Comparative Study of Electrochemical Biosensors Based on Highly Efficient Mesoporous ZrO₂-Ag-G-SiO₂ and In₂O₃-G-SiO₂ for Rapid Recognition of E. coliO157:H7

Abstract

Here, we reported an innovative and electrochemical biosensor for the rapid detection of Escherichia coliO157:H7. We fabricated the mesoporous ZrO₂-Ag-G-SiO₂ (ZAGS) and In₂O₃-G-SiO₂ (IGS) sensors, and cyclic voltammetry (CV) was employed to detect the bacteria. The development of these portable sensors addresses the challenges of conventional time-consuming and more expensive laboratory-based analyses. Hence, the biosensors were highly selective to detect E. coli. The sensor could recognize an individual E. coli cell in 1 μL of sample volume within 30 s. E. coli live cells tied down on sample nanoparticles worked toward the definite acquirement of E. coli. The high thickness of negative charge on the surface of E. coli cells effectively regulated the concentration of dominant part charge carriers in the mesoporous channel, allowing a continuous check of E. coli concentration in a known sample. The signal current decreased linearly, while the E. coli concentration increased from 1.0 × 10¹ to 1.0 × 10¹⁰ CFU/mL. ZAGS and IGS biosensors could detect E. coli in the range from 10¹ to 10¹⁰ CFU/mL. ZAGS and IGS biosensors in this investigation showed great specificity, reproducibility, stability, and selectivity and are expected to have a great impact on applications in the detection of foodborne pathogens.

1. Introduction

Escherichia coliO157:H7 is the most widely recognized Shiga toxin-producing strain of E. coli and can cause disease with a minimum concentration of cells. Manifestations comprise severe stomach spasms, looseness of the bowels, nausea, or even lethal hemolytic uremic syndrome (HUS).^1,2 Electrochemical biosensors, a branch that is used for rapid detection, good sensitivity, miniaturization potential, and mass production, have been demonstrated to be exceptionally encouraging in the biosensor field, with continuous progress in nanomaterials, for instance, nanoporous films,^3,4 nanochains,⁵ nanotubes,^6,7 microelectrodes,⁸ and screen-printed electrodes.^9,10 Among the different nanomaterials considered,¹¹ graphene and its various forms, for example, graphene oxide (GO), reduced graphene oxide (RGO), and graphene quantum dot (GQD), have attracted attention for the progression of biosensors. Graphene is a single-atom-thick sheet of hexagonally organized, sp²-bonded carbon atoms occurring within a carbon material structure.¹² The two-dimensional (2D) properties of graphene with ultrahigh charge mobility give excellent electronic properties, having a large surface area (2630 m²/g), which accommodates a high possibility of active sites for charge–biomolecular interactions, leading to sensing enhancement as well as supporting the desired functionalization to target biomolecules to improve selectivity.¹³⁻¹⁵ Meanwhile, functionalized graphene is achieved by synthesis and preparation, for example, the carbon core structure can be oxidized forming GO, the reduced structure with vacancy defects is RGO, and structures a few nanometers in size with quantum phenomena are GQDs.¹⁶⁻¹⁸ ZrO₂ as a transducer is a promising material in the fabrication of a biosensor due to biocompatibility, phenomenal electrical and surface charge properties, and oxygen moieties.¹⁹ Silver nanoparticles (AgNPs) have low cost, nontoxicity, biocompatibility, and steady and high catalytic activity, helping us utilize it for biological sensing. In₂O₃ has been used for technological applications in optoelectronic devices and biosensors, owing to its high electric conductance, high transparency to visible light, and the strong interaction between certain biomolecules with In₂O₃ surfaces.²⁰ Mesoporous SiO₂ exhibits many advantageous characteristics, such as large surface and free volume, pore sizes that can be controlled, and tunable optical properties, and mesoporous SiO₂-based biosensor directly captures the target bacteria cells onto the porous surface. Therefore, monoclonal antibodies, which are specific to target bacteria, are immobilized onto the porous surface of a biosensor, which induces changes in the amplitude (intensity) of the sensor.²¹⁻²⁷ The nanocomposites combining two or several different components are expected to further improve the deficient characteristics of each component, leading to a promising application in biosensing. In this comparative study, we fabricated two mesoporous nanocomposites, In₂O₃-G-SiO₂ (IGS) (ternary) and ZrO₂-Ag-G-SiO₂ (ZAGS) (quaternary), for detecting E. coli. We focus on the difference between the two mesoporous materials in ternary and quaternary type, which can be achieved through biosensing application individually. Considering that ZrO₂-Ag-G-SiO₂ and In₂O₃-G-SiO₂ have excellent physical properties, electrical properties, and also good electrochemical stability, they can make a steady sensing framework for the preparation of graphene-based quaternary nanocomposites. The objective of the present study was to set up a novel nonenzymatic sensor for the sensing of E. coli. Its sensitivity, linear range, detection limit, and stability were examined. The ZrO₂-Ag-G-SiO₂ and In₂O₃-G-SiO₂ nonenzyme sensors were very sensitive, entirely stable, and financially savvy. Also, samples have been effectively analyzed through developed sensors for E. coli sensing. This new concept has opened a new direction to construct rapid, delicate, and highly efficient electrochemical biosensors.

2. Experimental Section

2.1. Materials

Graphite powder flakes (99%), zirconium(IV) isopropoxide (70 wt % 1-propanol), and Pluronic F127 were procured from Sigma-Aldrich; ethylene glycol, AgNO₃, HCl, phosphate buffer, NaOH, KOH, ethylene glycol, tetraethyl orthosilicate (TEOS), indium(III) chloride (InCl₃), urea, and sodium dodecylbenzene sulfonate (SDBS) were procured from Dae-Jung Chemical, Korea. Deionized water (18.2 MΩ/cm) was utilized throughout the work. All other chemicals were of investigative grade and were used without further purification.

2.2. Synthesis Procedure

2.2.1. Mesoporous SiO₂ Synthesis

Initially, 1.0 g of triblock copolymer Pluronic F127 surfactant (Sigma-Aldrich) was added to a solution containing 15 mL of deionized water and 60 mL of 2 M HCl at 313 K with stirring until the copolymer was dissolved. At that point, 3.20 mL of tetraethyl orthosilicate (TEOS, Acros Organics) was added and stirred for 12 h at 313 K. The mixture was moved to a sealed container and heated to 373 K in an oven for 20 h. The resulting white precipitate was filtered, washed with water and ethanol, and dried at 338 K overnight. Finally, the copolymer was removed by calcination in air at 823 K for 3 h.

2.2.2. Synthesis of In₂O₃-G-SiO₂

The mesoporous In₂O₃ nanoparticles were dispersed in 7 mL of deionized water, followed by the addition of 0.07 wt % GO water suspension considering the proportion of coordinated In₂O₃ nanoparticles. GO was obtained from graphite using Hummer’s method. The mixed solution was ultrasonicated for 20 min. This In₂O₃–GO solution was then added dropwise to a 100 mL beaker containing silica powder at 10 and 20%. After stirring for 48 h, it was filtered, washed with 1 mL of methanol, and dried at 343 K. The furnace temperature was increased to 973 K at a speed of 10 °C/min and held at 973 K for 4 h. A light-yellow sample was obtained.

2.2.3. Synthesis of ZrO₂-Ag-G-SiO₂

First, 6.5 g of Pluronic F127 was liquefied in 30.5 mL of ethanol, then 30.5 mL of the zirconium(IV) isopropoxide solution was added to 30.5 mL of ethanol and ethylene glycol with vigorous stirring. At this point, both solutions were mixed and stirred at 314 K for 1 h by adding 20.5 mL of H₂O dropwise to this mixture. Further, 3.5 g of AgNO₃ was liquefied in 10.5 mL of deionized water and then poured dropwise to the ZrO₂ solution under vigorous stirring in the dark; the mixed solution was constantly stirred till the gel was formed. First, 0.333 g of graphene oxide (GO) was added into 250 mL of water and ultrasonicated for 35 min. Sonicated graphene oxide was transferred to the ZrO₂–Ag solution and then stirred for 2 h at 374 K. The color of the precursor turned into a coffee color, showing the effective combination of G with Ag-combined ZrO₂ solution, forming a solution named E. The solution E was added dropwise to a beaker containing 0.3 g of SiO₂ powder and stirred at 374 K for 24 h and ultrasonicated for 1 h and 30 min. Then, the powder was filtered, washed with 1.5 mL of methanol, and dried at 338 K overnight. Next, it was calcined at 974 K at a speed of 283 K/min and held at 974 K for 5 h. Black color products were formed (Scheme 1).

Scheme 1. Synthesis Process of Graphene-Based Quaternary Mesoporous Nanocomposites.

2.3. Preparation of IGS and ZAGS Electrode

IGS and ZAGS thin films were developed using a traditional doctor-blade method.²⁸⁻³⁰ For the modified doctor-blade method, the synthesized ZrO₂ (ZS), ZrO₂–Ag (ZA), ZrO₂-Ag-G (ZAG), and ZrO₂-Ag-G-SiO₂ (ZAGS) (Table S1) paste was prepared as follows: first, the synthesized material powder (1.1 g) was mixed with ethylcellulose and acetone (1.5 mL) in a mortar for 15 min. Fluorine-doped tin oxide (FTO) glass was shielded through the sample paste to make a thin film. Afterward, it was dried in the open air for 35 min. One drop of lubricating oil was put onto the film surface and stabilized at 374 K in the oven for 25 min to diminish cracks. A greasing oil/lubricating oil on the film will the enable sensor via prevention of contamination of surfaces at points where films may break.³¹

2.4. Characterization of the Materials

The phase structure and purity of the as-synthesized products were examined by X-ray diffraction (XRD; Rigaku, X-ray Diffractometer) with Cu Kα radiation (λ = 1.5406 Å) at 40 kV, 30 mA over the 2θ range 20–70°. Morphologies were studied utilizing scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis by utilizing SEM (JSM-76710F, JEOL, Tokyo, Japan), transmission electron microscopy (TEM) (JEM-4010, JEOL, Tokyo, Japan), and high-resolution TEM (HRTEM) (JSM-76710F, JEOL, Tokyo, Japan) operating at a 300 kV accelerating voltage. X-ray photoelectron spectroscopy (XPS), differential reflectance spectroscopy (DRS), and Raman spectroscopy (RAMAN) analyses were performed utilizing WI Tec. alpha300 series. Porous characterizations of ZAG and ZAGS structures were obtained with a full analysis of N₂ adsorption–desorption tests (BELSORP-max, BEL Japan Inc.). (PG201, Potentiostat, Galvanostat, VoltaLab, Radiometer, Denmark.)

2.5. Electrochemical Measurements

Cyclic voltammetry (CV) and measurements were performed under a three-electrode electrochemical setup to check the current and voltage profiles, where ZS, ZA, ZAG, and ZAGS were utilized as working electrodes, while platinum cathode and Ag/AgCl were used as the counter and reference electrodes, respectively. Electrochemical properties in commercial urine were examined at pH 7.0. As electrolytes, a phosphate buffer aqueous solution was used. The limit of detection (LOD) of the analyte was determined by the following equation^32,33

1

where SD is the standard deviation of the analyte concentration determined from the current response of progressively including E. coli into the electrolyte and N is the slope of the calibration curve, which demonstrated the sensitivity of the electrode with a signal-to-noise ratio (SNR) of 3. All analyses were completed by voltammetry (PG201, Potentiostat, Galvanostat, VoltaLab, Radiometer, Denmark).

3. Results and Discussion

3.1. Characterization of the IGS and ZAGS Sample

The average crystallite size of the as-provided sample was determined by the Scherrer equation demonstrated as follows

2

where D is the grain size, K is the Scherrer constant (generally 0.89), λ is the X-ray wavelength, β is the width of half of the diffraction peak, and θ is the Bragg diffraction angle. To determine the crystalline phase, structure, and chemical compositions of IGS and ZAGS structures, we utilized XRD analysis. Figure 1a shows the XRD patterns of the IGS sample. It showed sharp peaks and no miscellaneous peaks. With the improvement of crystallites and the enhancement of crystallization, the peak intensities increased, which is designated to the great crystallinity of these samples.

Figure 1.

(a) XRD patterns of In₂O₃ (A), In₂O₃–G (B), In₂O₃-G-SiO₂-10% (C), and In₂O₃-G-SiO₂-20% (D) and (b) commercial ZrO₂ (a), synthesized ZrO₂ (b), ZrO₂–Ag (c), ZrO₂-Ag-G (d), and ZrO₂-Ag-G-SiO₂ (e).

Figure 1b shows the crystalline characteristic pattern of the ZAGS sample confirmed by the X-ray diffraction (XRD) method. As-synthesized ZrO₂ was shown at 24.8° with a lattice plane of (110). All diffraction peaks were well- indexed (JCPDS 37-1484). After being modified with Ag and SiO₂, the ZrO₂-Ag-G-SiO₂ spectrum demonstrated all diffraction peaks along the extra peaks, corresponding to (JCPDS 65-2871) and (JCPDS 39-1425), respectively, approving the pure crystalline nature of the material.

The morphology of IGS and ZAGS was considered by scanning electron microscopy (SEM). Figure 2a,b shows SEM images of IGS. The results showed that a heterogeneous structure could be formed by controlling the particle size and morphology. Figure 2a demonstrates pure In₂O₃ and G comprised of expansive sporadic spheres. After introducing SiO₂, large amounts of nanocrystals were present on In₂O₃ surfaces. Figure 2b shows that the particle size was increased slightly, and the total of nanoparticles became sporadic. Figure 2c,d confirmed that ZAGS was combined. These figures revealed that nano-flake-like structures of ZAGS were uniformly distributed. Such flake-like nanostructured geometry prompted a rough surface of the electrode, which enhances the electrode performance on account of its high surface area, better surface-to-volume ratio, and disclosure of more electrocatalytically active sites on ZAGS. Figure 2e shows the TEM image of Ag nanoparticle and shows continuous single directional lattice fringes. HRTEM image is shown in Figure 2f. The marked d-spacing of 0.205 nm corresponds to the interplanar distance of Ag{200} lattice planes. The HRTEM results further confirmed that the Ag nanoparticles adopt a single-crystal structure. Ag nanoparticles show equal distribution in TEM micrographs. They can increase the surface area of the sensor and are highly conductive, facilitating the charge transfer process. After the addition of AgNPs on the electrode surface with a large surface area, the sensor was used to detect E. coli with high sensitivity, where E. coli is immobilized on the biosensor surface through its mesoporous structure.

Figure 2.

SEM images of In₂O₃-G-SiO₂ (a, b) and ZrO₂-Ag-G-SiO₂ (c, d); TEM and HRTEM image of Ag nanoparticle (e, f).

Figure 3a shows that the Raman spectra of IGS and ZAGS structures are polarized parallel (E) and perpendicular (E_⊥) to their growth direction. The crossed polarization configuration demonstrates the cooperation among In₂O₃, ZrO₂, and G. This was affirmed by the Raman spectral analysis. For IGS, two distinct peaks were situated at 1450 and 1550 cm^–1. These intensity peaks could be recognized at unadulterated In₂O₃ vibrational modes. As expected, G showed two prominent G and D bands generally attributed to the E_2g photon of sp² bonds of carbon atoms and a breathing method of κ-point phonons of A_1g symmetry, respectively.³⁴ Lorentzian line shape analysis of these polarization-dependent relative intensities in In₂O₃ spectra showed that the Raman determination rules remained unaltered. The validity of these determination guidelines for this situation is related to the nanowire’s great crystalline quality. For single crystals, phonons are expected to experience a long-range order, causing phonons to conserve the translational symmetry. As shown in Figure 3b, as-synthesized G indicated two peaks at 1331 and 1573 cm^–1 corresponding to the D band and the C–C bond stretching frequency (G band), respectively. Generally, the intensity proportion of the D and G bands (I_D/I_G) is utilized to assess the degree of disorder and the typical size of sp² areas. In this study, the value of I_D/I_G was calculated to be 0.94.

Figure 3.

(a) Raman spectra of In₂O₃–G (IG), In₂O₃-G-SiO₂ 10% (IGS10), In₂O₃-G-SiO₂ 20% (IGS20) and (b) ZrO₂–Ag (ZA), ZrO₂-Ag-G (ZAG), and ZrO₂-Ag-G-SiO₂ (ZAGS).

Figure 4 shows the resultant absorbance of UV-DRS. DRS data recorded for IGS and ZAGS structures were changed over to absorption spectra by the Kubelka–Munk (K–M) theory. Their respective band gaps (E_g) were evaluated using the following equation³⁵

3

where α is the molar absorption coefficient determined as α = (1 – R)²/2R, hv is the incident light frequency, A is the proportionality constant, and E_g is the band gap of the material. Table 1 illustrates the data from (αhv)^1/2 as a function of photon energy. Figure 4a demonstrates that band gap in IGS nanocomposite was in the range of 1.25 eV, which was smaller than that of In₂O₃, IG, or IGS (i.e., 3.0, 2.10, and 2.0 eV, respectively). Figure 4b revealed that band gaps decreased from 4.79 eV for ZC to 3.25 eV for the ZS sample. On further investigation, the band gap of A_g-modified ZA decreased to 3.11 eV. After progressively combining with graphene, the band gap of the ZAG changed to 2.61 eV. Surprisingly, the band gaps remarkably change to 2.00 eV in the ZAGS after combining through mesoporous SiO₂. Valence band (VB) and conduction band (CB) potentials of all of the samples were calculated based on the following equations³⁶

4

5

Here, E_VB and E_CB are valence and conduction band edge potentials, respectively. X is the electronegativity of the semiconductor, E_e is the energy of free electrons on the hydrogen scale, and E_g is the band gap energy of the semiconductor.

Figure 4.

(a) DRS data of In₂O₃, In₂O₃–G (IG), In₂O₃-G-SiO₂ 10% (IGS10), and In₂O₃-G-SiO₂ 20% (IGS20) and (b) commercial ZrO₂ (ZC), synthesized ZrO₂ (ZS), ZrO₂–Ag (ZA), ZrO₂-Ag-G (ZAG), and ZrO₂-Ag-G-SiO₂ (ZAGS).

Table 1. Texture Properties of the Obtained IG, IGS10, IGS20, ZC, ZS, ZAG, and ZAGS.

samples	BET surface area (m2/g)	total pore volume (cm3/g)	average pore diameter (nm)	band gap energy (eV)
IG	29.688	0.1039	13.995	2.10
IGS20	253.79	0.281	4.4284	1.25
ZC				4.79
ZS				3.25
ZA				3.11
ZAG	8.6593	0.012273	5.6691	2.61
ZAGS	9.1703	0.020549	8.9632	2.00

Figure 5 demonstrates N₂ adsorption–desorption isotherms of mesoporous IGS and ZAGS. The perceived Brunauer–Emmett–Teller (BET) surface area was 253.79 m²/g for IGS mesoporous materials. The absolute pore volume of IGS was 0.281 cm³/g and the average pore diameter was 4.4284 nm. It demonstrated that the surface area parameters of the IGS sample were most elevated among these samples (Table 2). Figure 5b shows that the ZAGS samples exhibit typical type IV isotherms, demonstrating that the materials had mesopores. Isotherms of samples exhibit a steep H2 type hysteresis loop at a relative pressure (P/P₀) in the range between 0.6 and 0.9. The hysteresis loops gradually shift to a higher relative pressure (P/P₀) from ZAG to ZAGS, suggesting that these mesopores increased including mesoporous SiO₂. A mesopore diameter as large as 5.67 nm could be observed for ZAG. When combining through mesoporous SiO₂, it continues to increase to 8.96 nm in ZAGS and the BET surface area also increased from 8.66 to 9.17 m²/g (Table 2).

Figure 5.

(a) Nitrogen adsorption–desorption isotherms of In₂O₃–G (IG), In₂O₃-G-SiO₂ 10% (IGS10), and In₂O₃-G-SiO₂ 20% (IGS20) and (b) ZrO₂-Ag-G (ZAG) and ZrO₂-Ag-G-SiO₂ (ZAGS).

Table 2. Performance Comparison between Biosensors for the Detection of E. coliO157:H7.

developed sensor	particles	targeted bacteria	LOD (CFU/mL)	ref
colorimetric sensor	dopamine Fe3O4 NPs	E. coliXL 1	104	(37)
electrochemical immunosensor	GNPs chitosan	Salmonella and E. coli	105	(38)
electrochemical impedance	GNPs antibody	E. coli	103	(39)
mass spectroscopy	magnetic Fe3O4	Gram-positive/negative bacteria E. coliO157:H7	104	(40)
electrochemical immunofunctionalized	magnetic nanoparticle	E. coliO157:H7	103	(41)
electrochemical biosensor	l-Cyst-Fe3O4 NPs	E. coli	105	(42)
mesoporous electrochemical biosensor	ZrO2-Ag-G-SiO2 and In2O3-G-SiO2	E. coliO157:H7	1010	this work

Figure 6 shows the X-ray photoelectron spectroscopic (XPS) investigation results, which further affirm the above theory. It is revealed that In, O, C, and Si components exist in all samples. The XPS survey spectrum of IGS (Figure 6a) showed peaks corresponding to In, O, C, and Si atoms. Mass concentrations of these four atoms (In, O, C, and Si) were 277.6342, 15.9994, 12.0107, and 28.0855, respectively. These results suggested that In³⁺ ions bonded with unsaturated oxygen and Cl^– ions. Thus, the O–In–Clx (x = 1 or 2) structure was formed on the surface of the In₂O₃–SiO₂ sample. The XPS spectrum for In 3d is shown in Figure 8e. Spin–orbital splits for In 3d_5/2 and In 3d_3/2 XPS peaks also had characteristic double peaks centered at binding energies of 444.1 and 451.7 eV, respectively. These were relegated to In³⁺ ions.⁴³Figure 6b shows that the complete spectrum of ZrO₂-Ag-G-SiO₂ shows the presence of Si, Zr, C, Ag, and O atoms attributed to effective modification. The corresponding high-resolution spectra with respect to C 1s signal 284.5 eV as a reference binding energy in Figure S1a recognized to C–C, bonds of graphene. As shown in Figure S1b, Si 2p peaks were found at 103.2 eV. These peaks located at 184.3 eV were harmonized to Zr 3d in Figure S1c. The total results of the XPS study confirmed that all surface chemical compositions of ZAGS were found in the as-prepared nanocomposite.

Figure 6.

(a) XPS spectra of In₂O₃-G-SiO₂ 20% (IGS) and (b) ZrO₂-Ag-G-SiO₂ (ZAGS).

Figure 8.

(a) Cyclic voltammogram measurement in the presence of E. coli for In₂O₃ (IN), In₂O₃–G (ING), and In₂O₃-G-SiO₂ 20% (INGS20) and (b) synthesized ZrO₂ (ZS), ZrO₂–Ag (ZA), ZrO₂-Ag-G (ZAG), and ZrO₂-Ag-G-SiO₂ (ZAGS).

3.2. Electrochemical Property Measurements of Electrodes

The electrochemical analysis for two types of working electrodes (IGS and ZAGS electrodes) performed under a three-electrode cell system with Pt wire as the counter electrode and Ag/AgCl as a reference electrode within the potential range of −0.3 to +0.2 V is shown in Figure 7. The anodic part of the voltammogram was characterized by the occurrence of the anodic peaks corresponding to the electro-oxidation of the phosphate-buffered saline (PBS), while the cathodic part of the cyclic voltammogram was characterized by the occurrence of cathodic peaks corresponding to the electroreduction of the electrolyte. The IGS electrode is measured in the electrolyte and an oxidation peak was observed, which confirms that these electrodes have specific catalytic properties. IGS electrodes showed a well-defined oxidation peak at the potential of +0.1 V and presented a substantial rise in anodic current density of 0.0015 mA/cm². This indicates that the redox peaks in the curve can be attributed to the direct electron transfer behaviors of IGS and ZAGS nanocomposite films.

Figure 7.

Cyclic voltammogram measurement with In₂O₃ (IN), In₂O₃–G (ING), and In₂O₃-G-SiO₂ 20% (INGS20) samples without E. coli.

3.3. Electrochemical Measurements on Immobilized Bacteria

Figure 8 shows that the current of ZAGS was lower than that of IGS because of the unique structure of the quaternary nanocomposite. Here, E. coli was immobilized on the electrodes’ surface. Figure 8a,b demonstrates a progression of CV estimations completed in IGS and ZAGS electrodes with E. coli immobilized from their solutions in lysogeny broth (LB) (stock solution). The two diagrams show IGS and ZAGS electrochemical responses related to characteristic oxidation and reduction peaks. The current was about 0.0002 mA/cm² for IGS and 0.0003 mA/cm² for ZAGS, which diminishes with the introduction of E. coli due to the bacteria-adsorbed surface acting as an insulator to decline the current. The current was lowest when there were E. coli. The reason was that the E. coli entrapped the surface of the electrode and blocked the permeating channel. As E. coli was not electroactive, it could hinder the electron transfer rate, resulting in bigger electron transfer resistance. Therefore, ZAGS had high sensitivity.

The previous investigations are based on the improvement of microbe-based inhibition sensors, which is far from actual sensor improvement. Real sensor advancement utilizes bacteria immobilized on the electrode surface. In this work, E. coli was immobilized on the surface of IGS and ZAGS electrodes. Figure 9 shows the current variation with different concentrations of E. coli by IGS and ZAGS electrodes with E. coli immobilized from their solutions of different concentrations in LB broth (stock solution concentration of 10¹–10¹⁰ CFU/mL). The two diagrams show the electrochemical responses in IGS and ZAGS. As shown in Figure 9a,b, the estimated current was gradually decreased on increasing the concentration of E. coli. Negative currents usually correspond to reduction peaks for cathodic reactions (negative currents). An anodic current is a type of partial current that refers to the electrons entering an electrode in an electrode reaction. To understand the full current of an electrode reaction, it is necessary to consider the cathodic current, which is the flow of electrons leaving an electrode. Here, i–t curve showed the baseline current that means the cathodic current (negative current) is constantly linear, which corresponds that the electrode does not tend to lose an electron. The spikes of current are in the positive range, which means that electrons enter the electrode. On the other hand, in each cycle of cyclic voltammogram, with increasing E. coli concentration the peak current started falling because E. coli hindered the electron transfer to the electrode. From the spike of current in Figure S2, the calibration plot linearity range of the biosensor is demonstrated. E. coli blocks the electron transfer of the electrode through the immobilization of the electrode, thus increasing the resistivity of the sensor with increasing concentration of E. coli rather than conductivity. The cathode current I_c at −0.3 V increases with a low concentration of E. coli. Estimations of ΔI_c/I_c for E. coli reduce progressively with the increase of concentration, while E. coli are unaffected by the electrode in a wide concentration.

Figure 9.

(a) Concentration dependency (i–t curve) of E. coli by In₂O₃-G-SiO₂ (IGS) and (b) ZrO₂-Ag-G-SiO₂ (ZAGS) sensor.

3.4. Specificity of the Sensor

Here, the selectivity of IGS and ZAGS sensor for E. coli, compared to other bacteria strains, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella enterica, (each at 10⁹ CFU/mL), was employed, and the PBS sample was set as the reagent blank. However, there was no significant variation in the current response between the bacterial samples and the reagent control except E. coli, which is shown in Figure 10. For E. coli, a significant change in the current response was observed. Therefore, we can conclude that the sensors have good specificity. Figure S3 demonstrates the SEM and TEM images of ZAGS and IGS sensors exposed after the electrochemical test. Compared to Figure 2, it was found that no observable structural changes occurred in the sensor surface of the composites.

Figure 10.

Selectivity test of E. coli with ZrO₂-Ag-G-SiO₂ (ZAGS) sensor (a) and In₂O₃-G-SiO₂ (IGS) sensor (b).

3.5. Sensing Performance

IGS and ZAGS sensors are made on FTO electrodes with a mesoporous material as the conducting channel. In IGS and ZAGS, the mesoporous material links the source and the channel electrodes. Under ordinary conditions, GO is electrically insulating because of the existence of immersed sp³ bonds, the high density of electronegative oxygen atoms attached to carbon, and different deformities that increase the energy gap in the electron density of states. The conductivity of GO depends on its chemical and atomic structure because of the degree of structural disorder arising due to substantial sp³ carbon fraction. GO-based films are found to be of an insulating nature with a sheet resistance of around 10 ′Ω/sq. The insulating nature of GO can be linked to the concentration of sp³ C–O bonds, resulting in a decrease of electron transfer and leading to the disruption of the percolating pathway of the sp² carbon cluster.⁴⁴ After thermal reduction, because of bigger sp² domains, the mesoporous material and graphene turn out to be electrically conducting and the IGS and ZAGS sensors demonstrate resistance between 50 and 150 kΩ. The sensing of E. coli is subjected to a balance of electrical conductivity of the mesoporous material and IGS and ZAGS sensors. It does not react to the increase of the analyte. We additionally considered the impact of the distinctive thickness of the FTO glass layer (1, 2, 3, and 4 nm) on the appearance of the IGS and ZAGS sensors. The dimension of the coating was 5 mm and the thickness of the coating was about 0.1, 0.3, and 0.5 mm and had uniform thickness and dimensions. The performance was quite different between the film thicknesses of 0.1 and 0.5 mm. IGS and ZAGS sensors with a film thickness of 0.3 mm demonstrated the biggest response.⁴⁵ For sensing tests, the sensor was exposed to different concentrations of E. coli in DI water (10¹–10¹⁰ CFU/mL). The test sample volume of 1 μL was utilized for experiments. The real number of E. coli identified on the sensor relies upon both the concentration of E. coli and the sample volume utilized for the experiment. Even though the lowest concentration of the E. coli sample utilized for detection was 10¹ CFU/mL, considering calculations, the sensor could identify a E. coli cell. The sensing of E. coli cells by IGS and ZAGS sensors from the sample is reflected by the alteration in the electrical conductivity of the mesoporous channel. There was a linear reduction in current after the expansion of E. coli aliquot from 10¹ to 10¹⁰ CFU/mL. The IGS and ZAGS sensors had a quick response time for E. coli identification, which is one of the significant benefits of sensors. This was recognized as the fast diffusion of ions from the liquid drop on the surface of the device to the contact region.⁴⁶ Although E. coli is bigger than ions, the low volume of the sample reduces the diffusion length and allows rapid investigation of samples. The described techniques for E. coli recognition generally include at least 30 min of incubation time after various sensor design steps.⁴⁷ The FTO glass/mesoporous IGS and ZAGS was used as a control analysis. The increase of E. coli did not produce any signal, demonstrating that the E. coli IGS and ZAGS sensors respond simply after binding with E. coli. The conductivity of sensing mechanism between the FTO glass layer and the mesoporous IGS and ZAGS channel is regulated after interacting with negatively charged E. coli cells. The mesoporous layer is the potential electron reservoir for free electrons since oxygen vacancies in FTO glass fail to locate the corresponding empty O₂ p conduction band states. The 1–2 μm measured E. coli, which is rich in negative charge on its surface, is in the region close to the surface of the electron-rich FTO glass layer. This puff of negative charge further decreases the electron-donating capacity of FTO glass. To re-establish its insulating property, the oxygen-deficient domain boundary of FTO glass carries on like a local electron donor and mesoporous IGS and ZAGS as recipients.⁴⁷ The majority carriers (i.e., holes) in the mesoporous IGS and ZAGS merge with these electrons. This decline in the concentration of majority charge carriers diminishes the current flow between sources and channels. Moreover, the sensing mechanism can be associated with the gating impact of E. coli cells present on the sensor surface. The cell loses positive charge, which is instigated by adsorption of negatively charged E. coli cells. The dimensions of the positive charge on cells diminish with the increasing concentration of E. coli. This diminished positive charge of cells shows that ZAGS and IGS are less negatively charged since the counteracting agent itself can function as a dielectric medium. Subsequently, the concentration of holes in the mesoporous IGS and ZAGS from the gate-insulator/semiconductor interface, i.e., the current flow between the source and channel, declines because of the adsorption of E. coli. Over an edge concentration of 10¹⁰ CFU/mL, there are abundant E. coli cells in the sample solution. As opposed to coming in direct contact with the FTO glass layer, E. coli cells can be stacked over one another. The viewed outcome is because of the increasing E. coli cells in the sample solution, which decreases the conductivity of the sensor. This decrease in conductivity can be ascribed to the surface layer of E. coli cells. This created more holes in the mesoporous IGS and ZAGS channels along these lines, increasing the active site of IGS and ZAGS sensors. In this way, there are contending impacts wherein it prompts a decline in the current (10¹–10¹⁰ CFU/mL). By following the graph for real-time detection of E. coli, the impact of solution resistivity on IGS and ZAGS sensor is due to the increasing concentration of E. coli that progressively declines after the increase of 10¹⁰ CFU/mL of cells. This implies that E. coli is adsorbed on the surface of electrodes and acts as an insulating layer, diminishing the current. The current response of the manufactured electrochemical sensor to various concentrations of E. coli and controlled phosphate buffer solution was analyzed. The targeted E. coli was recognized by estimating the electrochemical signal of the IGS and ZAGS samples. It has been described that the electrochemical recognition technique proposes rapid signal transduction requiring a low cost. The catalytic current diminished linearly with the increasing concentration of E. coli from the scope of 10¹ × 10¹⁰ with a coefficient (r² = 0.879). The absolute time needed from the manufacture of the biosensor to the detection of E. coli was 75 min. In this way, this technique sensed E. coli in a lower concentration of 10¹ CFU/mL and with the maximum range of 10¹⁰ CFU/mL. At the point when E. coli was added, it was observed that the state of the CV curve changed significantly. The E. coli were cultured for 16 h at 37 °C and then transferred with PBS buffer for sensing. Despite this, the E. coli were distinguished by the developed sensor directly after the culture was grown. The leaving time was 2 s, and scanning time for two portions was 10 s. It was affirmed that the electrochemical signal was diminished by adding various concentrations of E. coli. These results demonstrate that the IGS and ZAGS samples are very delicate and sensitive for E. coli detection. The linear correlation between distinctive bacterial concentration and signal-to-noise ratio is r² = 0.879. The SNR was determined with the peak current of IGS and ZAGS sample to each concentration of E. coli. Then, it was isolated by the current 0.1 V of the blank, which was utilized as the signal of the electrochemical sensor. The signal-to-noise proportions of 10¹, 10², 10³, 10⁴, and 10⁵ CFU/mL were 0.64, 1.27, 1.40, 1.50, and 1.68 μA, respectively. The manufacture and experimental time of the targeted E. coli is 75 min. Thus, a sustainable method takes less time in contrast to the recently announced report (Table 2).³⁷⁻⁴² Subsequently, the most minimal concentration was 10¹ and the highest concentration was 10¹⁰ CFU/mL. This study demonstrates that the IGS and ZAGS sample is an efficient method to deal with very sensitive recognition materials for selected bacteria. The 10 scans keep running on the blank electrode without E. coli concentration, at which point the standard deviation of σ = 0.0087 of 10 scan runs of blank electrode response was determined, and then, there was an equation of limit of detection (LOD) with three times of the standard deviation equation (3 × σ)/m, where m, the decided slope (0.879) of the determined outcomes, was a limited deviation. We also investigated the stability of the ZAGS and IGS sensor under ambient conditions, as shown in Figure 11. Furthermore, the ZAGS and IGS electrode permitted exact control of the consistency of the film, which thus added to the stability of the sensor. When conserved at a storage temperature of 4 °C, IGS and ZAGS sensors did not demonstrate any major fading in the experiment following 7, 14, 21, and 28 days of fabrication. PBS can help the total recovery of the sensor surface without distressing IGS and ZAGS activity. The stability of such E. coli IGS and ZAGS sensors as far as reusability goes needs further analysis. The sensing tests were done at 25 °C. Increase in temperature will particularly influence the conductivity of mesoporous nanocomposites. Since our sensor was prepared for room-temperature investigation, we did not expand our analysis for a range of temperatures.

Figure 11.

Stability test of ZrO₂-Ag-G-SiO₂ (ZAGS) and In₂O₃-G-SiO₂ (IGS) sensor.

3.6. Mechanism of E. coli Sensing

The electrocatalytic effect of electrodes on the electrochemical decline was examined. IGS and ZAGS sensors in 0.1 M phosphate buffer support at pH 7.4 at an applied potential of −0.2 V were studied. An alternate effect was seen when the IGS and ZAGS electrodes were stored overnight in 0.1 M phosphate buffer. This effect is supposed to be the alteration in the hydration of the thin film. To demonstrate that was not just a collection impact because of the existence of the thin film, a comparable investigation was made with a cellulose layer to immobilize the thin film at the surface of the electrode. At the point when the biosensor was left overnight in a buffer solution with E. coli (the response yet going), the difference between the first and the second determination was observed. Various concentrations of E. coli were immobilized at the surface. Increasing the concentration of E. coli at the surface of the electrode (10¹–10¹⁰ CFU/mL) improved the recognition of the location of 1.17 × 10^–7 M and the linear domain somewhere in the range of 1.96 × 10^–5 and 2.55 × 10^–4 M of the improved electrode. The loss of the biosensor’s conductivity in time could be due to the inactivation or to the loss of the redox reaction from the biosensor’s surface. Figure 7 shows the regular i–t curves of IGS and ZAGS to increase the resistivity. Several E. coli were captured at the surface of the electrode. E. coli blocks the ion change membrane through the immobilization of the electrode. Thus, electron transfer is disrupted and the resistivity of the sensor rather than conductivity is increased. Better residuals with the increase of time could be clarified by film hydration. Better repeatability with time was also observed (Scheme 2).

Scheme 2. (a) Bacteria-Sensing Mechanism.

Electron transfer disruption and increase of resistivity of the sensor due to E. coli captured at the surface of the electrode. (b) E. coli blocks the ion change membrane through the immobilization of the electrode.

4. Conclusions

In this study, we have demonstrated a comparative electrochemical-based label-free, specific, sensitive detection of the waterborne pathogen E. coli. The electrochemical method for the ZAGS and IGS sensors is more sensitive, faster, and simpler than the conventional methods used to detect living bacteria in solution. The ZAGS and IGS biosensors detect E. coli in a lower concentration of 10¹ CFU/mL and with the maximum range of 10¹⁰ CFU/mL and it outputs accurate quantitative results. Future developments of this electrode must include reducing detection limits, increasing the selectivity of resistant bacteria, integrating this analytical method into paper-based devices, and detecting drug resistance in real and complex samples to identify different bacteria. By observing the cells adhered to the ZAGS and IGS sensors, this method can provide a platform to study the properties of a single cell after separation and/or attachment onto the sensor surface. This approach can also be applied to study other biomolecules. We believe that this method provides a robust potential for simple and specific detection of E. coli and antibiotic-resistant E. coli.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c00895.

• XPS spectra of C 1s, O 1s, Si 2p, In 3d, Zr 3d, and Ag 3d (Figure S1); calibration curve of In₂O₃-G-SiO₂ (IGS) sensor and ZrO₂-Ag-G-SiO2 (ZAGS) sensor (Figure S2); SEM and TEM images of In₂O₃-G-SiO₂ and ZrO₂-Ag-G-SiO₂ (Figure S3); nomenclature samples (Table S1) (PDF)

The authors declare no competing financial interest.