A facile, sensitive and rapid sensing platform based on CoZnO for detection of fipronil; an environmental toxin.

Abstract

A sensitive detection of extremely toxic phenylpyrazole insecticide, ‘Fipronil’ is presented. Currently, the advancement of approaches for the detection of insecticides at low concentrations with less time is important for environmental safety assurance. Considering this fact, an effort has been made to develop an electrospun CoZnO nanofiber (NF) based label-free electrochemical system for the detection of fipronil. The CoZnO NF were characterized using different techniques including field emission scanning electron microscopy (FE-SEM), Energy Dispersive X-Ray Analysis (EDX), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and Raman Spectroscopy. Based on the experimental results, the proposed platform displayed a linear response for fipronil in the attogram/mL range despite the multiple interfering agents. The sensitivity of the device was found to be 3.99 Kῼ (g/ml)⁻¹ cm⁻². Limit of detection (LOD) and limit of quantification (LOQ) were calculated and found to be 112 ag mL⁻¹ and 340 ag mL⁻¹ respectively. Further, this proposed sensor will be implemented in the fields for the rapid and proficient detection of the real samples.

1. Introduction

Insecticides are commonly monitored environmental chemicals ^[1–3]. Fipronil is an insecticide that belongs to N-phenylpyrazole group and acts as an ectoparasiticide agent ^[4–6]. Fipronil is thought to exhibit selectivity for insects vs companion and domestic animals as a result of differences in the gamma-aminobutyric acid (GABA) receptor affinity ^[7]. In insects, fipronil non-competitively binds to GABA_A-gated chloride channels, and thus hinders the action of GABA_A in the central nervous system (CNS) ^[8]. Fipronil at low doses exhibits hyperexcitation, whereas at higher concentrations it causes paralysis leading to death. In the liver, Fipronil gets converted into the fipronil sulfone by cytochrome P₄₅₀. Fipronil sulfone can remain in tissues, due to its lipophilicity. Fipronil has been detected in tissues, for over a week ^[9–11]. The long-time required (7–8 weeks) for fipronil to be cleared from blood might be due to slow metabolism or slow discharge of metabolites from tissue pools refractory to metabolism. In vivo studies in the rats suggest that fipronil is excreted largely in the feces (50–70%) with less in the urine (10–30%). Detection of fipronil can be used as a biomarker previous of fipronil exposure when found in tissues, urine, skin, or hair ^[12–14]. Fipronil is extensively used as broad-spectrum insecticide in crop production ^[15]. Detection and validation of fipronil metabolites are typically performed using different chromatography techniques such as GC and HPLC ^[16–19]. Despite being effective, these techniques require multi-step analysis are time-consuming, expensive, and also need trained staff. Hence these techniques are not ideal for handheld point-of-care alternatives. Sensitive approaches, for the detection of small molecules such as fipronil, are critically important and can be achieved by analytical systems such as biosensors. Electrochemical techniques are used among different types of biosensors and have proven to have intrinsic advantages ^[20–22]. Electrochemical biosensors are being used widely for drug ^[23,24], phytochemical compound ^[25–29] and disease markers ^[30–33] due to their sensitivity, reproducibility, and ease of miniaturization ^[34–36]. Also, in contrast with many other methods, it is ideal for the detection of small molecules, due to its simplicity and low cost ^[29]. The immobilization of protein (antibodies or antigens) onto the electrode is a key factor for the development of electrochemical sensors. Immobilization using self-assembled monolayers (SAMs) is an attractive approach.

Biocompatible nanomaterials with their unique properties offer support to this technique. It provides conformational freedom for antibody immobilization, and also augmented surface activity. ^[37,38]. Among several nanostructures, nanofibers (NF) have arisen as prominent biosensing materials based on their exceptional properties ^[39]. NF can able to effectively accelerate electron transfer between electrode and biomolecules. This leads to a better performance of the sensor for the detection of the target molecule. Zinc oxide (ZnO) nanocomposites are predominantly attractive due to their free-exciton binding energy (60 me V), bandgap (3.37 eV), lack of toxicity, and also its ability of high electron transfer ^[40,41]. Also, ZnO’s high isoelectric point (IEP=9.5) provides extra advantage for direct antibody immobilization. This might be due to the exhibition of higher binding capacities ^[42]. To synthesize uniform ZnO NF and control their sizes and morphology, a variety of techniques have been used previously, including drawing, template synthesis, phase separation, and solid–vapor decomposition ^[43,44]. The use of electrospinning technology is increasing worldwide ^[45] because it can synthesize sensitive NF layer with increased surface area and greater porosity. The optical and electrical properties of ZnO nanostructures are essentially reliant on their morphology and composition^[46]. ZnO has shown substantial antibacterial activity and can strongly fight micro-organisms. Also, they are efficiently cast-off in a drug delivery system in several diseases targeting tissues and cells. Another prominent application of ZnO is in the field of sensing and imaging used to monitoring and track patient’s health and be a suitable bio-imaging tool ^[22]. ZnO nanostructures have been extensively reported in bio-sensing ^[22,25,42]. The addition of impurities into ZnO induces great changes in their properties. Doping certain elements into ZnO has thus become an important approach to enhancing their electrical, magnetic, and optical properties ^{[24,26,27,47–50]}. In this perspective, Using the electrospinning technique, nanofibers are prepared to improve surface stability and sensitivity ^[51]. Besides, the similarity in ionic radii of Co Co⁺² to the ionic radii of Zn Zn⁺² (0.58 Å vs 0.60 Å) might favor enhanced crystallization of the metal oxide ^[52]. The enhanced structural, optical, and electrical properties are essential for the application of biosensor devices. Nevertheless, there are only limited reports on behaviors of CoZnO NF. There is a gap in the literature in understanding the electrochemical behavior of CoZnO NF used for insecticides detection. The present work completely focuses on building a judicious and responsive approach for the attomolar label-free detection of fipronil based on the electro-catalytic activity of CoZnO NF. Further, this platform can be adopted for the investigation of the real samples in the field.

2. Experimental

2.1. Chemicals

Poly(acrylonitrile) (PAN) (Mw = 150,000), Cobalt(II) acetate (Co(CH₃COO)₂), Zinc acetate (Zn(CH₃COO)₂), N,N-dimethylformamide (DMF), 3-mercaptopropionic acid (MPA), N-(3-dimethyl-aminopropyl)-N-ethylcarbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS), phosphate-buffered saline (PBS) tablets (pH-7.4), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (USA). The fipronil antigen and antibody were obtained from Sigma-Aldrich (USA) and Abcam (U.K) respectively. Throughout the experiments, ultrapure water has been used.

2.2. Preparation of CO-ZnO NF

The CoZnO NF was synthesized by the electrospinning process technique using the following protocol ^[53]. In brief, Zinc acetate and cobalt(II) acetate were added to the 8 wt.% of PAN polymer and DMF solution at a molar ratio of 2:1 and was stirred at 65 °C for 2 hours to get homogenous precursor-polymer bend solution. The solution was fed through a 26G needle attached to a 5 mL syringe using a 0.9 mL h⁻¹ feed rate. The applied voltage was 18 kV, and the distance between needle and collector was set at 12 cm. The environmental conditions of the electrospinning chamber were set at the ambient temperature of 37 °C and 25 % relative humidity. After approximately 3 hours of electrospinning, fibers were extracted and annealed in a furnace at 400 °C for 2 hours in the air.

2.3. Preparation of CoZnO NF/GCE electrode

The glassy carbon electrode (GCE) was carefully polished with 0.05 μm alumina powders and continually washed with water and acetone. CoZnO NF was immobilized onto the surface of GCE with drop-casting. Then, 6 μL of CoZnO NF (1wt%) were subsequently dropped on the GCE surface and dried under controlled room temperature conditions. The NF modified GCE (GCE/CoZnONF) was then treated with 10 mM MPA followed by incubation overnight (15h) at RT. MPA includes a cluster of carboxylic (–COOH) form a self-assembled monolayer (SAM) on the surface of the electrode ^[30]. The carboxylic groups were activated using NHS-EDC cross-linking chemistry onto the electrode. The modified electrode was delicately cleaned with ultrapure water before the surface immobilization of the antibody. Later, surface immobilization of the fipronil antibody on the working electrode took place with 6 μL of anti-fipronil (10 μg mL⁻¹) antibody drop-casted, and the electrode then was incubated overnight (12h) at 4 °C. Covalent immobilization of antibody takes place onto the NF modified GCE (GCE/CoZnONF) through the formation of amide bonds. After chemisorption, the antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil) was extensively washed with ultra-pure water. BSA was used to minimize the nonspecific adsorptions of biomolecules. When not in use, the antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil) was stored at 4 °C. The preparation of the proposed biosensor device has been illustrated step by step in Fig. 1.

Fig. 1:

Schematic illustration of the proposed CoZnO NF based biosensor device for the detection of fipronil.

2.4. Electrochemical analysis

In this work, CHI 660E electrochemical workstation has used for all the electrochemical measurements at RT. With a voltage scanning rate of 80 mV / S, the cyclic voltammetry studies were performed over a wide potential range of −0.4 V to + 0.8 V.in the presence of 5.0 mM [Fe(CN)₆]^3-/^4-. All the biomolecules (such as antibodies and antigens) have surface charges, and disturbance of the charge produces a change in capacitance. The electrochemical impedance spectroscopy (EIS) can measure the change in the capacitance due to the binding of these molecules. The vital constraint that is observed in EIS is the charge transfer resistance (R_ct), which is reliant on the conformation of the electrode surface. For the targeted fipronil detection, EIS analysis was conducted using successive additions, wherein 100 μL of antigen solution added serially to the 10 mL of electrolyte solution (0.1M) in the electrochemical cell. In between the process of analyte addition to the electrolyte and EIS readings, the system was kept on standby for 10 minutes for the appropriate stability. Hence, the diffusion of the analyte occurs near to the working electrode where the antibody-antigen interaction took place.

2.5. Protocol for repeatability, interference and stability studies

The repeatability of the sensor was evaluated by calculating the electrode response to 10 ng mL⁻¹ of fipronil five times a day at 3-hour intervals. Where, after the analyte detection, the electrode was stored at 4 °C, and its response was documented throughout the day in a fixed interval. An interference study was also conducted to assess the proficiency of the suggested sensor. Specifically, the sensor’s response to equal concentrations of the interfering compounds, such as BSA (1 μg mL⁻¹) and atrazine (ATZ) (1 μg mL⁻¹) was recorded. Thereafter, response to an equal proportion of fipronil and the BSA as well as was fipronil and the ATZ recorded. The sensor’s stability was assessed by storing antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil) at 4 °C for 21 days and the response of the sensor was measured.

3. Results and discussion

3.1. Material characterization

A morphological study of the CoZnONF was conducted using field emission scanning electron microscopy. FESEM images of synthesized CoZnO NF before and after calcination are shown in Fig. 2(A) and (B) respectively. Fig. 2(A) indicates the morphology of the NF was sleek and uniform with the diameter in the range of 500 nm. Whereas, Fig. 2(B) shows crystalline NF, having a diameter in the range of 200 nm after calcination at 400 °C. Post calcination, there is a decrease in the diameter of the NF due to the evaporation of PAN at 400 °C.

Fig. 2:

FESEM images of CoZnO NF (A) Pre-calcination (B) Post-calcination at 400 ^oC (C) Elemental analysis of the CoZnO NF.

The elemental compositional analysis of the CoZnO NF is shown as the EDX spectrum in Fig. 2 (C). The compositional analysis shows the presence of oxygen (15.94%), carbon (39.19%), nitrogen (8.79%), cobalt (4.74%), and zinc (3.68%). Silicon (Si) element was being observed because the sample was transferred on a clean silicon wafer for elemental analysis.

Powder X-ray diffraction was used to study the structural parameters and phase purity. The XRD patterns of CoZnO samples are shown in Fig. 3(A). All the peaks are sharp, appropriately indexed, and are in accord with the standard datasheet (JCPDS-036–1451) ^[54]. The most intense peak (101) shows a strong shift concerning lower 2θ value with a decrease in intensity, representing the incorporation of Co in ZnO ^[55]. Also, characteristic peaks for co-doped ZnO were observed at angles 31.9, 34.8, 37.16, 48.22, 56.71, 63.42, 68.13, which corresponds to the crystal plane (100), (002), (101), (102), (110), (103), and (112). Also, XRD results show extra small peaks at 28.31, 67.24, 73.49, and 77.83 which were determined to be a co metal impurity phase possibly as a result of the clustering of cobalt ^[56].

Fig. 3:

(A) X-ray diffraction patterns of the synthesized CoZnO NF (B) FTIR spectra of the synthesized CoZnO NF (C) Raman spectroscopy analysis of CoZnO NF.

The FT-IR technique is used to identify the functional groups present in the synthesized material. Fig. 3(B) shows the FTIR spectra of the electrospun CoZnO NFs in the wavenumber range 500–4000 cm⁻¹. The broad peak between 3750–3000 cm⁻¹ is attributed to O-H stretching from water molecules ^[57]. The peak located at 2354 cm⁻¹ represents the stretching vibration of CH3COO+ groups. The peaks at 1534 and 1697 cm⁻¹ in the spectrum were attributed to the O-H bending. The 953 and 781 cm⁻¹ peaks represent the C-O stretching from the organic group such as ethyl ^[58]. The peaks at 570–530 cm⁻¹ are assigned to the Zn-O stretching. Fig. 3(C) shows Raman spectroscopic analysis of the electrospun CoZnO NFs taken at RT in the range 400–2000 cm⁻¹. The sharp and strong peak at 1100 cm⁻¹ shows the spectra of ZnO and might be attributed to the strongest E2 (high) mode of ZnO. The peak at 480 cm⁻¹ giving a significant feature and can be assigned to the local vibration mode associated with CO representing binding with the donor defects ^[59–61].

3.2. Electrochemical studies of antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil)

Electrochemical analysis of the GCE, NF modified GCE (GCE/CoZnONF), and antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil) were investigated using CV and EIS. Fig. 4(A) represents the cyclic voltammograms, where peak-to-peak voltage difference (ΔE) is 98 mV, and peak current has decreased from GCE (64.5 μA) to GCE/CoZnONF (42.75 μA) which indicates the decreased rate of electron transfer at the GCE/CoZnONF surface. This is a quasi-reversible behavior that represents the semiconductive nature of NF ^[62]. EIS was also used to investigate the effects of surface modification, antibody immobilization, and the electron transfer properties shown in Fig. 4(B). Here, the Nyquist plots obtained for GCE, GCE/CoZnO, GCE/ZnO, and GCE/CoZnO/Ab adhere to a similar trend in terms of the changes in Rct. Rct is an indicator of the overall reaction kinetics, and an increase in the same symbolizes a reduction in the electron transfer rate at the electrode-electrolyte interface. This suggests that NF coating has improved the rate kinetics.

Fig. 4:

(A) Cyclic voltammogram of electrodes at each step EIS, (B) EIS studies of electrodes at each step, (C) Impedance response of anti-fipronil Ab immobilized electrode for different concentrations of fipronil (Inset: calibration curve with linear fitting), (D) Fitting curve (E) Circuit model.

3.3. Detection of fipronil

The electrochemical detection of the targeted fipronil was carried out using the antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil). To assess the analytical performance of the sensor, a serial dilution was performed with different concentrations of the target analyte (100 ag mL⁻¹ to 100 μg mL⁻¹). A Nyquist plot of the sensor is shown in Fig. 4(C), it demonstrated that the Rct value is dependent on the target concentration. As the concentration of target analyte increased, the R_ct values increased; opposite to that, the peak currents decreased. The linear detection range of the sensor was calculated by using this equation.

$$\mathrm{\Delta }Rct=Rct(Ab)-Rct\left(Targetconcentration\right)$$

The R_ct values for each concentration were calculated using a standard Randles circuit with the implementation of the curve fitting method ^[63]. The modeled circuit with its parameters for the sensor is shown in Fig. 4(E). The calibration curve in Fig. 4(D) clearly showed that there was a linear relationship between the ΔR_ct and target concentration over a wide detection range from 100 ag mL⁻¹ to 100 μg mL⁻¹ with an r² = 0.997. Here, the error bars represent the standard deviation of 4 devices. The limit of detection (LOD) for target analyte was calculated using the typical computational formula.

$$LOD=\frac{\frac{S}{N}\times \mathrm{\sigma }}{S}$$

Where,  S/N = signal-to-noise ratio (3.3)

σ = standard deviation of blank

S = slope of the calibration curve

The limit of quantification (LOQ) for target analyte was calculated using the typical computational formula.

$$LOQ=\frac{10\times \mathrm{\sigma }}{S}$$

Limit of detection (LOD) and limit of quantification (LOQ) were calculated and found to be 112 ag mL⁻¹ and 340 ag mL⁻¹ respectively.

Further, the sensitivity of the sensor was calculated using the following formula.

$$Sensitivity=\frac{Slope}{area}$$

Likewise, the calculated sensitivity was found to be 3.99 KΩ (g mL⁻¹)⁻¹ cm⁻². The biosensor showed high sensitivity and low LOD. The LOD reported at different techniques is tabulated in Table 1 which represents the superiority of the present method.

Table 1:

Comparison of detection limits of fipronil by different techniques.

Techniques	LOD	References
Electrochemical sensor	0.00120 μM	[64]
MS analysis	0.012–0.055 μg/kg d.w	[65]
High-Performance Liquid Chromatography – Diode Array Detector (HPLC-DAD	0.45 μg L−1	[66]
disk-based solid-phase extraction (SPE)	11.19 ng/L	[67]
Electrochemical sensor	112 ag/mL	This work

3.4. Repeatability, interference and stability studies

Following a protocol outlined above, the repeatability of the proposed sensor was carried out. The recorded impedance signal of the antibody immobilized bioelectrode (GCE/CoZnONF/Anti-fipronil) at the same concentration of fipronil (10 ng mL⁻¹). Fig. 5(A) represents the ΔRct values with the standard deviation (5.14) for four sensors. The low SD value showed that the repeatability of the proposed sensor for fipronil detection was satisfactory.

Fig. 5:

(A) Study of Repeatability of five identical bio electrodes for 1 ng/mL of fipronil (B) Interference study: Bar charts show ΔR_ct for each 1 nM of pure non-specific compounds (Fip, BSA, ATZ, Fip+BSA, and ATZ+BSA) (C) Stability analysis: Bar diagram representation of R_ct for 21 days storage of bioelectrode.

A key parameter, the interference study of the proposed sensor was investigated with other proteins included BSA, because it is an abundantly found protein in the blood. Also, atrazine has been used as a possible cross-reacting small molecule. The same concentration of fipronil, BSA, and atrazine and also mixture in equal proportions have been used. Fig. 5(B) showed the percentage normalized change in Rct for BSA is 11% and for atrazine 10% as opposed to 100% change for fipronil. The biosensor even showed a higher response when it was incubated in mixture solution Fig. 5(B). This sensor exhibits excellent selectivity for the fipronil antigen.

For the determination of stability, the (GCE/CoZnONF/Anti-fipronil) electrodes were kept at 4 °C for 21 days (Fig. 5(C)). The EIS response of the sensor was analyzed and after three weeks of storage, this sensor had a mere 5.4% change in the Rct. This suggests that the proposed sensor shows good long-term stability.

4. Conclusion

A CoZnO NF based electrochemical sensor was proposed for ultra-sensitive detection of fipronil. EIS technique was used to evaluate the electrochemical behavior of fipronil on the surface of the CoZnO NF modified glassy carbon electrode. Result exhibits that the process was adsorption controlled and The proposed biosensor is highly proficient in the selective and sensitive determination of fipronil in the electrode/electrolyte interface. This biosensor was able to respond to fipronil concentration changes within few minutes in an electrolyte solution with the detection limit of 112 ag mL⁻¹. It also exhibits a great sensitivity of 3.99 KΩ (g mL⁻¹)⁻¹ cm⁻². The biosensor demonstrated high selectivity towards fipronil detection obtained with spiked buffer samples with the stability of 21 days. Present work represents superiority in the limit of detection (LOD) as compared with earlier reported techniques. The future scope of the proposed work is aimed towards the transformation into a miniaturized device for the detection of fipronil in the field.