1 Introduction

A biological weapon is more fatal than a chemical weapon. Terrorists deliberately add disease-causing biological agents like bacteria, spores or viruses, etc. into the environment to infect a large section of society. A technique which is very quick and reliable is essentially required for the determination of BWAs so that life of thousands of people can saved by knowing the nature and type of infection. Several pathogenic bacteria fall under the category of possible biological warfare agents (BWAs) [1]. However, highly dangerous include Botulinum toxin, Francisella tularensis, Salmonella typhimurium, Staphylococcus epidermis, Vibrio vulnificus, etc. Others i.e., V. E. encephalitis, Ebola, Marburg, infectious viruses, etc. are rarely used as a biological weapon because they are not easily available.

There are different types of electrochemical biosensors i.e., impedance, potentiometer, conductance and amperometric [2,3,4,5]. Among these, amperometric biosensors are quick, reliable, sensitive, and most of them are composed of ion-membrane electrodes [6]. Si-Jing Huang and his co-workers have developed the wireless magnetoelastic sensor to detect S aureus in two different mediums i.e., cell culture medium and milk [7]. Yi Xu and his co-workers have investigated thermal parameters in a column filled with different size micro-particles as a reactor in thermal biosensor [8]. Hala Ghali and his co-workers have reported WGM (Whispering Gallery Mode) micro-resonators optical micro-disks-based biosensors for detection of S. aureus [9]. Chibli and his research group have immobilized specific phage proteins on silicon wafers to bind staphylococci with the help of micro-resonators optical disk [10]. Chang et al. have reported that S. aureus is a hypersensitive H2O2 sensor using Fe-mediated histidine oxidation and proved their results by using Fluorescence spectroscopy [11, 12]. Zhu et al. have reported carbon nanospheres coupled with Ag NPs and endonucleases to sense DNA by using the electrochemical stripping method [13]. Fenglei et al. have developed the electrochemical immune assay strategy to detect carcinoembryonic antigen by using a nucleic acid probe in presence of ferrocene [14]. Few others have reported electrochemical sensors for theophylline [15], acetaminophen [16], rifampicin [17], and pyrazinamide [18].

S. aureus is a major nosocomial pathogen worldwide. It is still in the list of major drug resistance bacteria and it is a frequent and important human pathogen. It was first reported in Britain in 1960 and later in the western countries in 1970. Infections caused by S. aureus is a very serious problem all over the world, as it is resistant to many antibiotics and transmit from patient to patient. The diseases caused by S. aureus include gastric, colorectal, recurrent cancer, and bowel obstruction followed by gastrostomy.

To the best of our knowledge, no researchers have reported the electrochemical sensor for S. aureus pathogenic bacteria at different temperatures by qualitative and quantitative manner without using any enzyme, antibody, or genetic material. In continuation to our study [19,20,21,22,23], here we have focused on three electrodes based novel electrochemical sensors for the quick, accurate, non-specific, and reliable detection of S. aureus pathogenic bacteria a potential BWAs. Reported work makes use of either enzyme, antibody, protein, or DNA moiety for immobilization over the surface of the working electrode for selective determination of the particular analyte. Different working electrodes have to be designed and fabricated for a specific analyte. But this novel electrochemical sensor solves this problem of specificity of the biosensors. This novel sensor can be used for the qualitative and quantitative estimation of different types of analytes, biological warfare agents, and environmental pollutants. The co-precipitation technique was used for the synthesis of CuO NPs. These NPs act as electron mediator in the working electrode. UV-visible, FTIR, TEM, TGA, and XRD techniques were used for the characterization of CuO nanoparticles. Cyclic voltammetry, linear resistance polarization, chronoamperometry, impedance spectroscopy, and Differential Pulse Voltammetry (DPV) techniques were used for the quick and correct detection of S. aureus pathogenic bacteria. The sensor was tested for 45 days for storage, stability and re-usability condition.

2 Materials and method

S. aureus bacteria (MTCC No. 3160) were collected from the Institute of Microbial Technology, Chandigarh, Haryana. Cu(NO3)2.3H2O (AR grade, 99.5%), CuSO4.5H2O (AR grade 99%) and ethyl acetate (AR grade 99.5%) were procured from Sisco Res. Lab. Pvt. Ltd. (SRL), India. Sodium phosphate buffer solution (99.5%) was procured from Sigma-Aldrich Pvt. Ltd.

The experimental technique was performed in five steps:

2.1 Bacterial strain preparation

The S. aureus was grown-up in nutrient broth in pre-sterilized Petri plates using laminar flow at a constant temperature of 37 °C. A sodium phosphate buffer solution (SPBS) was used for culturing the bacteria.

2.2 Co-precipitation method for CuO nanoparticles

Co-precipitation technique was used for the synthesis of CuO NPs. The copper nitrate and copper sulfate solutions were mixed together with constant stirring and sodium hydroxide solution was added with gentle heating. Ageing, nucleation, and the seedling process was carefully observed till the complete precipitation of CuO NPs.

2.3 Characterization of CuO NPs

Characterization of CuO NPs was carried out by UV-visible, X-ray diffraction, FTIR spectroscopy, Thermogravimetric analysis (TGA), and Transmission Electron Microscopy (TEM) techniques.

2.4 Designing of working electrode and assembling of electrochemical cell

For the detection of S. aureus pathogenic bacteria, three electrodes based electrochemical cell was used having a working electrode, Silver/Silver chloride (reference electrode), and a Pt electrode as an auxiliary electrode. Figure 1 shows a schematic diagram of an electrochemical cell for the electrochemical characterization of disease-causing pathogenic bacteria. The working electrode was specially designed and fabricated for the electrochemical characterization of pathogenic bacteria, or spores. A glass dropper was selected for this purpose. At its tip, a thick copper wire was inserted which was flattened to increase the surface area for electrochemical reactions. From the other open end, a slurry was added with the help of a syringe made up of butyl carbitol acetate + ethyl acetate mixture acting as conducting binder, 0.1 M sodium phosphate buffer solution (SPBS), CuO NPs, and graphite powder. CuO NPs here acts as redox mediator instead of Cytochrome-C or Ferrocene commonly used in CV measurements. A few advantages of using CuO NPs as redox mediator are: high order of conductivity, large surface area, small size, high catalytic activity, and high adsorption capacity. This newly designed working electrode along with reference and auxiliary electrodes are inserted into a double wall jacketed glass cell connected to a potentiostat (PGSTAT 128 N) acting as a transducer that converts the electrochemical response into an electrical signal.

Fig. 1
figure 1

Electrochemical sensor (Enzyme, antibody and DNA free) for the detection of S. aureus pathogenic bacteria in presence of CuO NPs as a potential biological warfare agent. Three electrodes i.e. working, reference and counter electrode are connected to potentiostat (PGSTAT 128 N) acting as transducer which convert electrochemical response in to an electrical signal

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2.5 Electrochemical characterization and detection of pathogenic bacteria

The electrochemical characterization experiments like LRP, CV, Chronoamperometry, Electrochemical Impedance Spectroscopy (EIS), and DPV were carried out on a potentiostat (PGSTAT 128 N) attached with a PC and digitally controlled water bath to maintain a constant temperature. SPBS was used for holding the bacterial culture. The effect of the addition of CuO NPs, heating, and stirring was thoroughly investigated. Change in Open Circuit Potential (OCP), current (μA/cm2) and oxidative anodic peak Epa, and reductive cathodic peak Epc, midpoint potential EMID, ΔEp, maximum current in anodic direction Ip, and formal potential Ecθ were recorded under different conditions. Definite values of an oxidative anodic peak, mid-point potential, formal potential, and OCP provides qualitative information i.e., nature and type of pathogens. The maximum current density on the anode provides quantitative information, i.e., concentration of BWAs.

1 × 107 CFU/mL of pathogenic bacteria was taken in 50 mL of SPBS. Four different samples were taken for study i.e. Sample 1: Pure SPBS, Sample 2: SPBS with 1.0 × 107 CFU/mL of bacteria, Sample 3: SPBS with CuO NPs, and sample 4: SPBS with CuO NPs and 1.0 × 107 CFU/mL of pathogenic bacteria. The effects of continuous stirring, heating (70 °C), and the effect of the addition of CuO NPs (100 ppm) was thoroughly investigated. The observations made after 4 h are current density (μA/cm2), equilibrium and non-equilibrium potential (mV), oxidative peak, reductive peak, EMID, ΔEp, Epa, Epc, Ecθ and variation in current values with time.

3 Results and discussion

Tables 1, 2 and 3 and Figs. 2, 3, 4, 5, 6, 7, 8 and 9 show the results of the electrochemical characterization of S. aureus pathogenic bacteria. Table 1 represents the OCP of stirred and unstirred samples of S. aureus with CuO NPs. Table 2 shows the OCP of heat-treated samples of S. aureus without CuO NPs. Table 3 shows the effect of stirring, heating, and addition of CuO NPs and their effect on the current density (μA/cm2), OCP, oxidative peak, reductive peak, Epa, EMID, ΔEp, Ecθ and limit of detection (LOD). Table 4 shows the results of the electrochemical impedance spectroscopy technique.

Table 1 Open circuit potential (OCP) values of unstirred and stirred samples of S. aureus with CuO nanoparticles
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Table 2 Open circuit potential (OCP) values of heat-treated samples of S. aureus with CuO nanoparticles
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Table 3 Effect of stirring, heating and addition of CuO nanoparticles (100 ppm) on the values of current (μA/cm2), potential (OCP), oxidative peak Epa, mid-point potential EMID, difference in potential ΔEp, and formal potential Ecθ
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Fig. 2
figure 2

Tafel plots for electrochemical characterization of S. aureus pathogenic bacteria. All potentials were recorded with a 3 mm graphite working electrode w.r.t. Ag/AgCl reference electrode in 1.0 × 107 CFU/mL of S. aureus in 50 mL of PBS buffer solution and at a scan rate of 5 mV/s. Utmost care has been taken while recording open circuit potential (OCP) when no current was flowing through the working electrode/solution interface.

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Fig. 3
figure 3

Cyclic voltammetry curves for the electrochemical characterization of S. aureus pathogenic bacteria. IUPAC convention was used for representing the Cyclic Voltammogram. Voltammogram were recorded in 1.0 × 107 CFU/mL of S. aureus in 50 mL of PBS buffer solution, 1 M KCl solution was used as supporting electrolyte. All the potentials were measured w.r.t. Ag/AgCl reference electrode at a scan rate of 5 mV/s

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Fig. 4
figure 4

Chronoamperometry curves for the electrochemical characterization of S. aureus pathogenic bacteria. Chroamperometric curves were recorded at anodic step potential of 100 mV from its equilibrium value. Potential were recorded with a 3 mm graphite working electrode w.r.t. Ag/AgCl reference electrode in 1.0 × 107 CFU/mL of S. aureus in 50 mL of PBS buffer solution in presence and absence of 100 ppm of CuO NPs at 25 °C and at constant pH of 7

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Fig. 5
figure 5

Chronoamperometric technique showing variation of current density (μA/cm2) with time (s) at different concentration of S. aureus pathogenic bacteria in presence of sodium phosphate buffer solution

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Fig. 6
figure 6

Current (μA/cm2) versus concentration (CFU/mL) response of electrochemical sensor exposed to different concentration of bacterial i.e., S. aureus in presence of 0.1 M sodium phosphate buffer solution at a constant pH of 7 and 4 °C. Standard assay conditions were used for each assay. Limit of detection observed by Ghali et al. was 5 × 106 CFU/mL [9], 5 × 103 CFU/mL by Chibli et al. [10] and 2 × 107 cells/mL by Haung et al. [7]

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Fig. 7
figure 7

Nyquist plots of S. aureus pathogenic bacteria at different concentrations. EIS experiments were performed at OCP in the frequency range of 0.1 to 10,000 Hz with an AC amplitude voltage of 0.05 V after a time interval of every 2 s. The blank solution contains SPBS and 0.1 M KCl solution

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Fig. 8
figure 8

Differential pulse voltammetry technique showing potential time function of S. aureus with a linear incremental scan rate i.e., 5 mV/s with a pulse of 50 mV at every second for a duration of 100 mS. 0.1 mol/L KCl solution was used as a supporting electrolyte

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Fig. 9
figure 9

Differential pulse voltammetry technique showing variation of current density with increase in potential at different concentration of S. aureus pathogenic bacteria. A linear incremental scan rate i.e., 5 mV/s was kept and a pulse of 50 mV was given at every second for a duration of 100 mS. 0.1 mol/L KCl solution was used as a supporting electrolyte. The blank solution contains SPBS and 0.1 M KCl solution

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Table 4 Electrochemical impedance parameters in absence and presence of different concentrations of S. aureus pathogenic bacteria
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3.1 Characterization of CuO NPs

CuO NPs were characterized by UV-visible, FTIR, XRD, TGA, and TEM techniques (Fig. 10). Figure 10(a) shows the UV-visible spectra of CuO NPs synthesized by the co-precipitation method. In the UV-visible spectra absorption peaks were observed at 250 and 300 nm. The FT-IR spectra shows two peaks at 668.05 and 620.93 cm−1 in the fingerprint region corresponding to CuO NPs Fig. 10(b). A broad peak from 3000 to 3500 cm−1 may be due to OH− ion present as impurity absorbed by the sample in the form of moisture from the atmosphere. Figure 10(c) shows the XRD pattern of CuO NPs. The peaks at 2θ values 24.74, 32.42, 34.46, and 35.72° are indexed at 021, 110, 002, and 111, respectively. It is clear from the X-ray diffraction pattern that synthesized CuO NPs are crystalline [24, 25]. The average particle size of CuO NPs as calculated from the Scherrer equation corresponding to a maximum intensity peak was 70 nm. Figure 10(d) shows the TEM image of CuO NPs. The average size of CuO NPs as observed from TEM image was 60–80 nm. Figure 10(e) shows thermogravimetric analysis (TGA) of CuO NPs. The NPs were found to be very stable up to 600 °C and most of the weight loss take place at 200 °C after that weight loss was very small. CuO NPs get completely vaporized at 800 °C.

Fig. 10
figure 10

Characterization of CuO NPs by UV-visible, FTIR, XRD, TEM and DTA techniques

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3.2 Linear resistance polarization (LPR) method

Figure 2 shows Tafel plots for the electrochemical polarization of the working electrode exposed to S. aureus pathogenic bacteria. Figure 2(a) shows Tafel plots of SPBS at the start and end (4 h) of stirring at 600 rpm obtained from LRP experiments. Figure 2(b) shows Tafel plots of SPBS + CuO NPs. Figure 2(c) shows Tafel plots of SPBS + S. aureus. Figure 2(d) shows Tafel plots of SPBS, S. aureus and CuO NPs. Although LRP method provides a little information about the qualitative estimation of pathogenic bacteria but it provides useful information about the presence and absence of quasi-equilibrium, direction of shift in OCP, current density (icorr), anodic and cathodic Tafel slopes (βa and βc) in presence and absence of pathogenic bacteria. The Tafel slopes tell us about the shifting of equilibrium and kinetics of electrochemical reaction at the surface of the working electrode [26, 27]. The addition of CuO NPs (100 ppm) results in the decrease in OCP and current density (icorr), i.e., a shift towards a more noble direction Fig. 2(a, b). The heating at 70 °C and addition of CuO NPs (100 ppm) have the same effect on the behavior of the electrochemical system and results in the killing of S. aureus pathogenic bacteria (Fig. 2(c, d) and Table 2). Results of LRP are in closed agreement with CV, chronoamperometry, impedance spectroscopy, and DPV techniques.

3.3 Cyclic voltammetry (CV) method

Figure 3 shows the cyclic voltammogram (CV) performed on pathogenic bacteria in presence and absence of NPs. Figure 3(a) shows the cyclic voltammogram of SPBS at 0 h and after 4 h of stirring at 600 rpm. Figure 3(b) shows the CV curves of SPBS + CuO NPs. Figure 3(c) shows the CV curves of SPBS and S. aureus. Figure 3(d) shows the CV curves of SPBS, S. aureus and CuO NPs.

The electrochemical process taking place at the surface of the working electrode is reversible or Nerstian in nature and is majorly diffusion controlled (Fig. 3). At a slow scan rate, the process was thermodynamically controlled where at a high scan rate the process was kinetically controlled [28, 29]. It was observed during our experiments that as we increase the scan rate (at constant temperature and fixed concentration, i.e., 1.0 × 107 CFU/mL of S. aureus) than activation energy or height of oxidative and reductive peaks in CV increases and hence electrochemical reactions becomes more and more kinetically controlled as compared to low scan rate (Fig. 3) where peak height was less and hence thermodynamically controlled. The shape of CV curves depends basically on two important laws: (i) Fick’s law of diffusion and (ii) Nernst’s law, Eqs. (1) and (2), respectively.

$$\frac{\partial \left[A\right]}{\partial t}=D\frac{\partial^2\left[A\right]}{\partial {x}^2}$$
(1)
$$\frac{{\left[A\right]}_{x=0}}{{\left[B\right]}_{x=0}}={e}^{\frac{nF}{RT}}\left({E}_t-{E}_c^{\theta}\right)$$
(2)

Here, [A]x=0 and [B]x=0 are the surface concentration of the oxidized and reduced form of electroactive species, respectively. n is the number of electrons exchanged, Et is the applied potential and \({E}_c^{\theta }\) is the formal potential. The n has a very small influence on the value of faradaic current but it has a large effect on the transport of electroactive species towards the working electrode.

The capacitive and faradaic current were recorded separately to an approximate level. At low concentration, the capacitive current, Ic is maximum and at high concentration faradaic current was maximum [30, 31]. The magnitude of the faradaic current, If was directly proportional to concentration of electroactive species present at the surface of the working electrode and hence provides quantitative information about pathogens. The value of capacitive current does not change with S. aureus concentration. The faradaic current is directly proportional to the rate of diffusion of S. aureus at the surface of the working electrode (Fick’s law of diffusion). The capacitive current results due to the formation of electrified interface and is given in Eq. (3) [32]:

$${I}_c=-q\frac{\partial A}{\partial t}-A\frac{\partial q}{\partial t}$$
(3)

Here, A is the surface area, q is the charge present at the interface and t is the time. Capacitive current can also be expressed in terms of the electrical potential gradient, Eq. (4):

$${I}_c=-q\frac{\partial A}{\partial t}-A{C}_{dl}\frac{\partial E}{\partial t}$$
(4)

Here, E is the potential created along with the interface due to the separation of charge. Hence, the capacitive current is dependent on the surface area gradient and potential gradient [33].

It was observed that the addition of CuO NPs sharpens the oxidative and reductive peak in CV experiments. Heating of the analyte at 70 °C and the addition of CuO NPs (100 ppm) has the same effect on CV. An oxidative anodic peak, Epa at −0.85 V, mid-point potential, EMID = −0.075 V, ΔEp = −0.15 V, maximum current intensity in anodic direction, Ip = 189.1 mA, reductive anodic peak, Epc = 0.75 V, formal potential, Ecθ = 0.075 V and OCP value of 0.347 mV (Table 3) are characteristics of S. aureus.

3.4 Chronoamperometric method

Figure 4(a) shows the chronoamperometry of SPBS at zero and after 4 h of continuous stirring. An increase in current density was observed after 4 h of stirring. Figure 4(b) shows the chronoamperometry curves of SPBS + CuO NPs. An increase in current density was observed after 4 h of continuous stirring. Figure 4(c) shows the chronoamperometry curve of SPBS + S. aureus after heating at 70 °C. Figure 4(d) shows the chronoamperometry curve of SPBS, S. aureus and CuO NPs. An increase in the current density was observed after 4 h of stirring in case of SPBS [19]. The current density decreases after 4 h of stirring in presence of CuO NPs (Fig. 4(a, b)). The 100 ppm CuO NPs in the system also results in the decrease in current density but the decrease is not as significant as compared to heating at 70 °C. Figure 5 shows the amperometric curves of S. aureus pathogenic bacteria at different concentrations. The values of current density decrease with an increase in bacterial concentration from 1 × 109 to 1 × 101 CFU/mL (Fig. 5). As more and more bacterial colonies were added in the system, interface resistance increases which results in a decrease in the current density. The value of the current density decreases appreciably after 1 × 107 CFU/mL. Figure 6 shows the variation of current density with S. aureus concentration. A decrease in current density was observed with S. aureus concentration. The value of current density decreases to 4 μA/cm2 (almost negligible) at 1 × 109 CFU/mL of S. aureus. A lower detection limit of 1 × 109 CFU/mL was shown by our sensor in comparison to 5 × 106 CFU/mL observed by Ghali et al. [9], 5 × 103 CFU/mL observed by Chibli et al. [10], and 2 × 107 cells/mL observed by Haung et al. [7]. In comparison with literature data, it was found that the lower and upper detection limit and concentration range for the detection of S. aureus by our electrochemical sensor was much better than reported in the literature.

3.5 Impedance spectroscopy technique

Impedance spectroscopic study was carried out at OCP in the frequency range 0.1–10,000 Hz with an AC amplitude voltage of 0.05 V after a time interval of 2 s. Figure 7 shows Nyquist plots in the presence and absence of different concentrations of S. aureus. The charge transfer resistance, Rct was found to increase with increase in S. aureus concentration. An increase in Rct indicates an increase in interface resistance. The thickness of the electrified interface increases as more and more pathogenic bacteria reaches the interface leading to an increase in charge transfer resistance [34, 35]. Table 4 shows the values of double-layer capacitance (Cdl), charge transfer resistance (Rct), and interaction parameter (λ) at different concentrations of S. aureus.

3.6 Differential pulse voltammetry (DPV) method

Since the concentration of BWAs used by the terrorist is very less. Hence, DPV technique was used for the quantitative estimation of S. aureus bacteria. A linear incremental scan rate i.e., 5 mV/s was kept and a pulse of 50 mV was given at every second for a duration of 100 mS. 0.1 mol/L KCl solution was used as a supporting electrolyte. The potential time function obtained from DPV is shown in Fig. 8. The potential was found to increase linearly with time and shows a linear potential pulse signal at every second. Figure 9 shows DPV curves between current density and potential at different concentrations of S. aureus. It is clear from Fig. 9 that the value of current density first increases and then decreases with an increase in potential from −0.2 to 0.5 V. It was observed that the value of limiting current density decreases from 2.25 to 0.5 mA/cm2 with an increase in the concentration of S. aureus at a constant potential. The lower detection limit of a novel electrochemical sensor was found to be 1 × 109 CFU/mL as observed from DPV (Figs. 6 and 9).

The CV (Fig. 3) and DPV potential time function plot (Fig. 8) tells us that peak potential, Epeak is almost equal to half-wave potential, E1/2. Although the value of the diffusion coefficient of the oxidized and reduced form of electroactive species is equal but oxidative and reductive current intensity peak is different [36, 37]. The formal potential, \({E}_C^{\theta }\), was also calculated which is equal to half of the sum of Epa and Epc and is termed as mid-peak potential in CV. Mid peak potential often coincides with half-wave potential. Mid peak potential is related to formal potential by the Eq. (5).

$${E}_{mid}={E}_C^{\theta }+\frac{RT}{2 nF}{\ln}\left(\frac{D_{Red}}{D_{Oxid}}\right)$$
(5)

Here, Dred and DOxid represent the diffusion coefficient for reduced and oxidized form present at the surface of the working electrode [38, 39].

The formal potential is characteristics for a particular electroactive species and helps in the qualitative estimation of species present at the surface of the working electrode. Ep is the peak maximum potential in the either anodic or cathodic direction from baseline in CV. Ep is the same as formal potential in alternating current voltammetry and square wave voltammetry but in DPV it is different and is given by the Eq. (6).

$${E}_P={E}_C^{\theta }-\frac{{\Delta E}_{pulse}}{2}$$
(6)

Formal potential allows us the determination of Gibbs free energy of oxidation/reduction process, acidity constants (pKa), stability constants of complex formed, solubility constant [40,41,42,43]. It was also observed that the working electrode used in CV and DPV acts as electrocatalysis as it increases electrode reactions without being consumed during the electrochemical reaction as proved by the stability test of the working electrode for 45 days.

4 Storage, stability, current response, and reusability of the electrochemical sensor

Figure 11 shows the storage, stability, and reusability of the electrochemical sensor tested for continuous 45 days. Figure 11(a) shows the storage behavior of specially designed and fabricated working electrode-based sensor in 0.1 M SPBS, pH 7, and at 4 °C. The same standard assay conditions were used for each experiment [44, 45]. Figure 11(b) shows the effect of pH on the response of the electrochemical sensor. The two buffers solutions used for maintain constant pH are sodium succinate (pH range 4.5–5.5) and sodium phosphate (pH range 6.0–8.0). Figure 11(c) illustrates the influence of temperature on the current response of a novel electrochemical sensor. The same standard test conditions were used except for the incubation temperature which was varied from 20 to 70 °C. The number of observations made was three. The electrochemical sensor lost 50% of its activity after its regular testing i.e. 100 times in 45 days when kept in 0.1 M SPBS, pH 7, and at 4 °C. The current response of the electrochemical sensor was checked at different CFU/mL in presence of 0.1 M SPBS at pH 7 and 4 °C (Fig. 6). The current response of the amperometric sensor decreases with increase in bacterial concentration at a constant potential (Fig. 6). The current response of the investigated electrochemical sensor was found linear with a correlation coefficient, R2 = 0.991.

Fig. 11
figure 11

Storage, stability and reusability of electrochemical sensor tested for continuous 45 days

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5 Conclusions

A novel electrochemical sensor free from enzyme, protein, and nucleic acid was developed for the quick, accurate, and reliable detection of disease-causing pathogenic bacteria. Different electrochemical characterization techniques were used for the qualitative and quantitative estimation of S. aureus pathogenic bacteria i.e., LRP, CV, chronoamperometry, EIS, and DPV. The definite values of OCP, oxidative and reductive peak, Epa, mid-point potential (EMID), ΔEp, limiting current intensity (Ip), reductive anodic peak (Epc), and formal potential (Ecθ) were recorded at different potential, concentration and time. Heating of samples at 70 °C for 4 h and addition of CuO NPs (100 ppm) has the same effect, i.e., both results in the decrease in current density, open-circuit potential, and killing of pathogenic bacteria. S. aureus shows a characteristic oxidative anodic peak at 189.1 mA, EMID = −0.075 V, ΔEp = −0.15 V, reductive anodic peak, Epc = 0.75 V, formal potential, Ecθ = 0.075 V and an OCP of 0.347 mV. A decrease in the current density was observed with time and concentration of S. aureus. The electrochemical sensor was tested for 45 days for its storage, stability, and reusability. The current response was found linear with a correlation coefficient of R2 = 0.991. The developed electrochemical sensor was proved very quick, accurate, and reliable in the estimation of pathogenic bacteria to be used as potential biological warfare agents.