DNA-Based Sensor for the Detection of an Organophosphorus Pesticide: Profenofos

In this work, we propose an electrochemical DNA aptasensor for the detection of profenofos, an organophosphorus pesticide, based on a competitive format and disposable graphite screen-printed electrodes (GSPEs). A thiol-tethered DNA capture probe, which results to be complementary to the chosen aptamer sequence, was immobilised on gold nanoparticles/polyaniline composite film-modified electrodes (AuNPs/PANI/GSPE). Different profenofos solutions containing a fixed amount of the biotinylated DNA aptamer were dropped onto the realized aptasensors. The hybridisation reaction was measured using a streptavidin-alkaline phosphatase enzyme conjugate, which catalyses the hydrolysis of 1-naphthyl -phosphate. The 1-naphtol enzymatic product was detected by means of differential pulse voltammetry (DPV). The aptasensor showed itself to work as a signal off sensor, according to the competitive format used. A dose response curve was obtained between 0.10 μM and 10 μM with a detection limit of 0.27 μM.


Introduction
"Pesticide" is a term used in a broad sense to address any substance or mixture of substances used to limit the growth of infesting species (e.g., insects, weeds, little mammals, fungi, etc.) that can compromise agricultural production. As stated by the Food and Agriculture Organization (FAO), a pesticide is defined as substances intended to kill or control pests, but, for the present purposes, it also embraces certain materials used to modify the behaviour or physiology of pests (e.g., insect repellents and synergists) or of crops during production or storage (herbicide safeners, germination inhibitors) [1]. Pesticide residues may gain access to the food chain through air, water and soil, which is an outstanding issue to face, since most of the substances used as pesticides are constituted of neurotoxic compounds.
Organophosphorus pesticides (OPs) like phorate, profenofos, and isocarbophos are highly toxic substances that irreversibly inhibit the enzyme acetylcholinesterase, an essential enzyme for the central nervous system to function properly both in humans and insects. This inhibition causes the accumulation of the acetylcholine neurotransmitter in nerves, which affects muscular activities and the normal functioning of vital organs, causing severe symptoms and even death [2,3]. Thus, the according to the competitive format used. Finally, preliminary experiments were carried out with fruit juices.
This innovative method combines the portability of screen-printed electrochemical cells and of a computer-controlled instrument to ensure the possibility of a disposable and cost-effective in situ analysis.
The DNA sequences were purchased from Eurofins Genomics (Ebersberg, Germany) and are listed below:

Apparatus
UV absorption measurements were carried out through a Varian Cary 100 Bio UVspectrophotometer equipped with a 6 + 6 peltier thermostatable multicell holder and built-in temperature probes. The results were analysed with the Thermal application provided in the Cary 100 Bio software suite (Agilent, Cernusco sul Naviglio Milan, Italy). Secondary structures of both the DNA sequences were predicted through the Mfold algorithm [18].
Electrochemical measurements were carried out with a portable potentiostat/galvanostat PalmSens electrochemical analyser (PalmSensGA Houten, The Netherlands), and the results analysed by PSTrace 2.3 software. All the reported potentials refer to the pseudo-reference silver screen-printed electrode and all the measurements were carried out at room temperature.
The aptasensors were realised using screen-printed cells formed by graphite working electrode (3 mm diameter), a silver pseudo-reference electrode and a graphite counter electrode (GSPEs). The screen-printed cells were purchased from EcoBioServices (Florence, Italy).

DNA Melting Curve Studies
The hybridisation reaction between the DNA capture aptamer (apt-BIO) and the selected complementary sequence (oligo-SH) was assessed by recording melting curves. Melting temperatures were obtained as first-order derivative plot of absorbance versus temperature.
Herein, 100 µL of 1.0 µM oligonucleotide solutions in immobilisation buffer were placed into quartz microcuvettes (1 cm path length), and the temperature was increased from 25 • C to 95 • C at a constant rate of 1.0 • C/min directly inside them through the immersed probe into the sample solutions. At the same time, the absorbance at 260 nm was monitored at 1 nm spectral bandwidth. Immobilisation buffer was used as a blank solution. The entity of the interaction between the DNA aptamer and the target pesticide was also investigated in the same conditions.

GSPEs Surface Modification by Electrodeposition of Polyaniline and Gold Nanoparticles
The developed DNA aptasensor assay was based on a competitive approach, as reported in Figure 1. solutions. At the same time, the absorbance at 260 nm was monitored at 1 nm spectral bandwidth. Immobilisation buffer was used as a blank solution. The entity of the interaction between the DNA aptamer and the target pesticide was also investigated in the same conditions.

GSPEs Surface Modification by Electrodeposition of Polyaniline and Gold Nanoparticles
The developed DNA aptasensor assay was based on a competitive approach, as reported in Figure 1.  Polyaniline (PANI) and gold nanoparticles (AuNPs) modified GSPEs were realised in accordance with the optimised procedure reported in our previous works [10,19].
Briefly, electropolymerisation of aniline was performed through cyclic voltammetry (CV) by dropping 50 μL of 2.5 mM aniline solution in 50 mM HClO4 onto GSPEs. The potential was scanned from −400 mV to +800 mV for 10 cycles at 50 mV/s scan rate.
After washing with 50 μL 0.5 M H2SO4, AuNPs were deposited through CV by dropping 50 μL of 0.5 mM HAuCl4 solution in 0.5 M H2SO4 onto the polymeric layer. The potential was varied from −200 mV to +1200 mV at 100 mV/s for 15 cycles.
The modified GSPEs were then washed three times with 50 μL milli-Q water to remove excess monomer and free ions from the surface. Polyaniline (PANI) and gold nanoparticles (AuNPs) modified GSPEs were realised in accordance with the optimised procedure reported in our previous works [10,19].
Briefly, electropolymerisation of aniline was performed through cyclic voltammetry (CV) by dropping 50 µL of 2.5 mM aniline solution in 50 mM HClO 4 onto GSPEs. The potential was scanned from −400 mV to +800 mV for 10 cycles at 50 mV/s scan rate.
After washing with 50 µL 0.5 M H 2 SO 4 , AuNPs were deposited through CV by dropping 50 µL of 0.5 mM HAuCl 4 solution in 0.5 M H 2 SO 4 onto the polymeric layer. The potential was varied from −200 mV to +1200 mV at 100 mV/s for 15 cycles.
The modified GSPEs were then washed three times with 50 µL milli-Q water to remove excess monomer and free ions from the surface.

Electrochemical Characterisation of the Modified GSPEs
Each modification step of the developed platform was electrochemically characterised through CV by dropping 50 µL of 5.0 mM [Fe(CN) 6 ] 3−/4− redox probe (equimolar solution in 0.1 M KCl) onto the screen-printed cells (SPCs). CV measurements were performed in the potential range from −500 mV to +800 mV at 150 mV/s scan rate. After the electrochemical analysis, the SPCs were discarded.

DNA Probe Immobilisation
The nanostructured GSPEs were modified by self-assembly of a mixed monolayer of thiolated DNA capture probe (oligo-SH) and MCH [20]. The purified thiolated complementary sequence was subjected to a thermal treatment by heating it at 90 • C for 5 min and cooling it down to room temperature. Then, 7.0 µL of the 2.0 µM capture probe solution were then deposited onto the modified surface of the working electrode and chemisorption was allowed to proceed overnight (≈16 h). During this period, the sensors were stored in petri dishes at 4 • C to protect the solution from evaporation. To remove unbound oligonucleotide sequences, the surface was washed three times with 15 µL of immobilisation buffer. This immobilisation step was followed by the formation of a self-assembled monolayer (SAM) formation by incubation with 7.0 µL of 1.0 mM MCH aqueous solution for 60 min. Finally, the aptasensors were washed with 15.0 µL immobilisation buffer for three times.

Profenofos Detection
To obtain a dose-response calibration curve, profenofos detection was performed by dropping a solution containing a proper concentration of biotinylated DNA aptamer (apt-BIO) containing the target pesticide onto the sensor surface and by allowing the competitive reaction to proceed.
In particular, the affinity reaction between 0.50 µM biotinylated DNA aptamer and target pesticide in the concentration range 0-10.0 µM was first performed into solution; then, after 40 min, 7.0 µL of these solutions were incubated for other 30 min onto the sensor surface.
The aptasensors were then rinsed for three times with 15.0 µL detection buffer.

Enzymatic Labelling and Electrochemical Measurements
The biotinylated hybrids formed onto the developed aptasensor surface were further incubated with 15.0 µL of a solution containing 1.0 U/mL of streptavidin-alkaline phosphatase conjugate and 8.0 mg/mL of BSA in detection buffer. After 10 min, each sensor was washed three times with 100 µL detection buffer for two cycles of washings.
Then, after this labelling step, 50.0 µL of 1.0 mg/mL 1-naphthyl-phosphate solution in the detection buffer were placed onto the disposable aptasensors. After 20 min, the electroactive enzymatic product thus formed (1-naphthol) was detected by differential pulse voltammetry (DPV) by scanning the potential from 0 mV to 600 mV at 40 mV/s (5 mV step potential, 70 mV modulation amplitude) [21].
The current peak height was taken as the electrochemical signal. The signal is expressed in relative percentage units as S x /S 0 (e.g., ratio between measured signal to blank signal) and plotted versus profenofos concentration. The obtained curve exhibits the typical sigmoidal shape of a competitive assay and was fitted by using OriginPro 8.5 software (OriginLab) with a Boltzmann-type sigmoidal equation [22]: where A 1 is the y value at the top plateau at the curve, A 2 is the y value at the bottom plateau, x 0 is the x value at which y is halfway between bottom and top and dx is the slope of the linear part of the curve.

Studies on the Affinity of the DNA Aptamer for the Target Pesticide
The aptamer was selected from a library of aptamers, built by SELEX that shows the highest ability to bind profenofos among other organophosphorus pesticides. The constant of dissociation was estimated K d = 1 µM [17]. The selective binding of the analytes is strongly influenced by the secondary structures of the DNA sequences, since aptamers themselves are subjected to conformational changes that create many weak bonds, in order to capture the target molecule. The secondary structures thus formed were predicted by using the MFold algorithm and are shown in Figure 2.

Studies on the Affinity of the DNA Aptamer for the Target Pesticide
The aptamer was selected from a library of aptamers, built by SELEX that shows the highest ability to bind profenofos among other organophosphorus pesticides. The constant of dissociation was estimated Kd = 1 μM [17]. The selective binding of the analytes is strongly influenced by the secondary structures of the DNA sequences, since aptamers themselves are subjected to conformational changes that create many weak bonds, in order to capture the target molecule. The secondary structures thus formed were predicted by using the MFold algorithm and are shown in Figure 2. The prediction temperature was set at 25 °C, and the ionic strength was regulated according to the buffer solution used. The drawing mode was set to untangle with loop fix, while the other parameters were left as default settings. From the predicted conformations, apt-BIO prevalently shows a single-stranded structure characterised by three differently sized loops, while oligo-SH shows a single-loop structure in the same conditions because of its shorter length. For this reason, the hybridisation reaction between the two DNA strands will be more efficient compared to the one that would occur in presence of any other sequence with a more stable secondary structure.
The affinity of the receptor for the analyte and for the chosen complementary sequence was assessed by comparing these structures with preliminary studies on the melting temperatures of the aptamer alone and in presence of its complementary sequence or the pesticide ( Table 1).
The obtained Tm values for the biotinylated aptamer in presence of the thiolated capture probe or profenofos are close to the one related to the aptamer alone; moreover, the overlapping region of the two oligonucleotides is located on the medium-size loop. This suggests that also the interaction with profenofos takes place in the same region. The prediction temperature was set at 25 • C, and the ionic strength was regulated according to the buffer solution used. The drawing mode was set to untangle with loop fix, while the other parameters were left as default settings. From the predicted conformations, apt-BIO prevalently shows a single-stranded structure characterised by three differently sized loops, while oligo-SH shows a single-loop structure in the same conditions because of its shorter length. For this reason, the hybridisation reaction between the two DNA strands will be more efficient compared to the one that would occur in presence of any other sequence with a more stable secondary structure.
The affinity of the receptor for the analyte and for the chosen complementary sequence was assessed by comparing these structures with preliminary studies on the melting temperatures of the aptamer alone and in presence of its complementary sequence or the pesticide ( Table 1).
The obtained T m values for the biotinylated aptamer in presence of the thiolated capture probe or profenofos are close to the one related to the aptamer alone; moreover, the overlapping region of the two oligonucleotides is located on the medium-size loop. This suggests that also the interaction with profenofos takes place in the same region. The GSPEs were first modified with a layer of the conducting polymer polyaniline. Since conducting polymers show numerous features suitable for their application in sensing and biosensing (e.g., low cost, flexibility and biocompatibility), they have recently attracted a lot of attention in this field.
Here, the polymer is used to provide higher electroactive area (compared to the bare electrode), protection versus fouling of the electrode surface and scaffold for dispersing and anchoring the metal particles. Inclusion of AuNPs in conductive polymers can enhance electron transfer through a direct or mediated mechanism with improved conductivity and enhanced stability [14]. Further, the AuNPs are an excellent substrate for the immobilisation of thiolated bioreceptor.
The polymerisation profile shows that, for potentials values around +0.65 V, the formation of the monomer radical decreases with the number of cycles, probably due to the fact that the polymer formed during the first cycles prevents the monomer from reaching the surface of the electrode. Thus, the growth of chains already formed is mainly occurring.
These voltammograms still show, at about 0.14 V vs. Ag/AgCl, the redox pair corresponding to the oxidation/reduction of PANI, the peak increasing in height with the number of cycles as the PANI is formed (Figure 3).

Modification of GSPEs with Polyaniline and Gold Nanoparticles
The GSPEs were first modified with a layer of the conducting polymer polyaniline. Since conducting polymers show numerous features suitable for their application in sensing and biosensing (e.g., low cost, flexibility and biocompatibility), they have recently attracted a lot of attention in this field.
Here, the polymer is used to provide higher electroactive area (compared to the bare electrode), protection versus fouling of the electrode surface and scaffold for dispersing and anchoring the metal particles. Inclusion of AuNPs in conductive polymers can enhance electron transfer through a direct or mediated mechanism with improved conductivity and enhanced stability [14]. Further, the AuNPs are an excellent substrate for the immobilisation of thiolated bioreceptor.
The polymerisation profile shows that, for potentials values around +0.65 V, the formation of the monomer radical decreases with the number of cycles, probably due to the fact that the polymer formed during the first cycles prevents the monomer from reaching the surface of the electrode. Thus, the growth of chains already formed is mainly occurring.
These voltammograms still show, at about 0.14 V vs. Ag/AgCl, the redox pair corresponding to the oxidation/reduction of PANI, the peak increasing in height with the number of cycles as the PANI is formed (Figure 3). The number of cycles of electropolymerisation was chosen according to the data from the cyclic voltammetry, considering the growth of the faradaic current with each cycle. However, the application of 10 cycles to obtain the PANI/GSPEs was more adequate for the desired purpose, since this gave a more stable signal. When AuNPs were electrodeposited on the PANI/GSPEs by cyclic voltammetry, the current peak dramatically increased (Figure 4). The number of cycles of electropolymerisation was chosen according to the data from the cyclic voltammetry, considering the growth of the faradaic current with each cycle. However, the application of 10 cycles to obtain the PANI/GSPEs was more adequate for the desired purpose, since this gave a more stable signal. When AuNPs were electrodeposited on the PANI/GSPEs by cyclic voltammetry, the current peak dramatically increased (Figure 4). In particular, the cooperation of PANI and AuNPs in the modified film amplified the peak current and reversibility of redox peaks, which can be related to the larger electroactive surface area of AuNPs/PANI/GSPEs and electrocatalytic behaviour of gold nanoparticles ( Figure 5) than PANI/GSPEs. AuNPs are excellent electrical conductors; their incorporation in the polymer generated multiple active sites, which facilitated the electron transfer across the PANI matrix during the electrochemical processes.

Competitive Assay
In order to develop the competitive aptamer-based assay for profenofos detection, the thiolated DNA capture probe (oligo-SH) was immobilised on modified graphite screen-printed working electrodes, and the hybridisation reaction with the concentration of biotinylated aptamer sequence (apt-BIO) was performed in accordance with previously reported studies [7,14].
Apt-BIO is functionalised with biotin in 5′ end, in order to interact with streptavidin-alkaline phosphatase enzyme conjugate. The current peak increased linearly with apt-BIO concentration up to 0.50 μM, where a plateau was reached ( Figure 6).  In particular, the cooperation of PANI and AuNPs in the modified film amplified the peak current and reversibility of redox peaks, which can be related to the larger electroactive surface area of AuNPs/PANI/GSPEs and electrocatalytic behaviour of gold nanoparticles ( Figure 5) than PANI/GSPEs. In particular, the cooperation of PANI and AuNPs in the modified film amplified the peak current and reversibility of redox peaks, which can be related to the larger electroactive surface area of AuNPs/PANI/GSPEs and electrocatalytic behaviour of gold nanoparticles ( Figure 5) than PANI/GSPEs. AuNPs are excellent electrical conductors; their incorporation in the polymer generated multiple active sites, which facilitated the electron transfer across the PANI matrix during the electrochemical processes.

Competitive Assay
In order to develop the competitive aptamer-based assay for profenofos detection, the thiolated DNA capture probe (oligo-SH) was immobilised on modified graphite screen-printed working electrodes, and the hybridisation reaction with the concentration of biotinylated aptamer sequence (apt-BIO) was performed in accordance with previously reported studies [7,14].
Apt-BIO is functionalised with biotin in 5′ end, in order to interact with streptavidin-alkaline phosphatase enzyme conjugate. The current peak increased linearly with apt-BIO concentration up to 0.50 μM, where a plateau was reached ( Figure 6). AuNPs are excellent electrical conductors; their incorporation in the polymer generated multiple active sites, which facilitated the electron transfer across the PANI matrix during the electrochemical processes.

Competitive Assay
In order to develop the competitive aptamer-based assay for profenofos detection, the thiolated DNA capture probe (oligo-SH) was immobilised on modified graphite screen-printed working electrodes, and the hybridisation reaction with the concentration of biotinylated aptamer sequence (apt-BIO) was performed in accordance with previously reported studies [7,14].
Apt-BIO is functionalised with biotin in 5 end, in order to interact with streptavidin-alkaline phosphatase enzyme conjugate. The current peak increased linearly with apt-BIO concentration up to 0.50 µM, where a plateau was reached ( Figure 6). This behaviour is due to the limited number of biorecognition sites that were bound onto the sensor surfaces. The concentration of 0.50 μM apt-BIO was thus used for all experiments involving profenofos, since to perform the competitive assay, it is necessary to work in saturation conditions to obtain the maximum signal value in absence of the analyte.

Profenofos Detection
The pesticide detection was performed by competitive assay. In order to check if the pesticide itself, as an organophosphorus compound, could constitute an inhibition element for the phosphatase enzyme used in the labelling step, spectrophotometric measurements were carried out. Absorbance at 405 nm was collected for biotinylated aptamer sequence, profenofos and enzyme in the presence of p-nitrophenyl-phosphate enzymatic substrate. No significantly different values were obtained compared to the ones collected without profenofos, confirming that the pesticide is not interfering with the enzymatic activity (data not shown).
Various profenofos concentrations containing 0.50 μM apt-BIO were thus evaluated. A doseresponse curve of profenofos was obtained. The signal is reported as Sx/S0 percent units, that is the percentage of the signal decrease with respect to the blank value. A decrement of the current peak height was recorded by increasing the concentration of the pesticide in the range 0.10-10 μM at a fixed concentration of the aptamer sequence (apt-BIO) (Figure 7).
The fitting of experimental data values gives the equation A correlation coefficient R 2 of 0.997 was found. The detection limit (DL) was calculated as previously reported by Taylor et al. [23], and a value of 0.27 μM was obtained. The reproducibility of the aptasensor was also evaluated by multiple analysis of each standard profenofos solution.
Aptamer cross-reactivity of the proposed aptasensor was investigated in the presence of paraoxon, another OP pesticide. Paraoxon is the active metabolite of parathion, a molecule with a strong insecticidal and acaricidal effect, and the exposure to this molecule produces high mortality [24]. A value of 10% Sx/S0 was observed when 1.0 μM of parathion solution was tested. The described aptasensor was confirmed as a promising analytical tool for pesticide detection, since the tested concentrations gave an average 0.2% RSD value for ten repetitions of each standard solution. This behaviour is due to the limited number of biorecognition sites that were bound onto the sensor surfaces. The concentration of 0.50 µM apt-BIO was thus used for all experiments involving profenofos, since to perform the competitive assay, it is necessary to work in saturation conditions to obtain the maximum signal value in absence of the analyte.

Profenofos Detection
The pesticide detection was performed by competitive assay. In order to check if the pesticide itself, as an organophosphorus compound, could constitute an inhibition element for the phosphatase enzyme used in the labelling step, spectrophotometric measurements were carried out. Absorbance at 405 nm was collected for biotinylated aptamer sequence, profenofos and enzyme in the presence of p-nitrophenyl-phosphate enzymatic substrate. No significantly different values were obtained compared to the ones collected without profenofos, confirming that the pesticide is not interfering with the enzymatic activity (data not shown).
Various profenofos concentrations containing 0.50 µM apt-BIO were thus evaluated. A dose-response curve of profenofos was obtained. The signal is reported as S x /S 0 percent units, that is the percentage of the signal decrease with respect to the blank value. A decrement of the current peak height was recorded by increasing the concentration of the pesticide in the range 0.10-10 µM at a fixed concentration of the aptamer sequence (apt-BIO) (Figure 7).
The fitting of experimental data values gives the equation A correlation coefficient R 2 of 0.997 was found. The detection limit (DL) was calculated as previously reported by Taylor et al. [23], and a value of 0.27 µM was obtained. The reproducibility of the aptasensor was also evaluated by multiple analysis of each standard profenofos solution.
Aptamer cross-reactivity of the proposed aptasensor was investigated in the presence of paraoxon, another OP pesticide. Paraoxon is the active metabolite of parathion, a molecule with a strong insecticidal and acaricidal effect, and the exposure to this molecule produces high mortality [24]. A value of 10% S x /S 0 was observed when 1.0 µM of parathion solution was tested. The described aptasensor was confirmed as a promising analytical tool for pesticide detection, since the tested concentrations gave an average 0.2% RSD value for ten repetitions of each standard solution.

Fruit Juice Samples Analysis
In order to evaluate the practicability of the aptasensor, the preliminary experiments were performed in commercially available pear juice samples were carried out. Profenofos standard solutions were added to the samples after dilution (1:10) in phosphate buffer. The aptasensor response was then determined by DPV measurements, in the same conditions used for the pesticide calibration curve (Figure 8). The signal was found to decrease with respect to the sample solution by increasing the profenofos spiked concentration in the analysed samples. The profenofos concentration in the pear juice samples was calculated through the obtained calibration curve. A little matrix effect was observed resulting the recovery value. The results obtained for the spiked samples with standard addition of profenofos are shown in Table 2.

Fruit Juice Samples Analysis
In order to evaluate the practicability of the aptasensor, the preliminary experiments were performed in commercially available pear juice samples were carried out. Profenofos standard solutions were added to the samples after dilution (1:10) in phosphate buffer. The aptasensor response was then determined by DPV measurements, in the same conditions used for the pesticide calibration curve (Figure 8).

Fruit Juice Samples Analysis
In order to evaluate the practicability of the aptasensor, the preliminary experiments were performed in commercially available pear juice samples were carried out. Profenofos standard solutions were added to the samples after dilution (1:10) in phosphate buffer. The aptasensor response was then determined by DPV measurements, in the same conditions used for the pesticide calibration curve (Figure 8). The signal was found to decrease with respect to the sample solution by increasing the profenofos spiked concentration in the analysed samples. The profenofos concentration in the pear juice samples was calculated through the obtained calibration curve. A little matrix effect was observed resulting the recovery value. The results obtained for the spiked samples with standard addition of profenofos are shown in Table 2. The signal was found to decrease with respect to the sample solution by increasing the profenofos spiked concentration in the analysed samples. The profenofos concentration in the pear juice samples was calculated through the obtained calibration curve. A little matrix effect was observed resulting the recovery value. The results obtained for the spiked samples with standard addition of profenofos are shown in Table 2.

Conclusions
Affinity-based biosensing can contribute to pesticide detection as a valid and innovative analytical approach. In this work, we developed, for the first time, a simple and cost-effective aptasensor for the direct determination of profenofos pesticide based on a competitive format and disposable screen-printed electrochemical cells. Preliminary experiments were performed with real samples. A dose response curve was obtained between 0.10 µM and 10 µM with a detection limit of 0.27 µM. The sensitivity achieved was adequate for the analysis of profenofos in real samples, although the highest level of a pesticide residue that is legally tolerated in or on food depends on the food nature. From the obtained results, this analytical tool has proven itself to be promising for application in real samples analysis, since it involved low amounts of reagents and easy-to-prepare portable aptasensors. All these features make it suitable for the realisation of a commercial kit. This allows in situ analysis that with traditional techniques such as chromatography is not achievable.