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(N-cyclohexy-N-(2-morpholinoethyl)carbodiimide)methyl-p-

toluenesulphonate by Pedrosa et al. [21,22]. An AChE biosensor

was also constructed by means of gold nanoparticles and cys-

teamine assembledon glassy carbonpaste [23] or by singlewalled

carbon nanotubes wrapped by thiol terminated single strand

oligonucleotide (ssDNA) on gold [24].

In all these cases, however, keeping in mind that the

organophosphorus insecticides are able to irreversibly inhibit

the AChE, after each measurement the biosensor need to be

re-preparedorreactivated.TheAChE biosensor, in fact, canbe reac-

tivated putting in contact the biosensor, after the measurement,

for several minutes with 2-PAM (pyridine-2-aldoxime methylio-

dide) solution [25] or, for example, obidoxime solution [26]. The

reactivation should be performed immediately after the mea-

surement, in order to avoid the enzyme phenomenon called

“ageing” that renders the inhibited enzyme more resistant to the

reactivationbecomingpermanentlyinhibited[27]. Inordertoavoid

these steps with drawbacks in term of time of analysis, the use of 

screenprinted electrodes (SPEs) canbe a successfully alternative.

ThegoldSPEs have theadvantagesto beminiaturized,mass pro-

duced, and cost-effective, thus suitable for a single measurement,

properties very useful in the case of irreversible inhibition based

biosensors. In the present work we report the development of a

novelamperometricmono-enzymatic AChEbiosensor inwhichthe

enzyme is immobilized via glutaraldehyde on a preformed SAM

of cysteamine onto Au-SPE. In order to have a mono-enzymatic

biosensor, the acetylthiocholine was used as substrate. The enzy-

matic product thiocholine was detected by using ferricyanide in

solution as electrochemicalmediator.

2. Materials and methods

 2.1. Apparatus

Amperometric measurements were carried out using a VA

641amperometric detector (Metrohm,Herisau, Switzerland), con-

nected to an X-t recorder (L250E, Linseis, Selb, Germany).Cyclic voltammetry (CV) was performed using an Autolab

electrochemical system (Eco Chemie, Utrecht, The Netherlands)

equippedwith PGSTAT-12andGPESsoftware (EcoChemie,Utrecht,

The Netherlands). Electrochemical impedance spectroscopy (EIS)

measurements were carried out in the same cell with a PC-

controlled Autolab. A sinusoidal voltage perturbation of 10mV

amplitude was applied over the frequency range of 100kHz to

0.1Hzwith 10measurement points per frequency decade. For the

fitting of thedataobtainedbyEIS, Z-views software (ScribnerAsso-

ciates, Inc.) was used.

 2.2. Electrodes

Au-SPEs were bought from Ecobioservice (Florence, Italy). Thediameterof theworkingelectrodewas0.3cmresultingin anappar-

ent geometric area of 0.07cm2. Before thiol measurements, the

referenceelectrodewas chlorinatedbyapplyinga potentialof 0.6V

between the silver and an external Ag/AgCl electrode for 20s in a

phosphate buffer solution in the presence of 0.1M KCl [28].

 2.3. Reagents

Allchemicals fromcommercial sourceswereof analyticalgrade.

Potassium ferricyanide from Carlo Erba (Milano, Italy), acetyl-

cholinesterase (AChE) fromelectric eel, acetylthiocholine chloride,

cysteamine, glutaraldehyde and paraoxon were purchased from

Sigma Chemical Company (St. Louis, USA).

 2.4. Thiocholinemeasurements

Thiocholine was produced enzymatically by AChE using

acetylthiocholine as substrate(because thiocholineisnot commer-

cially available). For this purpose, 1mL of 1M acetylthiocholine

solution was prepared in phosphate buffer 0.1M (pH= 8), and

100 units of AChE were added to this solution. After 1h, the

concentration of thiocholine produced by AChE was estimated

spectrophotometrically by Ellman’s method. For this purpose,

900L of phosphate buffer solution (0.1M, pH= 8), 100L of 

0.1MDTNB, and 5L thiocholine solution (diluted 1:100 inwater)

were put in a spectrophotometric cells. The absorbance was mea-

sured, and the real concentration was evaluated by using the

Lambert–Beer law with the known molar extinction coefficient

of TNB (ε=13,600M−1 cm−1) [29]. After 1h, the acetylthiocholine

hydrolysis is completed, and 1mL solution of 1M thiocholine is

obtained. The solution is stable for 1 day at 4 ◦C.

Thiocholinemeasurementswere carried out usingamperomet-

ric batch analysis at Au-SPE in a stirred 0.05M phosphate buffer

solution+ 0.1MKCl,pH7.4 (10mL)containing1 mMof ferricyanide

ions at an applied potential of +400mV vs. Ag/AgCl. After around

7min, time required for baseline stabilization, the thiocholinewas

added and the responsewas recorded.

 2.5. AChE biosensor 

The Au-SPE, as received by Ecobioservice, was immersed in

100mM cysteamine aqueous solution for 15h at room temper-

ature in darkness to form cysteamine monolayer. Then, it was

thoroughlywashedwithdoubledistilledwater toremovethephys-

icallyadsorbedcysteamine.Afterthat,the sensorwas coveredwith

an aqueous solution of glutaraldehyde 5% (v/v) for 30min, then,

it was thoroughly washed with double distilled water to remove

the unreacted glutaraldehyde. Finally the resulting electrode was

incubated with AChE solution (10U/mL) for 15h; after that, the

biosensor waswashed and maintained in phosphate buffer at 4 ◦C

(Scheme 1).

 2.6. Acetylthiocholine measurement 

The acetylthiocholine was measured using AChE biosensor at

an applied potential of +400mVvs. Ag/AgCl in stirred 0.05M phos-

phate buffer solution+ 0.1M KCl, pH 7.4 (10mL) containing 1mM

of ferricyanide ions. When a stable baseline current was reached,

the acetylthiocholine was added and the analyte response at the

steady statewas recorded.

 2.7. Inhibitionmeasurement using AChE biosensors

Paraoxon in aqueous solutionswasused as standard insecticidefor the AChE inhibition measurements. The enzymatic activity of 

the biosensor was measured before (i0) and after (ii) its exposure

to paraoxon for a certain time (incubation time) at 3mM sub-

strate (acetylthiocholine) concentration.After the incubationtime,

the biosensor was rinsed three times with distilled water and the

response towards the substratewasmeasured as described above.

The degree of inhibition was calculated as a relative decay of the

biosensor response.

I % =i0 − iii0

× 100 (1)

where i0  and ii represent the biosensor response before and after

the incubation procedure, respectively.

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hange_reaction?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/20394016_Tacrine_slows_the_rate_of_aging_of_sarin-inhibited_acetylcholinesterase?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/51195872_Acetylcholinesterase_biosensor_based_on_single-walled_carbon_nanotubes-Co_phtalocyanine_for_organophosphorus_pesticides_detection_Talanta?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==

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F.Arduini et al. / Sensors and Actuators B 179 (2013) 201–208 203

Scheme 1. The schemeof theassemblingof theAChE biosensor.

 2.8. Sample collection

Thedrinkingwater samplewascollectedat TorVergata Univer-

sity Laboratory, the Sacco river water sample was collected near

Ceccano;realsampleswere diluted1:2with0.1Mphosphatebuffersolution+ 0.2M KCl, pH 7.4 and directly analysed.

3. Results and discussion

 3.1. Amperometric thiocholine detection at Au-SPE by means of 

 ferricyanide

Themono-enzymaticAChEbiosensoruses theacetylthiocholine

as substrate, thus the first goal was the development of a highly

sensitive enzymatic product thiocholine (RSH) sensor. The major

drawback relative to the electrochemical detection of thiocholine

is the high overpotential required at most conventional electrode

surfaces (gold, platinum and carbon paste) and the fouling of theworking electrode surface. To overcome these problems, the elec-

trochemical detection of thiocholine could be performed using

nanostructured materials or redox mediators [30–35]. In the last

case, the electrochemical mediator can be adsorbed on the work-

ingelectrode surface such as in thecaseofPrussianBlue [33], cobalt

hexacyanoferrate [34] or itcanbedirectly mixed tothe ink usedto

print the working electrode, such as in the case of cobalt phthalo-

cyanine (CoPc) [35]. In the case of Au-SPEs modified with SAM,

our choice was to use the electrochemical mediator ferricyanide

in solution because it is able to electrocatalyse the oxidation of 

thiocholine [36].

The electrochemical behaviour of ferricyanide towards thio-

choline oxidationwasfirstlyinvestigatedusinga CV techniqueand

a Au-SPE. Before the thiocholine measurement, the Au-SPE wasconditioned by means of 30 CVs using ferricyanide 10mM pre-

pared in0.05M phosphate bufferplus0.1MKCl, pH7.4 atscanrate

of 50mV/s. This condition stepwas necessary in order to decrease

thepeak topeak separation from314mV to93mVafter30 scans as

wecansee inFig. 1, thisbehaviourcanbeascribed to theenhanced

kinetic rate after electrochemical pretreatment as reported in lit-

erature using carbon SPE [37]. Next, we have investigated the

response of the so conditioned Au-SPE towards ferricyanide by CV

over a scan rangeof0.010–0.200V/s. Thecurrentofboth anodicand

cathodic peaks increases linearly with the square root of the scan

rate (data not shown), indicating a semi-infinite linear diffusion-

controlled current. After that, the Au-SPE was studied to evaluate

the electrocatalytic effect of ferricyanide towards the thiocholine

oxidation.

Fig. 1. CVsof Au-SPE in 10mM ferricyanide in 0.05M phosphate buffer+ 0.1M KCl,

pH=7.4,scanrate50mV/s (continuousline= first scan,longdashed line =10thscan,short dashed line= 20th scan anddotted line =30th scan).

Fig. 2 shows a typical response of  ik   (kinetically controlled

current) obtained in presence of thiocholine and id (diffusion con-

trolled current) obtained in absence of thiocholine. The generic

reaction can be describedby the following equations:

ferricyanide+ RSH→ ferrocyanide+ RSSR (2)

Fig. 2. CVs of ferricyanide 4mM in 0.05M phosphate buffer+ 0.1M KCl, pH=7.4,

scanrate20mV/sin absence(continuousline)and inpresenceof thiocholine10mM

(dashed line) or 20mM (dotted line).

https://www.researchgate.net/publication/227689072_A_Disposable_Biosensor_for_Organophosphorus_Nerve_Agents_Based_on_Carbon_Nanotubes_Modified_Thick_Film_Strip_Electrode?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/239149629_Electrocatalytic_oxidation_of_thiocholine_at_chemically_modified_cobalt_hexacyanoferrate_screen-printed_electrodes_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/252134743_Voltammetric_and_amperometric_studies_of_thiocholine_at_a_screen-printed_carbon_electrode_chemically_modified_with_cobalt_phthalocyanine_Studies_towards_a_pesticide_sensor?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/223819315_Characterisation_of_Prussian_blue_modified_screen-printed_electrodes_for_thiol_detection_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/239149629_Electrocatalytic_oxidation_of_thiocholine_at_chemically_modified_cobalt_hexacyanoferrate_screen-printed_electrodes_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/252134743_Voltammetric_and_amperometric_studies_of_thiocholine_at_a_screen-printed_carbon_electrode_chemically_modified_with_cobalt_phthalocyanine_Studies_towards_a_pesticide_sensor?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/12111125_A_micro_flow_injection_electrochemical_biosensor_for_organophosphorous_pesticides_Biosen_Bioelectron?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/251697388_Electrochemically_pretreated_screen-printed_carbon_electrodes_for_the_simultaneous_determination_of_aminophenol_isomers?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/227689072_A_Disposable_Biosensor_for_Organophosphorus_Nerve_Agents_Based_on_Carbon_Nanotubes_Modified_Thick_Film_Strip_Electrode?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/252134743_Voltammetric_and_amperometric_studies_of_thiocholine_at_a_screen-printed_carbon_electrode_chemically_modified_with_cobalt_phthalocyanine_Studies_towards_a_pesticide_sensor?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/252134743_Voltammetric_and_amperometric_studies_of_thiocholine_at_a_screen-printed_carbon_electrode_chemically_modified_with_cobalt_phthalocyanine_Studies_towards_a_pesticide_sensor?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/251697388_Electrochemically_pretreated_screen-printed_carbon_electrodes_for_the_simultaneous_determination_of_aminophenol_isomers?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/12111125_A_micro_flow_injection_electrochemical_biosensor_for_organophosphorous_pesticides_Biosen_Bioelectron?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/239149629_Electrocatalytic_oxidation_of_thiocholine_at_chemically_modified_cobalt_hexacyanoferrate_screen-printed_electrodes_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/239149629_Electrocatalytic_oxidation_of_thiocholine_at_chemically_modified_cobalt_hexacyanoferrate_screen-printed_electrodes_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/223819315_Characterisation_of_Prussian_blue_modified_screen-printed_electrodes_for_thiol_detection_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/223819315_Characterisation_of_Prussian_blue_modified_screen-printed_electrodes_for_thiol_detection_J_Electroanal_Chem?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/229201301_Hg_detection_by_measuring_thiol_groups_with_a_highly_sensitive_screen-printed_electrode_modified_with_a_nanostructure_carbon_black_film?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/223433636_Sensitive_electrochemical_detection_of_enzymatically_generated_thiocholine_at_carbon_nanotube_modified_glassy_carbon_electrode?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==

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ferrocyanideelectrode←→ ferricyanide (3)

According to the above equations, the addition of thiocholine

causesan increase inconcentration of theferrocyanide, resultingin

anincreaseofthe anodicpeakcurrent.On thecontrary,the cathodic

peak current is proportional to the amount of ferricyanide that

decreases after the addition of RSH (thiocholine). Moreover, the

increase of thiocholine concentration leads to a further increase

of the anodic peak current and a decrease of the cathodic peak,confirming theproperty of ferricyanide as electrocatalyst for thio-

choline oxidation.

In order to calculate the second-order homogeneous rate con-

stant ks for the reaction between ferricyanide and thiocholine, the

theory of stationary electrode voltammetry developed by Nichol-

son and Shain [38] was used. Calculation of ks has been performed

by varying scan rates in the range of 5–100mV/s and thiocholine

concentration in the range of 10–20mM, fixing the concentration

of ferricyanide at 4mM. The reaction scheme can be described as:

ferrocyanide− e−→ ferricyanide (4)

thiocholine+ ferricyanide  kf ←→ferrocyanide (5)

where kf  is the pseudo-first order rate constant. Using the experi-mental values of ik/id  a value of (kf RT /nFv) can be obtained from

the working curve reported in Nicholson and Shain paper [38].

Plotting (kf RT /nFv) vs. 1/v  at each concentration of thiocholine, it

is possible to calculate kf . Then, a plot of  kf  vs. thiocholine con-

centration was carried out obtaining a linear behaviour whose

slope was equal to the second-order homogenous rate constant

ks  for reaction between ferricyanide and thiocholine. It was found

equal to (5.26±0.65)×104M−1 s−1, demonstrating the good elec-

tron transfer between thiocholine as thiol and ferricyanide [39].

This value is,for example,higherthantheone found fortheelectro-

catalyticreductionofnitriteusingferricyanide(2.75×103M−1 s−1)

[40].

 3.2. Amperometric thiocholine detection at Au-SPEmodified bymeans of ferricyanide

 3.2.1. Choice of applied potentialA plot of current/applied potential using a thiocholine and fer-

ricyanide concentration of 3×10−4M and 1mMrespectively [36],

wasconstructed in therange of theapplied potential0–800mV vs.

Ag/AgCl (datanot shown). As expected,the amperometricresponse

followed the behaviour of the oxidation current in the CV. The oxi-

dation current increased from0 to +100mV vs. Ag/AgCl reaching a

plateau at around+200mVvs. Ag/AgCl. However, we observed the

highest ratio signal/noise at +400mVvs. Ag/AgCl, thus this applied

potential was selected for the rest of work.

 3.2.2. Analytical features of thiocholinemeasurement The good electroanalytical performances of the developed sys-

tem were then confirmed by performing amperometric batch

measurements. In fact, ourpurpose was thedevelopment of a sen-

sor for thiocholine detection as a platform for an amperometric

biosensor for insecticide detection.

Using the previous selected applied potential (+400mV vs.

Ag/AgCl) and a ferricyanide concentration equal to 1mM [36],

calibration curves were carried out obtaining a detection limit

(S/N = 3) of 3×10−6M together with a linear range up to

1×10−3M (R2 =0.9964). The sensor showed also a sensitivity

equal to 113mAM−1 cm−2, which is higher than the one shown

by the SPE modified with CoPc (24mAM−1 cm−2) and compara-

ble with that obtained in the case of SPE modified with Prussian

Blue (143mAM−1

cm−2

) [13]. Moreover, the reproducibility was

Fig. 3. CVs of ferricyanide 4mM in 0.05M phosphate buffer+ KCl 0.1M, pH=7.4,

scan rate20mV/susinga baregoldSPE(continuousline) anda cysteaminemodified

gold SPE (dashed line).

evaluated by studying the response of 5×10−5M thiocholine,

founding a RSD% equal to 5% (n=4).

 3.3. Ferricyanide behaviour at Au-SPEmodifiedwith a SAM of 

cysteamine

Ourgoalwasthedevelopmentofbiosensorfor insecticidedetec-

tion immobilizing the AChE by SAM. In this case, cysteamine was

selected as alkanethiol taking in consideration thatglutaraldehyde

will be used to link the AChE to cysteamine. Firstly, the effect

of cysteamine coverage of Au-SPE on ferricyanide response was

investigated, because the electron transfer for redox reactions of 

different molecules at SAM can be controlled by electrostatic and

hydrophobic effects [41].

ACVstudywasperformedusing ferricyanideat bareAu-SPEand

Au-SPE modified with a SAM of cysteamine, prepared by dipping

the Au-SPE in a 100mM cysteamine solution overnight (Cyst–Au-

SPE).Weobserved that in the caseof bareAu-SPE, the peak topeak

separation was 314mV; in the case of cysteamine modified Au-

SPE, instead, it was 89mV (Fig. 3); it seems that the presence of 

cysteamine on thesurface of theworking electrodecan improve its

electrochemicalperformance. Thisbehaviour shouldbe ascribed to

the electrostatic interaction between cysteamine and ferricyanide.

The pK a of the cysteamine immobilized on the gold electrode was

estimated by Shervedani et al. to be equal to 7.6, thus in the pH

region lower than 7.6, the Au surface is positively charged [42].

Inour case, weworkedat pH= 7.4, thus the surface of goldelec-

trode modified by SAM of cysteamine should be characterized by

positive charge, reason for that probably the CV of ferricyanide,which is negatively charged, is characterized by a peak to peak

separation lower than at the bare Au-SPE.

In conclusion, in our case the cysteaminewill be used to immo-

bilize the AChE by cross-linking with glutaraldehyde, butwe have

also demonstratedthat a SAMof cysteamine canfacilitate theelec-

trontransferof ferricyanideatworkingelectrodesurfaceofAu-SPE.

 3.4. AChE biosensor 

 3.4.1. Optimization of bioactive layer 

Inorder todevelop a sensitiveAChE biosensor, theAChEandthe

cysteamine concentrations and the deposition time of cysteamine

on the gold electrode surface were investigated andoptimized.

https://www.researchgate.net/publication/277548424_Theory_of_Stationary_Electrode_Polarography_Single_Scan_and_Cyclic_Methods_Applied_to_Reversible_Irreversible_and_Kinetic_Systems?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/277548424_Theory_of_Stationary_Electrode_Polarography_Single_Scan_and_Cyclic_Methods_Applied_to_Reversible_Irreversible_and_Kinetic_Systems?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/230527447_The_Oxidation_of_Cysteine_by_Aqueous_Ferricyanide_A_Kinetic_Study_Using_Boron_Doped_Diamond_Electrode_Voltammetry?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/12111125_A_micro_flow_injection_electrochemical_biosensor_for_organophosphorous_pesticides_Biosen_Bioelectron?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/12111125_A_micro_flow_injection_electrochemical_biosensor_for_organophosphorous_pesticides_Biosen_Bioelectron?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/229169780_Studies_on_the_electrochemical_behavior_of_a_cystine_self-assembled_monolayer_modified_electrode_using_ferrocyanide_as_a_probe?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/245083892_Determination_of_dopamine_in_the_presence_of_high_concentration_of_ascorbic_acid_by_using_gold_cysteamine_self-assembled_monolayers_as_a_nanosensor?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/245083892_Determination_of_dopamine_in_the_presence_of_high_concentration_of_ascorbic_acid_by_using_gold_cysteamine_self-assembled_monolayers_as_a_nanosensor?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/230527447_The_Oxidation_of_Cysteine_by_Aqueous_Ferricyanide_A_Kinetic_Study_Using_Boron_Doped_Diamond_Electrode_Voltammetry?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/229169780_Studies_on_the_electrochemical_behavior_of_a_cystine_self-assembled_monolayer_modified_electrode_using_ferrocyanide_as_a_probe?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/277548424_Theory_of_Stationary_Electrode_Polarography_Single_Scan_and_Cyclic_Methods_Applied_to_Reversible_Irreversible_and_Kinetic_Systems?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/277548424_Theory_of_Stationary_Electrode_Polarography_Single_Scan_and_Cyclic_Methods_Applied_to_Reversible_Irreversible_and_Kinetic_Systems?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/12111125_A_micro_flow_injection_electrochemical_biosensor_for_organophosphorous_pesticides_Biosen_Bioelectron?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/12111125_A_micro_flow_injection_electrochemical_biosensor_for_organophosphorous_pesticides_Biosen_Bioelectron?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==

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F.Arduini et al. / Sensors and Actuators B 179 (2013) 201–208 205

 3.4.2. Effect of AChE concentration

The effect of AChE concentration on the biosensor response

was investigated. The AChE concentration was varied in a range

comprised between 0.1 and 10U/mL using a SAM prepared with

cysteamine 0.1mMand a deposition timeof 15h, and a glutaralde-

hyde solutionat5%(v/v). Theresponseof thebiosensorswastested

using acetylthiocholine 3×10−4M. We found that in the case of 

AChE 0.1U/mL, 1U/mL and 10U/mL, a current equal to 0, 116 and

209nA was observed, respectively. The results showed that the

responseof thebiosensorincreasedwiththeincreaseof theenzyme

amount as expected, but also the working stability (successively

measured) increased with the increase of the enzyme amount.

Keeping inmind that: (i) theAChE inhibition byorganophosphorus

insecticides is a irreversible, the lower possible amount of enzyme

should be used [6] and (ii) an enzyme loading lower than 10U/mL 

does not allow a satisfactory working stability, thus an enzyme

amount of 10U/mL was finally selected for further work.

 3.4.3. Effect of cysteamine concentration and deposition time on

 gold electrode surface

The density of hydrocarbon chains on gold electrode surface

wasdueto theconcentration of alkanethiolsused andtheduration

of SAM deposition. For the investigation of SAM time deposition,

Au-SPE was immersed for 1 and 15h in cysteamine solution at

concentration of 0.1mM, using AChE at concentration of 10U/mL 

and glutaraldehyde at 5% (v/v). The response of the biosensors

was tested usingacetylthiocholine 3×10−4M.Wehave observed a

response towards the substrate only in the case of biosensor prea-

pared with 15h as deposition time, thus this time was selected

for further experiments. In order to evaluate the effect of cys-

teamine concentration, the Au-SPE was immersed in cysteamine

solution at different concentrations: 0.1, 1, 10 and 100mM, using

AChE at concentration of 10U/mL and glutaraldehyde at 5% (v/v).

The response of the biosensors was tested using acetylthiocholine

3×10−4M.Thebiosensorspreparedusing cysteamine100mMhad

thehighestresponse (around 200nA) andthehighest working sta-

bility. Forelectrodes immersed in cysteamine 0.1, 1 and10mM the

biosensors allowed results characterized by lowworking stability.Looking at these results, we selected asmost suitable biosensor in

terms of working stability, reproducibility and sensitivity towards

acetythiocholine, theonedevelopedusing SAMpreparedwith cys-

teamine 100mM, a deposition time of 15h, AChE at 10U/mL and

glutaraldehyde at 5% (v/v). This biosensor was characterized by a

reproducibility (RSD%) intra- and inter-electrode of 2.3% and 16%,

respectively. The high value of RSD% in the case of inter-electrode

reproducibility does not affect the accuracy of insecticide mea-

surements because they were carried outmeasuring the response

before and after the exposure to the organophosphate, thus mea-

suringtherelativedecayofenzymaticactivityof a singlebiosensor.

 3.5. Electrochemical impedance spectroscopymeasurements

Electrochemical impedance spectroscopy can provide useful

information on the impedance changes of the electrode surface

during the fabrication process of biosensors, giving information

about how the interfacial region of the gold electrode surface in

presence of SAM of cysteamine changes before and after the AChE

immobilization,measuringthevalueof electrontransfer resistance

(Rct). The Rct, estimatedaccording to thediameter of thesemicircle

present at the high frequency region, represents, in fact, the diffi-

cultyof electrontransferof ferro/ferricyanideredoxprobebetween

the solution and the electrode. Fig. 4 shows the typical Nyquist

plot obtained for cysteamine modified Au-SPE (Cyst–Au-SPE),

glutaraldehyde–Cyst–Au-SPE and AChE–glutaraldehyde–Cyst–Au-

SPE (biosensor). In this figure the Nyquist plot of the bare gold

SPE was omitted because characterized by a much higher Rct

Fig.4. Complexplane impedanceplotsat opencircuitpotentialfor Cyst–Au-SPE(a),

glutaraldehyde–Cyst–Au-SPE (b) and AChE-glutaraldehyde–Cyst–Au-SPE (biosen-

sor) (c) using 5mM ferro/ferricyanide in 0.1M KCl. Inset: Randles circuit.

(72,187±315) than the one obtained in the case of Cyst–Au-SPE(407±9), confirming the data obtained using the CV technique

that showed a better electron transfer of ferro/ferricyanide at

Cyst–Au-SPE than the at bare Au-SPE.

The fabrication process of biosensors was followed mea-

suring the Rct   values, observing that the Rct   increases in

the following order: Rct   AChE–glutaraldehyde–Cyst–Au-SPE

(2431±30) >glutaraldehyde–Cyst–Au-SPE (681±9) >RctCyst–Au-SPE (407±9), confirming also the deposition of a

glutaraldehyde layer andof theAChE enzymeon theCyst–Au-SPE.

 3.6. AChE biosensor for insecticide detection

The optimized biosensor was challenged with the enzy-

matic substrate acetylthiocholine. Fig. 5 shows the calibration

curve obtained for different substrate concentrations described

by Michaelis Menten equation. It was possible to calculate the

apparent Michaelis Menten constant (K Mapp), found equal to

1.1±0.2mM. A substrate concentration of 3.0mMwas chosen for

the inhibitionmeasurements.

Fig. 5. Calibration plot of acetylthiocholine chloride using the AChE biosensor.

Applied potential: +400mV vs. Ag/AgCl. 0.05M phosphate buffer+ 0.1M KCl, pH

7.4+ 1mM ferricyanide.

https://www.researchgate.net/publication/225630189_Biosensors_based_on_cholinesterase_inhibition_for_insecticides_nerve_agents_and_aflatoxin_B1_detection_review_Microchim_Acta?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==https://www.researchgate.net/publication/225630189_Biosensors_based_on_cholinesterase_inhibition_for_insecticides_nerve_agents_and_aflatoxin_B1_detection_review_Microchim_Acta?el=1_x_8&enrichId=rgreq-2813ba8b-9c38-424f-abd5-a616bef312e4&enrichSource=Y292ZXJQYWdlOzI1NzM1NDY5ODtBUzoxNjQ5ODg4ODI3OTI0NDhAMTQxNjM0NzgyMzM1Mw==

8/16/2019 Sensor and Actuators

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8/16/2019 Sensor and Actuators

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Biographies

FabianaArduini isa researcherin analytical chemistryat theChemical andTechnol-ogyDepartmentof theUniversityof Rome“TorVergata”. Shegraduated“cumlaude”in chemistry (2004) andobtained her Ph.D. degreein chemical science (2007). Herresearch interests include the development of electrochemical sensors andbiosen-sors and their application in thefield of clinical,food and environmental analyticalchemistry. Shewas involvedin severalNational andEuropeanprojects. Sheis authorof more than 30 papers in international andnational scientific journals and booksand of more than 50 poster and oral presentations at National and InternationalCongresses.

Simone Guidone is graduated in chemistry at University of Rome “Tor Vergata” in2010with the Thesis: development of biosensors for pesticidedetection.

 AzizAmine is a professor ofbiochemistry in theFacultyof Sciences and Techniquesof the University of HassanII-Mohammedia (Morocco). He received Ph.D. degree

from the Free University of Brussels in 1993. Professor Amine’s research over thelast20 years hasfocusedon sensorsand biosensorsandtheirusein analytical chem-istry. He is author of more than 100 papers and scientific contributions and hasserved ascoordinator of severalnationaland international researchprojects. He is areviewerfor several scientificinternational journals. Hewasco-ordinatorof variousco-operationprojects.

Giuseppe Palleschi, full professor of analytical chemistry, hasbeenthe Head of theDepartment of Chemical Science and Technology of the University of Rome “TorVergata” for 1995–2007. In 2000 he obtained the “Laurea Honoris Causa” from theUniversity of Bucharest. Prof. Palleschi’s research over the last 30 years has been

focusedon thedevelopmentof chemicalsensorsas wellas bio-and immunosensorsforuse inthe areas ofbiomedicine,foodandenvironmentalanalysis.Heis theauthorof more than 200papers in international scientific journals andan invited speakerin many International Congresses.

Danila Moscone is full professor in analytical chemistry at the Chemical andTechnology Department of the University of Rome “Tor Vergata”. She has beeninvolved in the field of biosensors for about 30 years, and is an expert in elec-trochemical biosensor and immunosensors assembling, their analytical evaluationand application for solving analytical problems. She is actually involved in Euro-pean projects and is responsible for National projects. Her scientific activity issummarized inmore than 150 papers on international andnationalscientific jour-nals and books, and in more than 300 oral and poster presentations at scientificmeetings.

All i f d li d i bl li k d bli i R hG l i d d h i di l