Polythiophene/multiwalled carbon nanotubes/nitrate reductase deposited glassy carbon electrode (GCE/PTH/MWCNT/NR): a novel biosensor for the detection of nitrate in aqueous solution Open Access

Contamination of water resources has become a severe threat to the environment. With the rapid growth of industrialization in the past few decades, the release of by-products into water bodies has increased enormously. The most prominent pollutants released from industrial, domestic and agricultural activities include pesticides, dyes, heavy metals, etc. (Lakherwal 2014; Qamruzzaman & Nasar 2014, 2015; Shakoor & Nasar 2016; Azimi et al. 2017; Sharma & Bhattacharya 2017; Nasar & Mashkoor 2019; Mashkoor & Nasar 2020, Mashkoor et al. 2020; Nasar 2021). Besides the man-made contributions, natural contaminants are also present in water which depend on the nature of the geographical location through which the groundwater passes. The water flowing through sedimentary rocks and soils may contain higher concentrations of contaminants like calcium, sodium, magnesium, iron, halides, arsenate, nitrate, sulfate, bicarbonate, etc. (Ghrefat et al. 2014). Even the naturally occurring elements present in water beyond the acceptable limits behave as a pollutant, and such water cannot be acceptable for drinking and other purposes. In the same way, the presence of nitrogen in water is essential for the survival of plants and animals while it becomes toxic beyond the permissible limit (Umar & Nasar 2018). Nitrogen is a part of many cells and also involved in amino acids, proteins, and nucleic acids. Even DNA and RNA, as the most important biomolecules in all known forms of life, contain nitrogenous bases (Wu 2009; Zhang et al. 2009). It is also needed to make chlorophyll in plants, which plants generally use in photosynthesis to make their food and energy. Further it is an essential constituent for normal growth, cell replacement and tissue repair in all life. While nitrogen is abundant in the environment, humans cannot directly use it from the air. Humans and animals get nitrogen from drinking water, fruits, vegetables and other animals which consume vegetation. However, if the concentration of nitrogen reaches beyond a safe limit, this nutrient has a serious impact on health. The high level of nitrate and its reduced form, i.e., nitrite, in drinking water could cause serious human diseases like methemoglobinemia and stomach cancer by the production of N-nitrosamines (Kim et al. 2007; Alimohammadi et al. 2018), gastric cancer (Reyter et al. 2008), goiter (Raoof et al. 2009; Santos et al. 2009) and congenital disabilities (Ashok Kumar et al. 2009). Due to the excessive use of nitrate chemical fertilizers to accelerate the growth of intended crops, some agricultural products, especially vegetable crops, do not use the whole of the fertilizers applied. The remaining inorganic minerals leach into surface waters or groundwater and get attached to soil particles, or contribute to pollution (Bord et al. 2005). Therefore, the quality of food and drinking water has been adversely affected by excessive fertilization (Silva et al. 1996). It has been considered that nitrates and nitrites are an important contamination in water, food products and environmental matrices (Morales-Suarez-Varela et al. 1995; Sambol et al. 2017). The World Health Organization (WHO) and European Economic Community (EEC) have established the values of 44.3 and 50.0 mg/L as the maximum acceptable limits of nitrate in drinking water, respectively (Kleinjans et al. 1991). Thus, a high level of nitrate (typically >44 mg/L) can lead to the formation of other toxic substances such as nitrite, nitric oxide and N-nitroso compounds and is not acceptable (Sohail & Adeloju 2016). Because of the extensive environmental concerns of nitrate, its removal from the aquatic ecosystem and other food matrices is necessary. The recognition of such a threat compels scientists to develop a simple, easy, cost-effective and efficient method to detect, monitor, and remove the excessive nitrate present in water and other different food items. The efficient elimination and control of any pollutant are based on exact information of its quantitative presence. Thus, the development of a suitable method for the detection of nitrate concentration in different environmental matrices is of utmost importance. Several techniques such as spectrophotometry (Armstrong 1963; Edwards et al. 2001; Yue et al. 2004; López Pasquali et al. 2007; Tsoulfanidis et al. 2008), gas chromatography coupled with mass spectrometry (Tsikas et al. 1997), high-performance liquid chromatography (Jedličková et al. 2002), ion chromatography coupled with electrospray ionization tandem mass spectrometry (Blount & Valentin-Blasini 2006), fluorescence (Mohr et al. 1997; Huber et al. 2001), electrochemical (Hussein & Guilbault 1975; de Beer & Sweerts 1989), disc electrode alert system (Soropogui et al. 2006), surface-enhanced Raman spectroscopy (Gajaraj et al. 2013), electrophoresis (Gao et al. 2004), hydrodynamic sequential injection (Somnam et al. 2008), microplate fluorimetry (Ciulu et al. 2018), etc. have been employed to detect nitrate in a variety of samples. However, most of these methods are costly, and in many cases, they are not suitable for prompt field analysis. In contrast to this, biosensors have the potential for the development of a simple, convenient tool having the capability for the detection of nitrate present even in a very low to high concentrations. The use of biosensors reduces the effect of cross-contamination, drift, surface fouling and carry-over (Wang et al. 2008). Recently, a nanometal-decorated exfoliated graphite nanoplatelet-based glucose biosensor with high sensitivity and fast response has been developed (Lu et al. 2008). Determination of nitrate by employing the enzymatic biosensor has been widely used (Moorcroft et al. 2001). The nitrate reductase (NR), a catalyst suitable for encouraging a reduction in the hazardous nitrate species, has been utilized as the bio-recognition component for the manufacturing of nitrate-distinguishing biosensors (Barbier et al. 2004; Sohail et al. 2012; Sohail & Adeloju 2016). The detection of nitrate employing NR for the reduction of nitrate into nitrite has reflected a superior approach (Kiang et al. 1978a, 1978b; Atmeh & Alcock-Earley 2011; Umar & Nasar 2018). This is due to the fact that the NR efficiently increases the electrode sensitivity and selectivity toward nitrate reduction (Cosnier et al. 2008). Further, NR can be simply obtained by extraction from animal, plant, and microorganism. Further, the efficiency of biosensors based on NR can be improved by incorporating nanoparticles (Sohail & Adeloju 2016). In the present work, a novel biosensor (GCE/PTH/MWCNT/NR) based on the composite of polythiophene (PTH), multi-walled carbon nanotube (MWCNT) and NR was developed on a glassy carbon electrode (GCE). The efficacy of the biosensor was studied in artificially polluted wastewater containing nitrate ions.

Multi-walled carbon nanotubes (Alfa Aesar) and nitrate reductase obtained from Aspergillus niger (Sigma Aldrich/Roche Diagnostics GmbH) were used as received. The following analytical reagent grade chemicals were used: chloroform (CDH), ferric chloride (CDH), methanol (CDH), N,N-dimethylformamide (SRL, AR), thiophene (Loba Chemie, synthetic grade), and potassium ferrocyanide (CDH). The water used throughout the experiment was obtained by deionization followed by double distillation. This doubly distilled deionized water (DDW) was used throughout the experiment.

PTH and PTH/MWCNT were prepared by in situ oxidative polymerization of thiophene by employing the well-established method (Karim et al. 2006; Guo et al. 2008). The polymerization was carried out by stirring 2 ml thiophene in 100 ml CHCl₃ for about 30 min. Simultaneously, a solution of FeCl₃ (2.0 g) in CHCl₃ (100 mL) was kept for ultrasonication for 1 h. This oxidant solution was added to thiophene solution and further agitated for 12 h. Thereafter, the precipitate was filtered and washed properly, initially by the methanol and then by distilled water. In this way, the PTH was so obtained, and then dried in an oven at 70 °C for 10 h and stored in an air-tight container for further use. By adopting a similar method, PTH/MWCNT composite was prepared. In this case, a dispersion of 5 wt% MWCNT along with thiophene in CHCl₃ was agitated on a magnetic stirrer for 1 h and the FeCl₃ solution was poured slowly. After agitation for 12 h, the composite mass was filtered, washed and stored for experimental use.

Three different types of working electrodes, viz, GCE/PTH, GCE/PTH/MWCNT and GCE/PTH/MWCNT/NR were prepared by depositing the respective species on glassy carbon electrodes. Initially, the GCE was washed and then electrochemically cleaned in a 1.0 M H₂SO₄ solution by cycling the potential between −1 and +1 V using a reference Ag/AgCl, at a sweep rate of 100 mVs⁻¹ as described elsewhere (Umar & Nasar 2018). The electrode was finally prepared by the drop casting of a 6 μL dispersion of PTH or PTH/MWCNT in N,N-dimethylformamide. In order to increase biocatalytic activity, the PTH/MWCNT composite was immobilized by nitrate reductase (NR). This was achieved by applying 8.0 μL of NR (in phosphate buffer solution of pH 7.3) on the dried GCE/PTH/MWCNT electrode and keeping it room at room temperature until it is dried.

The LSV curves show that the catalytic current increases with the increase in nitrate concentration up to 8.0 mM, beyond which the current density starts to decrease because the reaction rate tends to decrease at higher concentrations of nitrate. This may be attributed to the saturation of the active sites of the enzyme after some period of time on further increase in the concentration of substrate, leading to a reduction in reaction rate.

The high response of the developed GCE/PTH/MWCNT/NR biosensor electrode towards nitrate shows its potential application in the detection of toxic nitrate ions in water and wastewaters. The nanocomposite of PTH and MWCNT has been established to be a good matrix for the immobilization of NR enzyme. The prepared electrode showed outstanding electrocatalytic performance for nitrate reduction and offered good selectivity for nitrate ions. The nitrate reductase present as immobilizer on the surface of PTH/MWCNT composite is involved in direct electron transfer from the active site of the electrode surface leading to the enhancement of current density up to 5.12 mAcm⁻².

The authors are grateful to the chairperson of the Department of Applied Chemistry, Faculty of Engineering and Technology, Aligarh Muslim University, for extending laboratory facilities. They are thankful to the Council of Science and Technology, Government of Uttar Pradesh, for providing financial assistance under the research Project No. CST/SERPD/D-282 dated 14-05-2015.

Council of Science and Technology, Government of Uttar Pradesh provided financial assistance under the research Project No. CST/SERPD/D-282 dated 14-05-2015.

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The authors declare there is no conflict.