DOI: 10.1002/elan.202000051

A Facile, Sensitive and Rapid Sensing Platform based onCoZnO for Detection of Fipronil; an Environmental ToxinSanni Kumar,[a] Natalia Vasylieva,[b] Vikrant Singh,[c] Bruce Hammock,[b] and Shiv Govind Singh*[a]

Abstract: A sensitive detection of extremely toxic phenyl-pyrazole insecticide, ‘Fipronil’ is presented. Currently, theadvancement of approaches for the detection of insecti-cides at low concentrations with less time is important forenvironmental safety assurance. Considering this fact, aneffort has been made to develop an electrospun CoZnOnanofiber (NF) based label-free electrochemical systemfor the detection of fipronil. The CoZnO NF werecharacterized using different techniques including fieldemission scanning electron microscopy (FE-SEM), En-ergy Dispersive X-Ray Analysis (EDX), X-ray diffraction

(XRD), Fourier-transform infrared spectroscopy (FTIR),and Raman Spectroscopy. Based on the experimentalresults, the proposed platform displayed a linear responsefor fipronil in the attogram/mL range despite the multipleinterfering agents. The sensitivity of the device was foundto be 3.99 KΩ (g/ml)� 1 cm� 2. Limit of detection (LOD)and limit of quantification (LOQ) were calculated andfound to be 112 agmL� 1 and 340 agmL� 1 respectively.Further, this proposed sensor will be implemented in thefields for the rapid and proficient detection of the realsamples.

Keywords: Biosensors · Insecticides · Nanofibers

1 Introduction

Insecticides are commonly monitored environmentalchemicals [1–3]. Fipronil is an insecticide that belongs toN-phenylpyrazole group and acts as an ectoparasiticideagent [4–6]. Fipronil is thought to exhibit selectivity forinsects vs companion and domestic animals as a result ofdifferences in the gamma-aminobutyric acid (GABA)receptor affinity [7]. In insects, fipronil non-competitivelybinds to GABAA-gated chloride channels, and thushinders the action of GABAA in the central nervoussystem (CNS) [8]. Fipronil at low doses exhibits hyper-excitation, whereas at higher concentrations it causesparalysis leading to death. In the liver, Fipronil getsconverted into the fipronil sulfone by cytochrome P450.Fipronil sulfone can remain in tissues, due to its lip-ophilicity. Fipronil has been detected in tissues, for over aweek [9–11]. The long-time required (7–8 weeks) forfipronil to be cleared from blood might be due to slowmetabolism or slow discharge of metabolites from tissuepools refractory to metabolism. In vivo studies in the ratssuggest that fipronil is excreted largely in the feces (50–70%) with less in the urine (10–30%). Detection offipronil can be used as a biomarker previous of fipronilexposure when found in tissues, urine, skin, or hair [12–14]. Fipronil is extensively used as broad-spectruminsecticide in crop production [15]. Detection and valida-tion of fipronil metabolites are typically performed usingdifferent chromatography techniques such as GC andHPLC [16–19]. Despite being effective, these techniquesrequire multi-step analysis are time-consuming, expensive,and also need trained staff. Hence these techniques arenot ideal for handheld point-of-care alternatives. Sensitiveapproaches, for the detection of small molecules such as

fipronil, are critically important and can be achieved byanalytical systems such as biosensors. Electrochemicaltechniques are used among different types of biosensorsand have proven to have intrinsic advantages [20–22].Electrochemical biosensors are being used widely fordrug [23,24], phytochemical compound [25–29] and dis-ease markers [30–33] due to their sensitivity, reproduci-bility, and ease of miniaturization [34–36]. Also, incontrast with many other methods, it is ideal for thedetection of small molecules, due to its simplicity and lowcost [29]. The immobilization of protein (antibodies orantigens) onto the electrode is a key factor for thedevelopment of electrochemical sensors. Immobilizationusing self-assembled monolayers (SAMs) is an attractiveapproach.

Biocompatible nanomaterials with their unique prop-erties offer support to this technique. It provides con-formational freedom for antibody immobilization, andalso augmented surface activity [37,38]. Among severalnanostructures, nanofibers (NF) have arisen as prominentbiosensing materials based on their exceptional properties[39]. NF can able to effectively accelerate electron trans-

[a] S. Kumar, S. G. SinghDepartment of Electrical Engineering, Indian Institute ofTechnology Hyderabad, Telangana, India 502285Tel.: +91 40-2302-6076, Fax- +91 40-2301-6032E-mail: sgsingh@iith.ac.in

[b] N. Vasylieva, B. HammockDepartment of Entomology & Nematology, University ofCalifornia, Davis, USA

[c] V. SinghSchool of Medicine, University of California, Davis, USA

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fer between electrode and biomolecules. This leads to abetter performance of the sensor for the detection of thetarget molecule. Zinc oxide (ZnO) nanocomposites arepredominantly attractive due to their free-exciton bindingenergy (60 meV), bandgap (3.37 eV), lack of toxicity, andalso its ability of high electron transfer [40,41]. Also,ZnO’s high isoelectric point (IEP=9.5) provides extraadvantage for direct antibody immobilization. This mightbe due to the exhibition of higher binding capacities [42].To synthesize uniform ZnO NF and control their sizesand morphology, a variety of techniques have been usedpreviously, including drawing, template synthesis, phaseseparation, and solid-vapor decomposition [43,44]. Theuse of electrospinning technology is increasing worldwide[45] because it can synthesize sensitive NF layer withincreased surface area and greater porosity. The opticaland electrical properties of ZnO nanostructures areessentially reliant on their morphology and composition[46]. ZnO has shown substantial antibacterial activity andcan strongly fight micro-organisms. Also, they are effi-ciently cast-off in a drug delivery system in severaldiseases targeting tissues and cells. Another prominentapplication of ZnO is in the field of sensing and imagingused to monitoring and track patient‘s health and be asuitable bio-imaging tool [22]. ZnO nanostructures havebeen extensively reported in bio-sensing [22,25,42]. Theaddition of impurities into ZnO induces great changes intheir properties. Doping certain elements into ZnO hasthus become an important approach to enhancing theirelectrical, magnetic, and optical properties [24,26,27,47–50]. In this perspective, Using the electrospinning techni-que, nanofibers are prepared to improve surface stabilityand sensitivity [51]. Besides, the similarity in ionic radii ofCo 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 electricalproperties are essential for the application of biosensordevices. Nevertheless, there are only limited reports onbehaviors of CoZnO NF. There is a gap in the literaturein understanding the electrochemical behavior of CoZnONF used for insecticides detection. The present workcompletely focuses on building a judicious and responsiveapproach for the attomolar label-free detection of fipronilbased on the electro-catalytic activity of CoZnO NF.Further, this platform can be adopted for the investigationof 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-mercaptopropionicacid (MPA), N-(3-dimethyl-aminopropyl)-N-ethylcarbo-diimidehydrochloride (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 antibodywere obtained from Sigma-Aldrich (USA) and Abcam(U.K) respectively. Throughout the experiments, ultra-pure water has been used.

2.2 Preparation of CO� ZnO NF

The CoZnO NF was synthesized by the electrospinningprocess technique using the following protocol [53]. Inbrief, Zinc acetate and cobalt(II) acetate were added tothe 8 wt.% of PAN polymer and DMF solution at a molarratio of 2 :1 and was stirred at 65 °C for 2 hours to gethomogenous precursor-polymer bend solution. The solu-tion was fed through a 26G needle attached to a 5 mLsyringe using a 0.9 mLh� 1 feed rate. The applied voltagewas 18 kV, and the distance between needle and collectorwas set at 12 cm. The environmental conditions of theelectrospinning chamber were set at the ambient temper-ature of 37 °C and 25% relative humidity. After approx-imately 3 hours of electrospinning, fibers were extractedand 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 polishedwith 0.05 μm alumina powders and continually washedwith water and acetone. CoZnO NF was immobilizedonto the surface of GCE with drop-casting. Then, 6 μL ofCoZnO NF (1 wt%) were subsequently dropped on theGCE surface and dried under controlled room temper-ature conditions. The NF modified GCE (GCE/CoZ-nONF) was then treated with 10 mM MPA followed byincubation overnight (15 h) at RT. MPA includes a clusterof carboxylic (� COOH) form a self-assembled monolayer(SAM) on the surface of the electrode [30]. Thecarboxylic groups were activated using NHS-EDC cross-linking chemistry onto the electrode. The modifiedelectrode was delicately cleaned with ultrapure waterbefore the surface immobilization of the antibody. Later,surface immobilization of the fipronil antibody on theworking electrode took place with 6 μL of anti-fipronil(10 μgmL� 1) antibody drop-casted, and the electrode thenwas incubated overnight (12 h) at 4 °C. Covalent immobi-lization of antibody takes place onto the NF modifiedGCE (GCE/CoZnONF) through the formation of amidebonds. After chemisorption, the antibody immobilizedbioelectrode (GCE/CoZnONF/Anti-fipronil) was exten-sively washed with ultra-pure water. BSA was used tominimize the nonspecific adsorptions of biomolecules.When not in use, the antibody immobilized bioelectrode(GCE/CoZnONF/Anti-fipronil) was stored at 4 °C. Thepreparation of the proposed biosensor device has beenillustrated step by step in Figure 1.

2.4 Electrochemical Analysis

In this work, CHI 660E electrochemical workstation hasused for all the electrochemical measurements at RT.

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With a voltage scanning rate of 80 mV/S, the cyclicvoltammetry studies were performed over a wide poten-tial 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 antibodiesand antigens) have surface charges, and disturbance ofthe charge produces a change in capacitance. The electro-chemical impedance spectroscopy (EIS) can measure thechange in the capacitance due to the binding of thesemolecules. The vital constraint that is observed in EIS isthe charge transfer resistance (Rct), which is reliant on theconformation of the electrode surface. For the targetedfipronil detection, EIS analysis was conducted usingsuccessive additions, wherein 100 μL of antigen solutionadded serially to the 10 mL of electrolyte solution (0.1 M)in the electrochemical cell. In between the process ofanalyte addition to the electrolyte and EIS readings, thesystem was kept on standby for 10 minutes for theappropriate stability. Hence, the diffusion of the analyteoccurs near to the working electrode where the antibody-antigen interaction took place.

2.5 Protocol for Repeatability, Interference and StabilityStudies

The repeatability of the sensor was evaluated by calculat-ing the electrode response to 10 ngmL� 1 of fipronil fivetimes a day at 3-hour intervals. Where, after the analytedetection, the electrode was stored at 4 °C, and itsresponse was documented throughout the day in a fixedinterval. An interference study was also conducted toassess the proficiency of the suggested sensor. Specifically,the sensor‘s response to equal concentrations of theinterfering compounds, such as BSA (1 μgmL� 1) andatrazine (ATZ) (1 μgmL� 1) was recorded. Thereafter,

response to an equal proportion of fipronil and the BSAas well as was fipronil and the ATZ recorded. Thesensor’s stability was assessed by storing antibody immo-bilized bioelectrode (GCE/CoZnONF/Anti-fipronil) at4 °C for 21 days and the response of the sensor wasmeasured.

3 Results and Discussion

3.1 Material Characterization

A morphological study of the CoZnONF was conductedusing field emission scanning electron microscopy. FES-EM images of synthesized CoZnO NF before and aftercalcination are shown in Figure 2(A) and (B) respectively.Figure 2(A) indicates the morphology of the NF was sleekand uniform with the diameter in the range of 500 nm.Whereas, Figure 2(B) shows crystalline NF, having adiameter in the range of 200 nm after calcination at400 °C. Post calcination, there is a decrease in thediameter of the NF due to the evaporation of PAN at400 °C.

The elemental compositional analysis of the CoZnONF is shown as the EDX spectrum in Figure 2(C). Thecompositional 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 beingobserved because the sample was transferred on a cleansilicon wafer for elemental analysis.

Powder X-ray diffraction was used to study thestructural parameters and phase purity. The XRDpatterns of CoZnO samples are shown in Figure 3(A). Allthe peaks are sharp, appropriately indexed, and are inaccord with the standard datasheet (JCPDS-036-1451)

Fig. 1. Schematic illustration of the proposed CoZnO NF based biosensor device for the detection of fipronil.

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[54]. The most intense peak (101) shows a strong shiftconcerning 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 atangles 31.9, 34.8, 37.16, 48.22, 56.71, 63.42, 68.13, whichcorresponds to the crystal plane (100), (002), (101), (102),(110), (103), and (112). Also, XRD results show extrasmall peaks at 28.31, 67.24, 73.49, and 77.83 which weredetermined to be a co metal impurity phase possibly as aresult of the clustering of cobalt [56].

The FT-IR technique is used to identify the functionalgroups present in the synthesized material. Figure 3(B)shows the FTIR spectra of the electrospun CoZnO NFs inthe wavenumber range 500–4000 cm� 1. The broad peakbetween 3750–3000 cm� 1 is attributed to O� H stretchingfrom 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 wereattributed to the O� H bending. The 953 and 781 cm� 1

peaks represent the C� O stretching from the organicgroup such as ethyl [58]. The peaks at 570–530 cm� 1 areassigned to the Zn� O stretching. Figure 3(C) showsRaman spectroscopic analysis of the electrospun CoZnONFs taken at RT in the range 400–2000 cm� 1. The sharp

and strong peak at 1100 cm� 1 shows the spectra of ZnOand might be attributed to the strongest E2 (high) modeof ZnO. The peak at 480 cm� 1 giving a significant featureand can be assigned to the local vibration mode associatedwith CO representing binding with the donor defects [59–61].

3.2 Electrochemical Studies of Antibody ImmobilizedBioelectrode (GCE/CoZnONF/Anti-fipronil)

Electrochemical analysis of the GCE, NF modified GCE(GCE/CoZnONF), and antibody immobilized bioelec-trode (GCE/CoZnONF/Anti-fipronil) were investigatedusing CV and EIS. Figure 4(A) represents the cyclicvoltammograms, where peak-to-peak voltage difference(ΔE) is 98 mV, and peak current has decreased fromGCE (64.5 μA) to GCE/CoZnONF (42.75 μA) whichindicates the decreased rate of electron transfer at theGCE/CoZnONF surface. This is a quasi-reversible behav-ior that represents the semiconductive nature of NF [62].EIS was also used to investigate the effects of surfacemodification, antibody immobilization, and the electrontransfer properties shown in Figure 4(B). Here, theNyquist plots obtained for GCE, GCE/CoZnO, GCE/

Fig. 2. FESEM images of CoZnO NF (A) Pre-calcination (B) Post-calcination at 400 °C (C) Elemental analysis of the CoZnO NF.

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ZnO, and GCE/CoZnO/Ab adhere to a similar trend interms of the changes in Rct. Rct is an indicator of theoverall reaction kinetics, and an increase in the samesymbolizes a reduction in the electron transfer rate at theelectrode-electrolyte interface. This suggests that NFcoating has improved the rate kinetics.

3.3 Detection of Fipronil

The electrochemical detection of the targeted fipronil wascarried out using the antibody immobilized bioelectrode(GCE/CoZnONF/Anti-fipronil). To assess the analyticalperformance of the sensor, a serial dilution was performedwith different concentrations of the target analyte(100 agmL� 1 to 100 μgmL� 1). A Nyquist plot of the sensoris shown in Figure 4(C), it demonstrated that the Rctvalue is dependent on the target concentration. As theconcentration of target analyte increased, the Rct valuesincreased; opposite to that, the peak currents decreased.The linear detection range of the sensor was calculated byusing this equation.

DRct ¼ Rct Abð Þ � Rct Target concentrationð Þ

The Rct values for each concentration were calculatedusing a standard Randles circuit with the implementationof the curve fitting method [63]. The modeled circuit withits parameters for the sensor is shown in Figure 4(E). Thecalibration curve in Figure 4(D) clearly showed that therewas a linear relationship between the ΔRct and targetconcentration over a wide detection range from100 agmL� 1 to 100 μgmL� 1 with an r2=0.997. Here, theerror bars represent the standard deviation of 4 devices.The limit of detection (LOD) for target analyte wascalculated using the typical computational formula.

LOD ¼

S

N � s

S

Where, S/N= signal-to-noise ratio (3.3), σ= standarddeviation of blank, S= slope of the calibration curve.

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

Fig. 3. (A) X-ray diffraction patterns of the synthesized CoZnO NF (B) FTIR spectra of the synthesized CoZnO NF (C) Ramanspectroscopy analysis of CoZnO NF.

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LOQ ¼10 � s

S

Limit of detection (LOD) and limit of quantification(LOQ) were calculated and found to be 112 agmL� 1 and340 agmL� 1 respectively.

Further, the sensitivity of the sensor was calculatedusing the following formula.

Sensitivity ¼Slope

area

Likewise, the calculated sensitivity was found to be3.99 KΩ (gmL� 1)� 1 cm� 2. The biosensor showed highsensitivity and low LOD. The LOD reported at differenttechniques is tabulated in Table 1 which represents thesuperiority of the present method.

3.4 Repeatability, Interference and Stability Studies

Following a protocol outlined above, the repeatability ofthe proposed sensor was carried out. The recordedimpedance signal of the antibody immobilized bioelec-

trode (GCE/CoZnONF/Anti-fipronil) at the same con-centration of fipronil (10 ngmL� 1). Figure 5(A) representsthe ΔRct values with the standard deviation (5.14) forfour sensors. The low SD value showed that the repeat-ability of the proposed sensor for fipronil detection wassatisfactory.

A key parameter, the interference study of theproposed sensor was investigated with other proteinsincluded BSA, because it is an abundantly found proteinin the blood. Also, atrazine has been used as a possible

Fig. 4. (A) Cyclic voltammogram of electrodes at each step EIS, (B) EIS studies of electrodes at each step, (C) Impedance response ofanti-fipronil Ab immobilized electrode for different concentrations of fipronil (Inset: calibration curve with linear fitting), (D) Fittingcurve (E) Circuit model.

Table 1. Comparison of detection limits of fipronil by differenttechniques.

Techniques LOD References

Electrochemical sensor 0.0012 μM [64]MS analysis 0.012–0.055 μg/kg d.w [65]High-Performance LiquidChromatography – Diode ArrayDetector (HPLC-DAD)

0.45 μgL� 1 [66]

disk-based solid-phaseextraction (SPE)

11.19 ng/L [67]

Electrochemical sensor 112 ag/mL This work

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cross-reacting small molecule. The same concentration offipronil, BSA, and atrazine and also mixture in equalproportions have been used. Figure 5(B) showed thepercentage normalized change in Rct for BSA is 11% andfor atrazine 10% as opposed to 100% change for fipronil.The biosensor even showed a higher response when it wasincubated in mixture solution Figure 5(B). This sensorexhibits excellent selectivity for the fipronil antigen.

For the determination of stability, the (GCE/CoZ-nONF/Anti-fipronil) electrodes were kept at 4 °C for21 days (Figure 5(C)). The EIS response of the sensor wasanalyzed and after three weeks of storage, this sensor hada mere 5.4% change in the Rct. This suggests that theproposed sensor shows good long-term stability.

4 Conclusion

A CoZnO NF based electrochemical sensor was proposedfor ultra-sensitive detection of fipronil. EIS technique wasused to evaluate the electrochemical behavior of fipronilon the surface of the CoZnO NF modified glassy carbonelectrode. Result exhibits that the process was adsorptioncontrolled and The proposed biosensor is highly proficientin the selective and sensitive determination of fipronil inthe electrode/electrolyte interface. This biosensor was

able to respond to fipronil concentration changes withinfew minutes in an electrolyte solution with the detectionlimit of 112 agmL� 1

. It also exhibits a great sensitivity of3.99 KΩ (gmL� 1)� 1 cm� 2. The biosensor demonstratedhigh selectivity towards fipronil detection obtained withspiked buffer samples with the stability of 21 days. Presentwork represents superiority in the limit of detection(LOD) as compared with earlier reported techniques. Thefuture scope of the proposed work is aimed towards thetransformation into a miniaturized device for the detec-tion of fipronil in the field.

Acknowledgments

Financial support to Shiv Govind Singh from the DST-AMT, DST-FIST, and Partial support to Bruce Hammockby the NIEHS Superfund Research Program (P42ES04699) and the NIEHS RIVER Award (R35ES030443-01) are highly acknowledged.

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Fig. 5. (A) Study of Repeatability of five identical bio electrodes for 1 ng/mL of fipronil (B) Interference study: Bar charts show ΔRct

for each 1 nM of pure non-specific compounds (Fip, BSA, ATZ, Fip+BSA, and ATZ+BSA) (C) Stability analysis: Bar diagramrepresentation of Rct for 21 days storage of bioelectrode.

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A Facile, Sensitive and RapidSensing Platform based onCoZnO for Detection of Fipronil;an Environmental Toxin