocholine oxidation are better when compared with other carbon nanomaterials. In the case of CNTs, in fact, the applied potential is low as in the case of CBNPs, but the detection limit is higher than the one obtained using CBNPs [21, 22]. This behaviour is probably ascribed to the better signal/noise ratio in the case of CBNPs with respect to CNTs, as we previously observed in the case of other electroactive compounds [18]. In the case of graphene, the high applied potential required to detect thiocholine was reported in literature [23–25]. In fact, in the case of porousreduced graphene oxide modified AChE biosensor, 0.75 V as applied potential was necessary to detect thiocholine [23]; a similar potential was also found for AChE biosensor based on CdS–decorated graphene nanocomposite (0.68 V) [24] or 3carboxyphenylboronic acid/reduced graphene oxide–gold nanocomposites (0.7 V) [25]. The reason for the very good electrochemical performances of CBNPs can be ascribed to their high number of defect sites [15], which lead to detect the thiocholine at low applied potential, with high sensitivity without fouling problem. Furthermore, these results suggest that the CBNPs used in this work are competitive not only with CNTs [18] but also with graphene. Taking into consideration the results obtained using CBNPs-SPE in the enzymatic product thiocholine detection, this sensor was then used to immobilize the butyrylcholinesterase enzyme in order to produce a novel BChE biosensor for organophosphate detection.

BChE biosensor Very often in literature we found biosensors characterised by a very low detection limit but coupled with a satisfactory shelf

stability, obtained however only at low temperature, a characteristic that hampers a possible commercialisation of the biosensor [14, 26–31]. In order to construct a biosensor characterised by both high operating and shelf stability at room temperature, we have recently performed a study in which several different immobilisations procedures were investigated, demonstrating that the cross-linked BChE is characterised by high shelf stability using the SPE modified with electrochemical mediator Prussian Blue (PB-SPE) [32–34]. In this work, we tested the immobilisation of BChE by means of glutaraldehyde, Nafion and BSA on CBNPsmodified SPEs. Glutaraldehyde was necessary to link the enzyme, and Nafion was useful for the adherence of the enzymatic membrane on the CBPNs. Furthermore, the addition of the BSA in the enzymatic membrane was necessary to improve the enzyme shelf-life. To achieve satisfactory analytical performances, it is important that i) few enzymatic units are immobilised on the surface of the electrode, because in the case of irreversible inhibition, as for BChE inhibition by organophosphate insecticides, the degree of inhibition increases at enzyme units decrease, ii) however, it is also important to have a sufficient number of immobilised units, in order to have a thiocholine production adequate to be quantified. In the Fig. 2 the behaviour when increasing the enzymatic units is showed. As expected, we have observed the increase of current due to the increase of enzymatic thiocholine production and the decrease of degree of inhibition due to the fact that it is an irreversible inhibition type; thus, 26 mU were chosen as a compromise (Fig. 2a). Moreover, keeping in mind that it is an irreversible inhibition, the concentration of substrate selected for inhibition measurements should be suitable to reach the Vmax; thus the biosensor response in function of enzymatic substrate amount was evaluated. Once optimized, the biosensor was then tested towards butyrylthiocholine, the enzymatic substrate that was analysed in the range comprised between 1×10−5 M and 1× 10−2 M, observing a Michaelis Menten behaviour with a KMapp = (2.2 ± 0.3) mM (Fig. 2b). The minimum butyrylthiocholine concentration that gives Vmax was 5 mM, and it was chosen for the insecticide determination; in addition, the repeatability of the biosensor was tested at this Inhibition degree (%)

8e-6

6 5 4 3 2 1 0

80 60 40 20 0 10

20

30

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50

60

Enzymatic units (mU)

2,5 2,0 1,5 1,0 0,5 0,0 0,000

0,004

0,008

0,012

[butyrylthiocholine] M

Fig. 2 a Biosensor response towards 5 mM butyrylthiocholine and 20 μg L−1 paraoxon (as inhibition degree) in function of enzymatic units. b Calibration plot of butyrylthiocholine. Applied potential: +300 mV vs Ag/AgCl, 0.05 M phosphate buffer+0.1 M KCl, pH 7.4

Biosensor based on butyrylcholinesterase inhibition and for paraoxon determination

butyrylthiocholine level. The value obtained for six successive analyses was 1.56±0.06 μA with a RSD% equal to 3.9 %. The satisfactory repeatability of the biosensor is not only a good analytical property, but also shows the possibility of using the same biosensor several times without fouling problems that, instead, occur in the case of bare SPE. This behaviour is due to the presence of CBNPs. BChE biosensor characterization by means of electrochemical impedance spectroscopy and scanning electron microscopy Electrochemical Impedance Spectroscopy can provide useful information on the impedance changes of the electrode surface during the fabrication process of biosensors, by measuring the value of electron transfer resistance (Rct). The Rct, estimated according to the diameter of the semicircle present at the high frequency region, represents, in fact, the difficulty of electron transfer of ferro/ferricyanide redox probe between the solution and the electrode, giving information about the change of the electrode surface. The electrochemical impedance spectroscopy was performed with CBNPs-SPE in absence and presence of the enzymatic membrane (BChE-CBNPs-SPE) at open circuit potential (OCP). Fitting of spectra was done using the equivalent electrical circuit showed in Fig. 3 (inset) which comprises the electrolyte resistance, Re (around 150 Ω), in series with a parallel combination of Rct (interfacial charge transfer resistance), Zw (diffusion of the analytes in solution and corresponding to Warburg impedance straight line of the curves) and CPE (Constant Phase Element). Fig. 3 shows the Nyquist plots for CBNPs-SPE and BChE-CBNPs-SPE. The Rct for BChE-CBNPs-SPE was much higher (2842±89Ω) than the CBNPs-SPE (229±4Ω) confirming the deposition of the enzymatic layer that hampers the electron-transfer of the electrochemical probe (ferro/ferricyanide). The constant phase element determination, CPE, was necessary due to the nonhomogeneous surface of the working electrode and it is modeled as a non-ideal capacitor of capacitance C and roughness/non-uniformity factor α. The α resulted 0.70 for the CBNPs-SPE and 0.58 for BChE-CBNPs-SPE,

Fig. 3 Complex plane impedance plots at an open circuit potential for CBNPs-SPE a and BChE-CBNPs-SPE b using a 10 mM ferricyanide and 10 mM ferrocyanide solution in 0.1 M KCl. Inset: Randles circuit

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demonstrating the increase of the electrode surface roughness in the presence of the enzymatic membrane. These experimental evidences were supported and confirmed by the SEM analysis that was performed in order to investigate the morphological characteristics of the bare SPE and the CBNPsSPE before and after the enzyme immobilization (BChECBNPs-SPE). In fact, it is well known that the morphology of the substrate plays a pivotal role in the immobilization of the enzyme and strongly influences the biosensor performance. In Fig. 4 low and high magnification micrographs of bare SPE, CBNPs-SPE and BChE-CBNPs-SPE are compared. In all cases, the low magnification images testified the obtainment of a complete and uniform deposition on the working electrode (Fig. 4a, c and e). Considering the high magnification micrographs, the bare SPE showed a webbed surface with irregularly shaped and randomly orientated micrometer-sized flakes of graphite bound together with an inert polymeric binder and covered of small particles assigned to the cross-linking agents in the original ink (Fig. 4a and b). On the other hand, the SEM micrographs of the CBNPs-modified working electrode confirmed the homogeneous and uniform CBNPs deposition and revealed a rough and sponge-like structure, characterized by the presence of numerous and diffuse cauliflower aggregates of CBNPs (Fig. 4c and d). From the high magnification micrographs (Fig. 4d, inset) it is clear that the CBNPs film

Fig. 4 SEM micrographs of bare SPE a-b, CBNPs-SPE c-d and BChECBNPs-SPE e-f (insets: high magnification SEM micrographs)

Linear range

LOD

10−12 M 3 μg L−1 (≅1 10−8 M)

10−13–10−8 M 5–50μg L−1

Amperometry at +0.1 V

Amperometry at +0.2 V

Electrochemical detection mode

4 °C 4 °C 4 °C – RT 4 °C 4 °C

15 min 30 min 15 min 20 min – –

4 °C

4 °C

15 min

20 min

10 min

[14] [25] This work

[13]

[24]

[23]

[22]

References

– [32] During one-month test the current signals [33] still remained 70 % of initial response

Around 60 % f the initial activity after 1 week in buffer The response was stable for 7 days – At least 80 days in dry condition

At least 3 months

97 % of the enzyme activity over 4 week storage in 0.01 M PBS –

Incubation Storage stability time Temperature Time

GC glass carbon electrode, GO graphene oxide, NTA nitriloacetic acid, AChE acetylcholinesterase, PBS phosphate buffer solution, SPE screen-printed electrode, SWCNTs single wall carbon nanotubes, CoPc Cobalt-Phtalocyanine, Cyst cysteamine, Glut glutaraldehyde, MWCNTs multiwall carbon-nanotubes, PtSPE SPE with platinum as working electrode, CBNPs carbon black nanoparticles, Nf Nafion, BSA, allbumine bovine serum, BChE, butylcholinesterase, MC carbon mesoporous, OPH organophosphorus hydrolase

Chrono-amperometry at +0.05 V 2 μg L−1 (≅7 10−9 M) Amperometry at +0.3 V using SPE/Cyst-Glut-AChE 5–20 μg L−1 ferricyanide in solution Amperometry at +0.2 V SPE/MWCNTs-AChE Up to 6.9× 10−9 M 5 10−10 M PtSPE/AChE-gelatine 2.5–10 μg L−1 2.5 μg L−1 (≅9 10−9 M) Amperometry at +0.41 V −1 SPE/CBNPs-Glut-Nf-BSA-BChE 5–30 μg L 5 μg L−1 (≅2 10−8 M) Amperometry at +0.3 V Substrate biosensors (Paraoxon is the enzyme substrate) GC/MC-CBNPs-OPH Up to 8×10−6 M 1.2 10−7 M Amperometry at +0.9 V −6 −9 GC/MC-OPH-bacteria 0.05–25×10 M 9 10 M Amperometry at +0.84 V

SPE/Ni-NiO nanoparticles His6-tagged AChE SPE/SWCNTs-CoPc-AChE

Biosensors based on enzyme inhibition (Paraoxon is the enzyme inhibitor) GC/GO-NTA- His6-tagged AChE 10−9–10−5 M 6.5 10−10 M

Type of biosensor

Table 1 Different electrochemical biosensors for paraoxon detection

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completely and uniformly covered the SPE surface, being not possible, in fact, to observe the presence of the graphite platelets. Significant morphology differences of the CBNPs-SPE working electrode before and after enzyme immobilization were detected, revealing the BChE-CBNPs-SPE a superficial uniform film (Fig. e and f), testifying the occurred enzyme immobilization. Paraoxon determination Fig. 5 Photograph of the analysed samples

inhibition. The use of CBNPs allows i) to use low applied potential, ii) to easily prepare a stable dispersion for massproduced modified sensors (e.g. using BioDot automatized low volume dispensing equipment, www.biodot.com) and iii) to employ a cost effective nanomaterial. Furthermore, this miniaturized sensor can be adaptable for integration in microfluidic platform [33]. The biosensor here proposed can be competitive with the ones integrated in the microfluidic platforms reported in literature [37, 38] because it employs an easy drop casting CBNPs modification procedure. On the contrary, the use of electrochemical mediators, such as CoPc, requires necessarily its incorporation in the ink before the SPE printing. Paraoxon determination in waste water samples In order to check the suitability of the developed biosensor in real samples, firstly it was applied for sensing in drinking water samples collected in our Department. In this case no inhibition was observed, demonstrating the absence of insecticide at 10 μg L−1 level. In order to evaluate the accuracy of the biosensor, the sample was fortified with 50 μg L−1 of paraoxon and diluted 1:2 (v/v)in phosphate buffer, obtaining a recovery value of (96±2) % demonstrating the accuracy of this biosensor in a drinking water matrix. In order to challenge the biosensor in the waste water samples, three different

(b)

20

Days

78

50

36

1 8 15 22

0

78

40

50

60

b

36

80

60 50 40 30 20 10 0

1 8 15 22

a

100

Degree of inhibition (%)

(a) Residual activity (%)

Our goal was the insecticide detection in waste water sample, thus in a complex matrix. In order to fine tune an analytical system capable to work well in waste water samples, without sophisticated sample pre-treatment and interference problem, the “medium exchange method” proposed by us in an our previous work was adopted [34]. This method consists in three steps; briefly, in the first step the enzymatic activity was measured in buffer solution in presence of the only enzymatic substrate. After, in the second step, the biosensor was put in contact with the sample contaminated with insecticides for a selected time, followed by the several times rinsing of the biosensor with distilled water. In the last step, the enzymatic residual activity was finally determined in a new buffer aliquot in presence of the only enzymatic substrate. In this way, it was possible to avoid electrochemical interferences such as ascorbic acid, phenolic compounds, etc., since the enzymatic activity was always quantified in phosphate buffer in absence of any electroactive interfering species. Furthermore, washing the biosensor with distillate water after the inhibition step, only irreversible inhibitors (e.g. organophosphate) able to link the enzyme by covalent bonk can be detected. In fact, the other types of inhibitor that could be present in waste water samples such as Cd2+, Cu2+, Fe3+ (reversible inhibitors) were avoided. In order to obtain a sensitive measurement, the incubation time was optimized. The degree of inhibition increases with the incubation time, due to the fact that the cholinesterase inhibition by organophosphate is an irreversible inhibition. In our case we chose 20 min for the incubation time as compromise between a sensitive measurement and no tedious analysis time (data not shown). Under optimized parameters, the biosensor was challenged with paraoxon, obtaining a linear range up to 30 μg L−1 with a calibration curve described by the following equation: y=(2.1±0.1)×+(3.6±1.8), R2 = 0.971. 5 μg L−1 was the detection limit, calculated as the amount of analyte that gives 10 % of inhibition. The developed biosensor allows the paraoxon detection at ppb levels and at low applied potential when compared with biosensors developed using carbon black and/or mesoporous carbon and organophosphorus hydrolase enzyme [35, 36] (Table 1). These results demonstrate for the first time the possibility to apply the CBNPs in biosensors based on butyrylcholinesterase

Days

Fig. 6 Storage stability evaluated by the residual activity a and by the degree of inhibition b using paraoxon 20 μg L−1. Applied potential: + 300 mV vs Ag/AgCl, 0.05 M phosphate buffer+0.1 M KCl, pH 7.4, 5 mM butyrylthiocholine as substrate, 20 min as incubation time (n=3)

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samples were analysed (Fig. 5); two of them were collected from a paint industry (sample A and C) and the other one (sample B) from a catalyst industry. In each case no inhibition was observed, thus the samples were fortified with 50 μg L−1 of paraoxon, a level lower than the legal limit of 100 μg L−1 for surface waste waters [39]. For the samples A, B and C the recovery values were (88±6) %, (96±9) % and (76±9) %, respectively, confirming the satisfactory accuracy of the biosensor and the capability to work even in a more complex matrix. Storage stability of biosensor and enzymatic substrate The storage stability is a key point for the commercialization of biosensors, probably the reason of the relevant gap between the research sector and the market. In order to test the practicability of the developed biosensor, the storage stability was tested. When the biosensor was not in use, it was stored at RT in dry conditions. The CBNPs-SPE sensor is stable at RT in dry conditions for at least 100 days, as demonstrated in our previous work [17]. In the case of biosensors, usually the low storage stability is mainly due to the biocomponent; however, by using the enzymatic membrane here described, the shelf life at RT in dry conditions is rather satisfactory (Fig. 6a), highlighting that the storage condition at 4 °C is not necessary, as in many papers present in the Literature [14, 26–31], and as highlighted in the Table 1. Until now, we have observed the same degree of inhibition for biosensors stored at RT in dry conditions within 78 days, with RSD% not higher of 6 % for all the analyses of inhibition performed (Fig. 6b). Also we want to underline that the inhibition measurements shown in Fig. 6b, did not belong to the same electrode, but to several biosensors stored at RT and tested at different times. Each electrode is used for just one inhibition measurement. Indeed, after each inhibition experiment, the biosensor lost about 50 % of its original activity. After few experiments done with the same electrode, the activity falls to zero, thus a single use of such biosensor is highly recommended. Concerning the substrate, it is better to maintain at 4 °C the butyrylthiocholine solution rather than the butyrylthiocholine powder, because this latter is highly hygroscopic. At 4 °C it is possible to maintain a 5 mM solution of the enzymatic substrate in working buffer for no more than 2 weeks; on the contrary, the powder should be stocked in Eppendorf vials in weighting amounts after flowing nitrogen for 1 min, to attain a storage stability for at least of 6 months at RT [32].

Conclusions We have demonstrated that the use of BChE cross-linked on SPE modified with carbon black nanoparticles showed a

F. Arduini et al.

detection limit of 5 μg L−1 with good storage stability at RT in dry conditions, making the so assembled biosensor competitive with the ones reported in Literature. The use of carbon black nanoparticles avoids the fouling problem during the thiocholine electrochemical detection, thanks to the high number of defect sites present in carbon black nanoparticles. The proposed biosensor may be very useful as alarm system in the case of neurotoxic agents (i.e. organophosphate, carbamate). Furthermore, the disposability of this biosensor is extremely useful in the case of organophosphorous insecticides quantification since the reactivation of the enzyme after its irreversible inhibition requires incubation time with specific reactivator. The developed biosensor based on carbon black nanoparticles and BChE inhibition allowed in situ paraoxon detection characterised by low detection limit and/or absence of electrochemical interferences due to the use of low applied potential and the “medium exchange method” even in a complex matrix like the waste water samples. Acknowledgments This work was supported by National Industria 2015 (MI01_00223) ACQUA-SENSE project and Marie Curie FP7PEOPLE-2011-IRSES, 294901 “Peptide Nanosensors”. The authors thank Prof. F. Cataldo (Actinium Chemical Research srl) for the CBNPs samples, Tover Italia s.r.l. (Rome) and BASF Italia Divisione Catalizzatori (Rome) for the waste water samples.

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