Electrochemical micro analytical device interfaced with portable potentiostat for rapid detection of chlorpyrifos using acetylcholinesterase conjugated metal organic framework using Internet of things

An Electrochemical micro Analytical Device (EµAD) was fabricated for sensitive detection of organophosphate pesticide chlorpyrifos in the food chain. Gold microelectrode (µE) modified with Zinc based Metal Organic Framework (MOF-Basolite Z1200) and Acetylcholinesterase (AChE) enzyme served as an excellent electro-analytical transducer for the detection of chlorpyrifos. Electrochemical techniques such as Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV) were performed for electrochemical analysis of the developed EµAD. The sensor needs only 2 µL of the analyte and it was tested within the linear range of 10 to 100 ng/L. The developed EµAD’s limit of detection (LoD) and sensitivity is 6 ng/L and 0.598 µ A/ng L−1/mm2 respectively. The applicability of the device for the detection of chlorpyrifos from the real vegetable sample was also tested within the range specified. The fabricated sensor showed good stability with a shelf-life of 20 days. The EµAD’s response time is of 50 s, including an incubation time of 20 s. The developed EµAD was also integrated with commercially available low-cost, handheld potentiostat (k-Stat) using Bluetooth and the results were comparable with a standard electrochemical workstation.

These methodologies are time-consuming, expensive, and require an analytical laboratory with skilled manpower for its operation. These constraints have paved way for the development of an on-field bio-sensing platform capable of monitoring pesticide levels with excellent sensitivity.
Researchers have been working for the development of biosensor platforms based optical [6][7][8] , electrochemical [9][10][11] , piezoelectric 12 properties. Electrochemical bio-sensors are advantageous when compared to currently available methods for OP detection, due to their low detection limit, rapid response, portability, and affordability 13 . Electrochemical transduction involves monitoring and measurement of redox (reduction-oxidation) reactions occurring at the surface of the electrode, which associates the transport of electrons between the members of the redox species for a particular value of applied potential 14,15 .
There are various types of electrodes reported in literature such as Indium Tin Oxide (ITO) 16 , Fluorine-doped Tin Oxide (FTO) 17 , Glassy Carbon electrodes (GCE) 18 , and Screen Printed electrodes (SPEs) 19 for electrochemical sensing of OPs. These electrodes have various disadvantages from a commercialization point of view like high cost, large sample volume requirement, surface imperfections, electrode fouling, short shelf-life etc. Carbon and noble metals based electrodes show some promising results with easier fabrication techniques 20 . The reusability factor in conjunction with low fabrication costs and accurate reproducible measurements makes gold microelectrodes (µE) the ideal choice.
Recently, various nanoparticles, functional nanomaterials, nanoporous materials, nanocomposites etc have been used as transducing elements in enzyme-based electrochemical biosensors to detect OPs. For example, a platinum working electrode was modified with ZnO nanoparticles in order to detect carbosulfan 21 , poly-(amidoamine) with cystamine core immobilized with AChE enzyme was developed for the detection of carbaryl 22 and nanocomposite composed of multi-walled carbon nanotubes, tin oxide, and chitosan constructed to detect chlorpyrifos 23 . Metal Organic Frameworks (MOFs) are currently being explored for their potential use as a transducing element for ultra-sensitive detection of OPs. MOFs can be classified based on various metals ions and their bridging ligands. Recently, the design and synthesis of zinc-based MOFs have been on the rise due to its widespread applications in sensing, gas storage, separation and catalysis owing to their captivating framework architectures and topologies [24][25][26][27] . MOFs are primarily employed for sensing applications because of its high surface area, tunable porosity and modifiable host-guest chemistry compared to traditional porous materials like zeolites, and activated carbon 28,29 . MOFs have also been explored as a bio-compatible matrix in bio-sensing applications. They act as carriers for the enzyme and gives a protective bio-compatible environment which helps in long term stability of the enzymes 30 . Fusion of the enzyme and the MOF results in the novel functional material which provides a robust substrate for both qualitative and quantitative measurement of pesticides.
In this work, a novel EµAD was developed using conjugated MOF with µEs assembled on a microfluidic platform for the detection of chlorpyrifos. MOFs were exploited as a matrix element to fabricate EµAD conjugated with the bio-recognition element (AChE enzyme) in order to facilitate the specificity and sensitivity of the sensor. The applicability of the sensor in the detection of real vegetable samples had also been investigated. The present work is the integration of EµAD with a portable potentiostat (k-Stat) having Bluetooth interface, which facilitated the overall device miniaturization for the detection of OPs in real time. Low sample volume requirement, portability, ease-of-use and response time is less than a minute, makes it an ideal candidate for potential commercialization.
Results and Discussion fabrication of eµAD. As purchased gold microelectrodes (µE) were cleaned using conventional RCA-1 cleaning protocol 31 . Following the cleaning process, MOF (2% in ethanol) was drop casted on the µE and kept for drying at room temperature for 30 minutes. In the next step, AChE enzyme (in PBS, pH 7) was immobilized on the MOF/µE. These electrodes were then stored in 4 °C for further use. The schematic of the step-wise electrode (EµAD) fabrication is shown in Fig. 1. The electrodes were electrochemically examined at every fabrication step using CV and EIS as a confirmatory measure.
Electrochemical characterization of EµAD. Cyclic voltammogram were obtained for the µE, MOF/µE and AChE/MOF/µE (EµAD). The gold µE facilitated significant peak current enhancement in the presence of redox couple, which was attributed due to the conductive and electrocatalytic property of µE. The peak current at −0.01 V decreased from 1.6 µA to 0.7 µA after casting MOF on µE. The AChE enzyme was immobilized on the MOF/µE and peak current was further decreased to 0.4 µA. This was due to the less conductive nature of the AChE enzyme as shown in Fig. 2(a) 13 . The electrochemical response of EµAD fabrication steps was further examined using EIS as shown in Fig. 2(b). It was observed that EµAD showed lower charge transfer resistance (R ct ) values of 4 KΩ due to the good conducting nature of µE, which further enabled the electron transfer between µE and electrolyte. After casting MOF and immobilizing AChE enzyme on the µE, the R ct values further increased to 35.8 KΩ and 40 KΩ respectively, which was a direct consequence of the hindrance in electron charge transfer between the modified electrode and electrolyte 24 . The results of CV and EIS correspond well with each other confirming the successful fabrication of EµAD. Scanning Electron Microscopy (SEM) was done to study the surface morphology of as purchased Basolite Z1200 MOF (Fig. 3). SEM image depicts a uniform size distribution with the rhombic dodecahedron topology. Fourier transform infrared (FTIR) spectroscopy was done at each step of modification of the electrode to confirm the successful fabrication of a sensor (Fig. 4). For the MOF/ µE, the peak at 1575 cm −1 was due to the C=N stretching vibration of imidazole, and the bands in the range of 1350−1500 cm −1 were associated with the imidazole ring stretching. The strong bands at 1145 and 991 cm −1 were attributed to the C−N stretching of the imidazole units. In AChE/MOF/µE the band at 1604 cm −1 represents NH bending and scissoring mode of AChE enzyme. At 1432 cm −1 the peak was attributed to C = C stretching vibrations. The peak around 3350 cm −1 is due to the presence of free hydroxyl stretching mode in AChE enzyme.
www.nature.com/scientificreports www.nature.com/scientificreports/ Optimization of sensor performance. The fabricated EµAD was optimized with various parameters such as incubation time, pH, temperature and substrate concentration (ATCl). DPV curve (Fig. 5(a)) depicts that at lower pH the current value increases, a maximum current of 2.1 µA was observed at pH 7. Further increase in pH decreases the peak current value, as lower pH has more H + which dominates the current response. Therefore, further experiments were carried out at pH 7. Figure 5(b) shows the effect of electrolyte temperature on EµAD. The results showed no significant change in current values at temperatures ranging from 25 °C to 45 °C. The current response may decrease at elevated temperatures beyond 45 °C owing to enzyme instability at high temperatures. All further experiments were performed at an ambient temperature of 25 °C. However, the sensor can provide an effective response up to 45 °C which makes it suitable for on-field detection. Figure 5(c) shows the effect of EµAD at different incubation time. The incubation time is the time taken for the reaction to complete once the analyte is added onto EµAD. From the DPV curve, it was observed no significant change in current response after 20 s. Therefore, the incubation time of 20 s was fixed for the subsequent experiments.
At optimized conditions, DPV current was recorded at various substrate concentrations of ATCl, which helps to study the influence of substrate concentration to the AChE enzyme inhibition. In Fig. 5(d), due to the increase in concentration of ATCl ranging 10 fM to 10 µM in 0.1 M PBS containing 5 mM K [Fe(CN) 6 ] 3−/4− the peak current also increased, which was a clear indication of ATCl oxidation due to the electrocatalytic reaction of the immobilized AChE enzyme and ATCl. The maximum current response was obtained at 10 µM substrate concentration, which showed the best enzyme activity and this concentration was used for subsequent experiments.   Fig. 7(a). The peak current of EµAD with and without chlorpyrifos was found to be 1.3 µA and 0.65 µA respectively. This sharp decrease in the current signifies blocking of active sites of the enzyme by chlorpyrifos, which inhibits the specific interaction of ATCl with an enzyme 32 . Figure 7(b) shows the standard curve of % Inhibition vs chlorpyrifos concentration. % inhibition values and concentration of chlorpyrifos were found to be directly proportional to each other within the linear concentration range 33 . The linear regression equation for DPV studies was formulated by the slope and intercept values from the standard calibration curve. Intercept and slope was calculated as 42.66 ± 0.76 and 0.47 ± 0.012 with equation % Inhibition = 0.47 × +42.66 (x = Concentration of chlorpyrifos (ng/L)) and R 2 = 0.99 for the concentration ranging from 10 to 100 ng/L. The developed EµAD has the linear detection range of 10 to 100 ng/L and LoD calculated using 3σ rule was 6 ng/L 34 . The sensitivity of the sensor calculated by the formula Sensitivity = Slope of calibration plot (µA/ngL −1 )/Active Surface Area (mm 2 ) is 0.598 µA/ng L −1 /mm 2, 35 .
The biosensor was also tested with real vegetable samples (cucumber, capsicum, and brinjal) prepared as explained in the previous section and % recovery study for chlorpyrifos in the real vegetable sample was shown in Table 1. For the chlorpyrifos added to real vegetable samples, the recovery ratios were within the range 94 to 99% ± 4%, and RSD values less than 5%. From the data, it is evident that the EµAD showed good accuracy in testing real vegetable samples. In order to show the selectivity, the sensor was exposed to cartap hydrochloride a  www.nature.com/scientificreports www.nature.com/scientificreports/ class of pesticide which is not having a phosphate group. 100 ng/L of cartap hydrochloride and chlorpyrifos was tested on the sensor and the % inhibition for chlorpyrifos and cartap hydrochloride was found to be 89.3% and 4.5% respectively, as shown in Fig. 8(a). It suggests the selective nature of the present sensor for chlorpyrifos specifically having a phosphate group. The permissible maximum residue limits (MRLs) of chlorpyrifos according to EU standards is 50 µg/L and the developed EµAD was able to detect a minimum value of 6 ng/L. The stability of the biosensor was tested up to 30 days, there was no major change in its response until 20 days and after that, there was a minimal decrease in current response which proved that the biosensor showed excellent stability till 20 days, Fig. 8(b).  www.nature.com/scientificreports www.nature.com/scientificreports/ integration of eµAD with portable potentiostat. Figure 9(a) represents the schematic diagram of the integration of EµAD with as purchased portable potentiostat (k-Stat). k-Stat can measure various electrochemical parameters using CV and DPV techniques. Integration of EµAD with k-Stat gives the flexibility of using the proposed sensor in real time environment by a person with minimum scientific knowledge. k-Stat is low powered, portable potentiostat which needs 5 V DC for its operation. It also has the capability of transferring data wirelessly. Figure 9(b) shows comparative data obtained from EµAD attached with conventional electrochemical workstation (ECW) and a portable potentiostat (k-Stat), the comparative data is in a close match with each other. This integration of EµAD with the portable electronics gives the feasibility of using EµAD for real-time on-field analysis for the detection of chlorpyrifos OP in low concentration levels available in the food chain.

Reagents and apparatus. Acetylcholinesterase (AChE) from Electrophorus electricus (electric eel), Acetyl
Thiocholine Chloride (ATCl), Pestenal Chlorpyrifos, Basolite Z1200 MOF were purchased from Sigma Aldrich. Sodium chloride (NaCl), potassium ferricyanide K 3 [Fe(CN) 6 ], potassium ferrocyanide K 4 [Fe(CN) 6 ], sodium dihydrogen orthophosphate (NaH 2 PO 4 ) and Disodium hydrogen orthophosphate (Na2HPO4) was purchased from Fisher Scientific. Cartap Hydrochloride 50% w/w was bought from Dhanuka Agritech Ltd. All experiments were performed using Milli-Q deionized water having a resistance value of 18.2 MΩ and analytical grade chemicals.   conclusion This work demonstrated the use of bio-conjugated MOF with AChE enzyme grafted on the gold microelectrode on a microfluidic platform integrated with portable electronics for the selective detection of chlorpyrifos. This platform is a portable tool for on-site analyses. Implementation of mechanism assures robust mechanical transfer of a solid phase sample in to a reservoir for detection. The EµAD exhibited a good recognition capability toward chlorpyrifos, within the linear range from 10 to 100 ng/L with a low limit of detection of 6 ng/L with a sensitivity of 0.598 µA/ng L −1 /mm 2 . The developed bio-sensor requires 2 µL of sample and remains stable for nearly a month.
The integration of EµAD with portable potentiostat enables it to be used in collecting real-time data from on-field samples with minimum scientific skills. In summary these advantages render the novel EµAD platform an attractive candidate for on-site analysis. Further work is underway using another category of pesticides to make the setup more viable so that it can be used by farmers and others without technical knowledge.