Polypyrrole functionalized (Ti₃C₂T_x–SnO₂ NPs) nanocomposite-based hybrid capacitive electrode for electrochemical detection of Klebsiella pneumoniae

Abstract

Bacterial detection is crucial for accurate clinical diagnostics and effective environmental monitoring. Particularly, Klebsiella pneumoniae, a pathogenic bacterium, can cause a wide range of infections, including meningitis, bloodstream infections, pneumonia, urinary tract infections, and wound or surgical site infections. Herein, a polypyrrole (PPy) functionalized Ti₃C₂T_x–tin dioxide nanoparticle (SnO₂ NPs) nanocomposite-based hybrid capacitive electrode for the electrochemical detection of Klebsiella pneumoniae ATCC 700603 is developed. The PPy layer was coated onto the Ti₃C₂T_x–SnO₂ NPs via drop-casting, followed by immobilization of bacteriophages through a potentiostatic, charge-directed chronoamperometric approach. The resulting Ti₃C₂T_x–SnO₂ NPs/PPy/phage biosensor exhibited a wide dynamic detection range of 10 to 10⁶ CFU mL⁻¹, with excellent linearity confirmed by differential pulse voltammetry and electrochemical impedance spectroscopy. The nanocomposite was characterized by using a suite of techniques including FTIR, XRD, elemental mapping, cyclic voltammetry, and galvanostatic charge–discharge to understand its composition, structure, and electrochemical properties. The developed Ti₃C₂T_x–SnO₂ NPs/PPy hybrid electrode demonstrated typical supercapacitor behavior with a specific capacitance of 806.67 F g⁻¹ at 2.0 A g⁻¹ of current density, and exhibited exceptional cycling stability, storing 98.3% of its capacitance after 10⁴ consecutive cycles. The selectivity of the modified electrode to detect K. pneumoniae while minimizing interference from various bacterial cells was assessed, exhibiting remarkable resilience, and remaining unaffected. Additionally, after eleven successive weeks of storage, the proposed sensor showed no discernible reduction current (∼98.1%), demonstrating an excellent stability. Despite the presence of background bacterial interference in the environmental sample, K. pneumoniae detection remained highly reliable and consistent with recovery efficiency ranging from 99.75 to 99.90%.

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1 Introduction

Klebsiella pneumoniae (K. pneumoniae), is an opportunistic pathogen that causes severe infections, particularly in immunocompromised individuals, hospitalized patients, and those with chronic conditions.¹ The ATCC 700603 strain, originally isolated from a hospitalized patient in Virginia, is known for producing the SHV-18 β-lactamase enzyme, contributing to its extended-spectrum β-lactamase (ESBL) activity. It serves as a reference strain for ESBL detection and antimicrobial susceptibility testing.² This strain commonly causes hospital-acquired pneumonia, especially in chronic respiratory patients, urinary tract infections, bloodstream infections, and other conditions like meningitis and liver abscesses.³ Other types of disease, including wound infections, endophthalmitis, and necrotizing fasciitis, are caused by these bacteria.⁴ Symptoms vary by infection site and include fever, cough with bloody sputum, shortness of breath, painful urination, abdominal pain, chills, and confusion.⁵ Therefore, there is a critical need for fast and precise detection of K. pneumoniae ATCC 700603 infections to enable timely antibiotic treatment. Early detection is crucial because these infections, especially those involving antibiotic-resistant strains, can be serious and require prompt intervention to improve patient outcomes. Furthermore, existing diagnostic methods often struggle with issues like inadequate sensitivity, selectivity, and long detection times.

Conventional detection of K. pneumoniae relies on microbiological techniques involving isolation and identification, which can be time-consuming and labor-intensive. DNA-based biosensors offer an alternative but have limitations such as complex fabrication, low stability, non-specific binding, high costs, and poor performance in complex samples like blood or environmental matrices.^6,7 These challenges restrict their practical use in clinical diagnostics and environmental monitoring. Enzyme-Linked Immunosorbent Assay (ELISA), widely used for antigen and antibody detection, is prone to cross-reactivity, which may cause false positives. Its accuracy depends heavily on the specificity of antibodies and the quality of reagents.⁸ Similarly, polymerase Rhain reaction (PCR) is a highly sensitive method for amplifying DNA, but DNA extraction from clinical samples can be laborious.⁹ Moreover, PCR cannot distinguish between live and dead pathogens, reducing its reliability in assessing active infections. In contrast, phage-based electrochemical biosensors present a promising alternative.¹⁰ These biosensors leverage bacteriophages' natural specificity for their host bacteria, enabling rapid, sensitive, and cost-effective detection of viable pathogens. They are also easier to fabricate and can operate effectively in diverse sample conditions, making them suitable for both clinical and environmental applications.

Charge-directed immobilization of bacteriophages using chronoamperometry is essential for biosensor fabrication. This process enables the effective construction of bacteriophage-immobilized biorecognition elements for detecting target whole-cell bacteria.^11–14 In this process, applying a positive voltage to the sensor surface promotes the preferred orientation and stable attachment of bacteriophages, particularly those with positively charged tails and negatively charged heads.¹⁵ Accordingly, the electrochemical capacitive properties promote efficient bacteriophage attachment and ensure its stable retention on the sensor surface.¹⁶ In this study, PPy-functionalized capacitive electrode was engineered for efficient bacteriophage immobilization to detect K. pneumoniae. Similarly, Umer Farooq et al.,¹⁷ used a bacterial cellulose-carbon nanotube nanocomposite modified with polyethyleneimine to detect S. aureus in milk at a 5.0 CFU mL⁻¹ limit. Another study used a polyacrylonitrile-derived carbon nanofiber SPE to detect E. coli, achieving a 36 CFU mL⁻¹ detection limit across 10²–10⁶ CFU mL⁻¹.¹⁸ In this study, the PPy-functionalized electrode surface was modified with bacteriophages using a charge-directed chronoamperometry method for targeting K. pneumoniae. This approach offers a more straightforward alternative to previously reported biosensors.

The PPy composites exhibit excellent electrical properties and have been widely explored for applications in sensors,¹⁹ energy storage,²⁰ electromagnetic interference (EMI) shielding,²¹ pharmaceuticals,²² and catalysis.²³ Enhancing PPy's electrochemical performance often involves “p-doping”-the introduction of dopant anions to improve conductivity and alter the material's morphology and physicochemical characteristics.²⁴ They are commonly synthesized by hydrothermal/solvothermal,²⁵ in situ growth,²⁶ solution mixing,²⁷ electrochemical deposition,²⁸ and layer-by-layer assembly.²⁹ Among the large family of 2D Ti₃C₂T_x MXene, and owing to its large surface area, high electrical conductivity, and unique surface chemistry, Ti₃C₂T_x is frequently cited as the most commonly used and versatile Ti₃C₂T_x material for sensor applications,³⁰ supercapacitors,³¹ Li–S batteries,³² and Li-ion batteries³³ because of the active T_x group (e.g., –O, –OH, and –F).³⁴ But, due to strong van der Waals interactions between the layers and hydrogen bonds, Ti₃C₂T_x forms a stacking and aggregation film. Thus, Ti₃C₂T_x is usually used to form composites with transition metals (e.g., SnO₂, TiO₂, ZnO) and other electrochemically active materials.^35,36 Tin dioxide (SnO₂) is a key n-type semiconductor, depicted by its wide band gap (3.6 eV), high electron mobility (100–200 cm² V⁻¹ s⁻¹), and large exciton binding energy (130 meV), making it suitable for optoelectronic and sensing applications.³⁷ This research utilized hydrothermal methods to synthesize the Ti₃C₂T_x–SnO₂ nanocomposite. Ti₃C₂T_x can act as a buffer layer to help move electrons across the composite and reduce the relatively low conductivity of SnO₂.¹⁸ Furthermore, SnO₂ can enhance the structural integrity and cycle life of the electrodes.

A Ti₃C₂T_x–metal oxide–PPy hybrid composite enhances supercapacitor flexibility and performance by combining high conductivity with structural support. Ti₃C₂T_x ensures conductivity and stability, while the metal oxide and PPy improve both conductivity and flexibility.³⁸ Ti₃C₂T_x can achieve a specific capacitance of ∼245 F g⁻¹ in H₂SO₄ due to pseudocapacitance when stacking is avoided.^39,40 However, its inherent capacitance is limited and can be significantly enhanced by integrating redox-active materials to form synergistic composites. For instance, Liang Tong et al.,⁴¹ developed a PPy/Ti₃C₂T_x composite demonstrating exceptional electrochemical performance, with a specific capacitance of 420.2 F g⁻¹ and remarkable cycling stability (86% capacitance retention after 10⁴ charge–discharge cycles), representing a significant enhancement over the individual components. Chen Zhao et al.,⁴² fabricated a Ti₃C₂T_x/SnO₂ composite via hydrothermal synthesis, achieving an exceptional specific capacity of 904 mA h g⁻¹. It shows that this synthetic technique is beneficial for energy storage applications. As a result, numerous strategies have been explored to improve the electrochemical performance of Ti₃C₂T_x, particularly targeting enhancement of its specific capacitance.

The Ti₃C₂T_x–SnO₂ NPs/PPy hybrid nanocomposite is electrochemically evaluated in this work as a hybrid capacitive electrode for electrochemical biosensing targeting the detection of K. pneumoniae. The formation mechanism of the Ti₃C₂T_x–SnO₂ NPs nanocomposite involves several key steps, including the preparation of Ti₃C₂T_x, the synthesis or incorporation of SnO₂ NPs. For this, pre-synthesized SnO₂ NPs were dispersed with Ti₃C₂T_x in a solvent (e.g., ethanol/water) and sonicated to achieve homogeneity. The Sn–O–Ti covalent bonds were formed between SnO₂ NPs and oxygen-terminated Ti₃C₂T_x. Hydrogen bonding was formed between –OH groups on SnO₂ NPs and Ti₃C₂T_x, and Ti₃C₂T_x acted as a support, inhibiting NPs agglomeration. Ti₃C₂T_x provided high conductivity, while SnO₂ contributes to catalytic/adsorptive properties. Enhanced charge transfer (SnO₂ NPs → Ti₃C₂T_x) improves performance in applications, like energy storage, electrochemical biosensors. Therefore, Ti₃C₂T_x–SnO₂ NPs can enhance surface area for electrochemical sensing, and these properties can be further improved by integrating PPy functional materials.

In this study, Ti₃C₂T_x–SnO₂ NPs nanocomposites were synthesized and incorporated into PPy to fabricate an electrochemical capacitive electrode for biosensing applications. The electrochemical capacitive nature of Ti₃C₂T_x–SnO₂ NPs/PPy was characterized by cyclic voltammetry (CV), galvanostatic charge–discharge (GCD), electrochemical impedance spectroscopy (EIS), and Dunn methods. The results revealed that Ti₃C₂T_x–SnO₂ NPs/PPy exhibited a capacitive (supercapacitor) electrode type. The capacitive electrode (Ti₃C₂T_x–SnO₂ NPs/PPy) was modified with bacteriophage using charge-directed immobilization by applying a potential of +1.0 V for 10 minutes. Afterwards, the Ti₃C₂T_x–SnO₂ NPs/PPy/Phage electrode demonstrates effective infection and binding specificity toward the host K. pneumoniae. The bacterial-infected electrochemical response was measured via differential pulse voltammetry (DPV) and impedance-increasing EIS techniques. This is the first reported research on PPy functionalized capacitive electrode for electrochemical detection of K. pneumoniae, explaining the capacitive nature towards immobilization of bacteriophages. In this research, the developed hybrid capacitive electrode proved to be an efficient, selective, and cost-efficient sensor for quantifying K. pneumoniae to overcome the gap of conventional detection methods.

2 Reagents & methods

2.1. Chemicals

Materials were all analytical grades, and they were used as received with no extra purification. Ammonium persulfate (APS) (98%), pyrrole monomer (99.7%), and stannous chloride dihydrate (SnCl₂·2H₂O) (99%) were obtained from Aladdin Reagent Co. (China). Ti₃AlC₂ MAX phase in powder form (particle size smaller than 0.04 nm) was obtained from Y-Carbon Ltd (Ukraine) as the Ti₃AlC₂ precursor, which was subsequently etched to produce Ti₃C₂T_x following the previously reported protocol.⁴³ Potassium ferrocyanide (K₄[Fe(CN)₆]) (99.0%), potassium ferricyanide (K₃[Fe(CN)₆])(99.0%), and potassium chloride (KCl) (99.5%) were purchased from Merck Life Science Pvt. Ltd Screen-printed carbon electrodes (SPEs, 3 mm diameter) were sourced from CH Instruments (Bee Cave, TX, USA). Type I-ultrapure deionized water (DI) with a resistance of 0.0182 Ω cm at room temperature was utilized for all solutions in this work. All glassware was thoroughly cleaned before use. K. pneumoniae was cultured by following the standard ATCC 700603. The SM buffer for bacteriophage storage was made by sodium chloride NaCl, magnesium sulfate (MgSO₄), and tris-hydrochloric acid (pH 7.5), and 10% gelatin (w/v) obtained from Aladdin Reagent Ltd (China). Furthermore, nutrient agar and Luria-Bertani were acquired from Oxoid (UK). 0.85% of NaCl was used to dilute the K. pneumoniae.

2.2. Instrumentation and characterization

The topology of the Ti₃C₂T_x flakes, SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite modified electrode was examined employing a scanning electron microscope (SEM, ZEISS Gemini SEM 500, UK) operated at 15 kV of accelerating voltage. The crystalline phases of Ti₃C₂T_x were analyzed with an X-ray diffractometer (D/teX Ultra2, EMPYREAN, PANalytical, Netherlands), using Cu Kα radiation (λ = 154 10⁻³ μm) over a 2θ range of 10°–95° at a scan rate of 2° min⁻¹ with texture mode. The working distance was maintained between 10.0 and 11.1 mm, and the emission current ranged from 9000 to 10 100 nA. EIS, CV, and DPV measurements were conducted utilizing two electrochemical workstations: Corrtest CS350 (China) and AUT 53154 (Metrohm, Switzerland). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, PerkinElmer, Waltham, MA, USA) was employed to analytically characterize the structure of the synthesized materials. EIS analysis was conducted across a frequency range from 0.1 MHz to 0.1 Hz in 0.1 mol per L KCl containing 0.005 mol per L [Fe(CN)₆]^3−/4− to evaluate the resistivity. For the electrochemical detection of Klebsiella pneumoniae, all media, buffers, and glassware were thoroughly sterilized before their use with both K. pneumoniae and its corresponding bacteriophage.

2.3. Fabrication of Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite

SnO₂ NPs were fabricated via a modified, template-free hydrothermal method, adapted from previously reported protocols.^44–46 In a typical procedure, 800 mg of stannous chloride dihydrate (SnCl₂·2H₂O) was dissolved in a solvent mixture of 0.04 L of ethanol and 80 mL of DI. The solution was mixed continuously and heated to 50 °C for 60 minutes. The resultant mixture was transferred to a Teflon-lined stainless-steel autoclave and was exposed to hydrothermal treatment at 180 °C for 8 hours. The white precipitate was collected by centrifugation, washed repeatedly with DI and ethanol, and vacuum-dried at 50 °C for 48 hours. For enhancing their electrochemical properties, the dried SnO₂ nanoparticles (SnO₂ NPs) underwent calcination in air at 600 °C for 3 hours, with a controlled heating rate of 10 °C per minute.

The as-prepared SnO₂ NPs were ultrasonically dispersed with Ti₃C₂T_x for 45 minutes, followed by mixing for 30 minutes to form the Ti₃C₂T_x–SnO₂ NPs nanocomposite. The synthesis of PPy was carried out based on previously reported procedures.^47–49 Specifically, 1.369 g (60 mM) of APS was dissolved in 0.1 L of DI, and 210.2 μL (0.03 M) of pyrrole monomer was added while maintaining the reaction temperature at 10 °C to obtain a black suspension. The solution was mixed for 8 hours to guarantee complete polymerization. The resulting PPy product was collected by filtration and purified using a Soxhlet extractor with water and ethanol to eliminate residual oxidant. Then, a vacuum oven set at 60 °C for 12 hours was used to dry the purified PPy powder. A graphic illustration of the fabrication and surface decoration strategy for the Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite is presented in Scheme 1. For the modification of the SPE, the Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite was deposited onto the electrode surface using a drop-casting technique. First, the Ti₃C₂T_x–SnO₂ NPs nanocomposite was dissolved in N,N-dimethylformamide and dropped-cast onto the SPE. Subsequently, PPy was dispersed in N,N-dimethylformamide, dropped-casted onto the SPE/Ti₃C₂T_x–SnO₂ NPs, and vacuum dried at 60 °C and stored for further usage.

Scheme 1 Graphic representation of the preparation of PPy and Ti₃C₂T_x–SnO₂ NPs powder for SPE/Ti₃C₂T_x–SnO₂ NPs/PPy fabrication.

2.4. Pneumoniae and phage preparation

2.4.1. Revival and preparation of K. pneumoniae. K. pneumoniae ATCC 700603, a well-characterized reference strain known for its sensitivity to meropenem, as reported in previous studies⁵⁰ was employed in this investigation. The strain was provided by the Department of Microbiology at Jashore University of Science and Technology. To revive the strain, 100 μL of glycerol stock was inoculated into 5 × 10³ μL of trypticase soy broth under standard conditions. Following enrichment, the culture was streaked onto MacConkey agar plates and incubated at 37 °C overnight to isolate individual colonies. Morphologically distinct colonies were examined microscopically and subjected to a series of standard biochemical tests to confirm their identity as K. pneumoniae before their use in downstream experiments. K. pneumoniae was isolated from hospital effluent water collected near a river in Jashore city, Bangladesh. For the isolation process, 10 mL of filtered effluent sample was inoculated into 0.9 L of Mueller–Hinton broth and incubated at 37 °C overnight. Following incubation, the enriched culture was spread onto MacConkey selective agar to allow colony formation. Individual colonies were then purified and subjected to microscopic examination and standard biochemical tests. Presumptive K. pneumoniae isolates, intended as host bacteria for bacteriophage studies, were further cultured in Mueller–Hinton broth and subsequently placed on Mueller–Hinton agar to obtain pure cultures. Biochemical characterization, along with SI data from a previously published study, confirmed that the isolated strain is identical to Klebsiella pneumoniae ATCC 700603.⁵⁰

2.4.2. Phage isolation and purification. For phage isolation, several sewage samples were gathered from hospital effluents. The samples were first centrifuged at 10⁴ rpm for 15 minutes. The supernatant was then sieved through a 220 nm syringe filter to isolate bacteriophages potentially targeting unknown bacterial hosts. For phage enrichment, 0.005 L of the filtered bacteriophage-containing solution was mixed with 10 mL of Luria–Bertani (LB) broth and inoculated with K. pneumoniae strain ATCC 700603. The solution was incubated for 24 hours at 37 °C with gentle shaking. Next, the culture was centrifuged for 15 minutes to lyse the bacterial cells, and the supernatant was again sieved using a 220 nm syringe filter to purify the bacteriophages. To enhance selectivity and purity, this infection, centrifugation, and filtration cycle was repeated ten times using fresh K. pneumoniae cultures. The presence of lytic bacteriophages was confirmed via spot tests, which involved spotting phage suspensions onto bacterial lawns and incubating overnight at 37 °C until zones of clearance were observed. Each phage isolate was subjected to five successive rounds of single plaque purification to obtain clonal bacteriophages. Then, the Bacteriophage titer was determined using the double-layer agar plate technique. Serial dilutions of the phage suspensions were prepared in SM buffer for accurate quantification. Furthermore, the host range and spot test were performed.

2.5. Electrode preparation and K. pneumoniae detection

The SPE was first activated via electrochemical pre-anodization in 1.0 M H₂SO₄ solution at +1.5 V for 15 minutes. Next, the electrode was ultrasonicated in DI for 5 minutes and then dried under a nitrogen stream. For surface modification, 5.0 μL of a freshly prepared homogeneous suspension of Ti₃C₂T_x–SnO₂ NPs nanocomposite was drop-casted onto the SPE and dried at 60 °C in a vacuum oven. Then, the second drop-casting step was performed using a PPy dispersion to complete the composite layering. The total amount of deposited electrode material was determined by direct weighing. The resulting SPE/Ti₃C₂T_x–SnO₂ NPs/PPy electrode was employed directly for supercapacitor characterization and subsequently functionalized with bacteriophages for biosensor applications. For phage immobilization, 5.0 μL of bacteriophage solution (10⁸ PFU mL⁻¹ in PBS) was drop-casted onto the electrode and immobilized anodically at +1.0 V for 15 minutes using a chronoamperometric method. After immobilization, the electrode was washed with PBS to get rid of unbound phages. To perform bacterial detection, the biosensor was incubated in a solution containing K. pneumoniae for 30 minutes, allowing sufficient interaction between the immobilized phages and the target bacteria. The electrode was then washed out again with PBS to eliminate nonspecifically adhered bacterial cells. Finally, electrochemical signals of the SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage/K. pneumoniae biosensor were measured in a [Fe(CN)₆]^3−/4− redox probe solution utilizing DPV and EIS.

3 Results and discussion

3.1. Analytical characterization: FTIR & XRD investigation

Fig. 1 shows the ATR-FTIR spectra of Ti₃C₂T_x, SnO₂ NPs, Ti₃C₂T_x–SnO₂ NPs, PPy, and Ti₃C₂T_x–SnO₂ NPs/PPy. In the spectrum, the absorption bands at 1130, 822, and 505 cm⁻¹ correspond to C–O, C–F, and Ti–O stretching vibrations.⁵¹ The C–F and Ti–O were confirmed by following the etching and exfoliation procedure, and they matched with previously published research.⁴³ The spectrum of the annealed SnO₂ NPs shows peaks at about 505 and 605 cm⁻¹, showing the vibrations connected to Sn–O bonds.⁵² For Ti₃C₂T_x–SnO₂ NPs, the peaks around 609 cm⁻¹ and 487 cm⁻¹ are for the vibration of Sn–O and Ti–O bonds, respectively, confirming the presence of Ti₃C₂T_x and SnO₂ NPs. During analysis of PPy spectrum, the peaks at 1689, 1552, and 1471 cm⁻¹ are attributed to stretching vibration of the pyrrole ring (C=H, C–N). Additionally, those at 1296 cm⁻¹ and 1042 cm⁻¹ are due to the in-plane vibrations of C–H.^53,54 The sharp peak observed at 1181 cm⁻¹ is because of the C–N stretching vibrations, and 1042 cm⁻¹ is for the in-plane deformation vibration.⁵⁴ The peak appearing at 901 cm⁻¹ relates to the C–C deformation vibrations, and the peaks at 788 cm⁻¹ and 675 cm⁻¹ are due to the aromatic ring bending vibrations.⁵⁵ Furthermore, PPy in Ti₃C₂T_x–SnO₂ NPs shows the symmetric peaks for PPy. However, all peaks are slightly shifted to the left side with increasing wavenumber. These shifts provide insight into interfacial bonding, the electronic environment, or structural reorganization, thereby confirming the successful formation of a composite. FTIR spectroscopy is a powerful tool for detecting such interactions, as it reveals changes in the vibrational environment of functional groups. Shifts in stretching frequencies, for example, in –OH, –NH, or –C=O groups, may signify hydrogen bonding or covalent interactions between the matrix and the filler.⁵⁶ Additionally, the appearance or disappearance of specific peaks can indicate new chemical bonding or functionalization events during composite formation. For instance, a redshift (shift to a lower wavenumber) in the –OH stretching band is typically associated with hydrogen bonding between polymer chains and nanoparticles, providing direct evidence of interfacial interaction.⁵⁷

Fig. 1 (A) ATR-FTIR of as-prepared (i) Ti₃C₂T_x, (ii) SnO₂ NPs, (iii) Ti₃C₂T_x–SnO₂ NPs, (iv) PPy, (v) Ti₃C₂T_x–SnO₂ NPs/PPy, (B) XRD of (i) Ti₃AlC₂, (ii) Ti₃C₂T_x, (iii) SnO₂ NPs, (iv) Ti₃C₂T_x–SnO₂ NPs, (v) Ti₃C₂T_x–SnO₂ NPs/PPy.

The XRD patterns of Ti₃AlC₂, Ti₃C₂T_x, SnO₂ NPs, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy are shown in Fig. 1(B). The peaks of Ti₃AlC₂ at 2θ values of 9.54°, 19.1°, 34.04°, 38.86°, 41.8°, 48.38°, 56.32°, 60.02°, 70.18°, and 73.86° are assigned to the (002), (004), (004), (104), (105), (107), (109), (110), (112), (118), respectively, according to JCPDS no. 520875.⁵⁸ The crystallinity and structural order of Ti₃AlC₂ changed by disappearing the Al layers at 38.86° with HF etching for Ti₃C₂T_x synthesis.⁴³ In Ti₃C₂T_x, the (002) peak is displaced to 5.46° for the removal of Al atoms. This peak shift is a well-established fingerprint of the successful Al layer removal and the formation of few-layered or delaminated Ti₃C₂T_x, often indicating increased interlayer spacing due to surface functionalization or intercalation. The XRD results of as-fabricated SnO₂ NPs shown in Fig. 1(B), display the diffraction patterns peaks at (110), (101), (200), (105), (211), (220), (202), and (321) at 2θ diffraction angles of 35.16°, 46.4°, 52.44°, 58.04°, 73.7°, 78.22°, 83.68°, and 89.22°, consecutively. These peaks are found in the tetragonal rutile structure of the cassiterite crystal phase (JCPDS no. 41-1445).^59,60 In the Ti₃C₂T_x–SnO₂ NPs spectrum, both Ti₃C₂T_x and SnO₂ NPs individually show the peak of diffraction planes; however, Ti₃C₂T_x peaks overlap with SnO₂ NPs. The diffraction planes of the peaks of (002), (004), (110), (101), (200), (105), (211), (220), (202), and (321) are displayed in Ti₃C₂T_x–SnO₂ NPs, which confirms the successful mixing of Ti₃C₂T_x–SnO₂ NPs nanocomposite. Eventually, the XRD pattern of Ti₃C₂T_x–SnO₂ NPs/PPy indicates the reflection plane of Ti₃C₂T_x–SnO₂ NPs with a new (100) plane. The peaks at (100) with 2θ diffraction angle indicate the presence of PPy in Ti₃C₂T_x–SnO₂ NPs/PPy. This analysis supports the results obtained from the FTIR analysis, indicating the successful formation of a composite among Ti₃C₂T_x, SnO₂ NPs, and PPy.

3.2. Morphological investigations

Field Emission Scanning Electron Microscopy (FE-SEM), Energy-Dispersive X-ray Spectroscopy (EDX), and elemental mapping were utilized to characterize the topology and elemental analysis of the modified electrode. The FE-SEM images were collected from hydrothermally synthesized SnO₂ NPs with a 100 nm scale, as shown in Fig. 2(A). The nanoparticle is nanosphere-like, and the diameter was found to be 24.97 nm, from the average of five particle sizes. The FE-SEM image supports the FT-IR and XRD results of SnO₂ NPs. Fig. 2(B) displays the microscopic image of PPy nanoparticles, which are around five times greater than the particle size of SnO₂ NPs. The FE-SEM images are like previously reported research work.^61–63 Fig. 2(C) displays a FE-SEM picture of multilayered, crystalline Ti₃C₂T_x. As demonstrated in previously reported work, each flake has a thickness of around 1.25 nm and a hexagonal structure.⁴³ Fig. 2(D) displays the FE-SEM image of Ti₃C₂T_x–SnO₂ NPs nanostructure, confirming the insertion of SnO₂ NPs in Ti₃C₂T_x, and increasing the porosity and surface area. The FT-IR and XRD results, along with recently published research, support the formation and electrochemical applications of Ti₃C₂T_x–SnO₂ NPs nanocomposite.^64,65 In Fig. 2(E), the FE-SEM image of Ti₃C₂T_x–SnO₂ NPs/PPy is presented, in which PPy is covered on the Ti₃C₂T_x–SnO₂ NPs nanostructure. The homogeneous dispersion of PPy confirms the successful attachment to the Ti₃C₂T_x–SnO₂ NPs structure. The SEM images in Fig. 2(A–E) demonstrate a progressive increase in surface porosity, attributed first to the incorporation of SnO₂ NPs into Ti₃C₂T_x, as evident in Fig. 2(D), and further enhanced by the subsequent drop-casting of circular PPy structures, as illustrated in Fig. 2(E). Therefore, this modification strategy led to the successive increase of active surface area.

Fig. 2 FE-SEM images of (A) SnO₂ NPs, (B) PPy, (C) Ti₃C₂T_x, (D) Ti₃C₂T_x–SnO₂ NPs, (E) Ti₃C₂T_x–SnO₂ NPs/PPy, (F) EDX of Ti₃C₂T_x–SnO₂ NPs/PPy, (G) elemental mapping of Ti₃C₂T_x–SnO₂ NPs/PPy, (H) carbon map, (I) nitrogen map, (J) oxygen map, (K) titanium map, and (L) tin map.

The Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode consists of 68.15% of C, 11.74% of O, 2.11% of Ti, and 16.70% of N, as shown in the EDX results represented in Fig. 2(F), indicating the C, O, Ti elements from Ti₃C₂T_x and N from PPy. Furthermore, Fig. 2(G–L) display the elemental mapping of Ti₃C₂T_x–SnO₂ NPs/PPy, where the C, N, O, Ti, and N are visually represented by distinct colors shown in Fig. 2(H–L). Therefore, the element shown in the EDX spectra was identified by elemental mapping.

3.3. Ti₃C₂T_x–SnO₂ NPs/PPy for electrochemical biosensing

In the phage-based biosensor, bacteriophage was attached to the Ti₃C₂T_x–SnO₂ NPs/PPy modified according to potential-driven chronoamperometry techniques, where the current follows a constant concerning time (s) and potential (V), as presented in Fig. 3(A and B). As the current remains constant around 1.0 V, the immobilization process feels like charging, as displayed in Fig. 3(C). As phage immobilization is a charging phenomenon, the charge holding time analysis is important. The charge retaining capability was studied utilizing the self-discharging data presented in Fig. 3(D–G) of Ti₃C₂T_x–SnO₂ NPs/PPy electrode in H₂SO₄. The H₂SO₄ electrolyte is an alternative to phage, where H₂SO₄ is mostly utilized as an ionic medium to explain the capacitive behavior of the electrode material.⁶⁶ While a well-capacitive surface can support bacteriophage immobilization, the instability of bacteriophages, particularly at room temperature, poses a significant challenge for long-term capacitive measurements in phage-based biosensors. Careful consideration of phage stability and the implementation of appropriate preservation strategies are crucial for developing reliable phage-based capacitive biosensors.

Fig. 3 (A) Potentiostatic (chronoamperometry) immobilization of bacteriophage, (B) corresponding current of potentiostatic immobilization at 1.0 V, (C) corresponding charge during potentiostatic immobilization, (D) self-discharge plot of Ti₃C₂T_x–SnO₂ NPs/PPy electrode in H₂SO₄ after charging (2.0 Ag⁻¹ up to 0.5 V), and corresponding plot of (E) log(potential) versus time of self-discharge, (F) potential vs. time^0.5, (G) voltage versus log(time), (H) DPV response, (I) Bode plot of showing Ti₃C₂T_x–SnO₂ NPs/PPy, after immobilization of bacteriophage, and attacking of K. pneumoniae.

Moreover, the stability of the bacteriophages under varying temperature and pH conditions is illustrated in Fig. S1(A and B) of the SI. At temperature values of −20, 4, 25, and 37 °C, the concentration of bacteriophage remains constant but decreases at over 56, 70, and 100 °C. Also, the bacteriophage was stable at an optimum pH of 7.0 without destroying any plaques but decreases at lower and higher pH levels of 4.0 and 10.0. To assess the self-discharge behavior of the Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode, it was initially charged up to 0.5 V with a 2 A g⁻¹ of current density. The corresponding self-discharge profile is presented in Fig. 3(D). The electrode exhibited a minor voltage drop during the early stage of the self-discharge activity, followed by a stable stage where no significant voltage loss was observed, indicating excellent charge retention. The self-discharge mechanism was interpreted based on methodologies previously reported.⁶⁷ The self-discharge of capacitive or supercapacitor electrodes can be attributed to three primary factors: (i) diffusion-controlled redox reactions, (ii) overcharging beyond the optimal voltage window, and (iii) ohmic leakage through the internal resistance of the device.⁶⁸ Fig. 3(E) presents the plot of the logarithm of (V) vs. time (t), displaying a consistent linear relationship indicative of a diffusion-controlled self-discharge process, with no evidence of ohmic leakage in the Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode.⁶⁹ The plot of voltage (V) vs. t^0.5 shown in Fig. 3(F) demonstrates linearity in the steady-state region, indicating the predominance of capacitive-controlled processes and the absence of ohmic leakage during self-discharge. Moreover, the linear profile observed in the V versus log(t) plot confirms that the self-discharge of the Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode is not governed by overcharging-induced charge redistribution.⁷⁰

A comparative oxidation peak of Ti₃C₂T_x–SnO₂ NPs/PPy, Ti₃C₂T_x–SnO₂ NPs/PPy/Phage, and Ti₃C₂T_x–SnO₂ NPs/PPy/Phage/K. pneumoniae was collected in 100 mmol per L KCl containing 5 mmol L⁻¹ of [Fe(CN)₆]^3−/4−. From the DPV study, the oxidation current was found to be 218.27 μA for SPE/Ti₃C₂T_x–SnO₂ NPs/PPy, 102.73 μA for SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage, and 61.11 μA for SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage/K. pneumoniae. The oxidation current gradually decreased from the developed SPE/Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode due to the successful immobilization of phage and hosting K. pneumoniae on the immobilized phage. Furthermore, impedance-based EIS data were collected for Ti₃C₂T_x–SnO₂ NPs/PPy, Ti₃C₂T_x–SnO₂ NPs/PPy/Phage, and Ti₃C₂T_x–SnO₂ NPs/PPy/Phage/K. pneumoniae functionalized electrodes, and the Bode plot is displayed in Fig. 3(I). Bode plot reveled that the charge transfer resistance (R_ct) was determined to be 200 Ω for SPE/Ti₃C₂T_x–SnO₂ NPs/PPy, 3424 Ω for Ti₃C₂T_x–SnO₂ NPs/PPy/Phage, and 6260 Ω for Ti₃C₂T_x–SnO₂ NPs/PPy/Phage/K. pneumoniae. The R_ct value gradually increased from the developed SPE/Ti₃C₂T_x–SnO₂ NPs/PPy due to immobilization of Phage and hosting K. pneumoniae on immobilized bacteriophage, confirming the presence of capacitive double layer (C_dL). Therefore, bacteriophage immobilization and K. pneumoniae hosting are certainly capacitive phenomena. The corresponding Nyquist plot of Fig. 3(I) is shown in Fig. S1(C). The phage and K. pneumoniae can successfully decrease the DPV current and increase the value of R_ct. So, the DPV and EIS results assured the successful immobilization of bacteriophage onto the surface of the Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode after hosting the targeted K. pneumoniae ATCC 700603. Also, the attacking ability of the isolates of bacteriophage to K. pneumoniae ATCC 700603 and the hospital effluent collected K. pneumoniae is presented in Fig. S2(A and B). The host range testing results towards Salmonella typhi (S. typhi), E. coli DH5α, E. coli 0157:H7, Pseudomonas aeruginosa (P. aeruginosa), Shigella flexneri (S. flexneri), Vibrio cholerae (V. cholera), Salmonella enterica (S. enterica), and Listeria monocytogenes (L. monocytogenes) are selective as shown in Fig. S2(C–J). This study confirms that the K. pneumoniae ATCC 700603 strain and the collected K. pneumoniae are identical.

3.4. Electrode characterization through supercapacitor studies

The power law equation controlling the correlation between peak current and scan rate is considered to comprehend the charge storage mechanism, as shown in eqn (1). The correlation between the logarithm of scan rate and the logarithm of peak current is presented in Fig. 4(A). Here, the slope is used to determine the b value.⁶⁸ In general, a b-value between 0.5 and 1 suggests a combination of diffusion-controlled and surface-controlled (pseudocapacitive) charge storage. A b-value closer to 0.5 indicates that diffusion-controlled processes dominate, while a value closer to 1 implies that capacitive behavior is more dominant. For PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy, the b value is 0.656, 0.456, 0.962, and 0.830, respectively, in which Ti₃C₂T_x–SnO₂ NPs show the highest b-value, and the b value for PPy increased from 0.656 to 0.830 in Ti₃C₂T_x–SnO₂ NPs/PPy. The results show that the hybrid electrode has both surface and diffusion processes. Furthermore, diffusion-controlled charge storage and surface capacitance are estimated using the widely recognized Dunn method, as outlined in eqn (3) and (4). In this case, I(V), k₂v^1/2 and k₁v stands for the total current at a given voltage, the diffusion contribution, and the surface control contribution, respectively. Fig. 4(B) represents the capacitive and surface-controlled contribution (%) plot of Ti₃C₂T_x–SnO₂ NPs/PPy. Based on eqn (4), the plot of I(V)/v^1/2 versus k₁v^1/2 for Ti₃C₂T_x–SnO₂ NPs/PPy was used to calculate the constant k₁ and k₂ at the potential of 0.335 V for the anodic sweep, with the scan rate varying from 10 to 60 mV s⁻¹. At a scan rate of 5 mV s⁻¹, the surface control contribution is 90% and reaches 96% at 60 mV s⁻¹. This is because there is not enough time for a diffusion layer to develop at a fast scan rate.

I = av^b (1)

log I = log a + b log v (2)

I(V) = k₁v + k₂v^1/2 (3)

I(V)/v^1/2 = k₁v^1/2 + k₂ (4)

Fig. 4 In 1.0 mol per L H₂SO₄ solution, (A) determination of b value of Ti₃C₂T_x–SnO₂ NPs/PPy and sub electrodes, (B) contribution (%) of capacitive controlled and diffusion controlled charge storage processes of Ti₃C₂T_x–SnO₂ NPs/PPy (C) CV and (D) GCD comparison profile of PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy, (E) comparison of corresponding specific capacitance of CV and (F) GCD, (G) Nyquist and (H) Bode plot of Ti₃C₂T_x–SnO₂ NPs/PPy, (I) GCD stability at the charging–discharging current density of 4.0 A g⁻¹ over 10 000 cycles.

To evaluate the electrochemical properties of the sythasized materials, PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy, CV and GCD measurements were conducted using a 3-electrode setup, with 1 mol per L H₂SO₄ as the aqueous electrolyte. Fig. 4(C) presents a comparison of the CV curves. They were recorded at a scan rate of 50 mV s⁻¹, with voltage range of −0.3–0.4 V for Ti₃C₂T_x–SnO₂ NPs, and −0.5–0.5 V for PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs/PPy (vs. Ag/AgCl). The recorded rectangular CV curves indicate ideal capacitive behavior with current response proportional to scan rate and independent of the potential. However, these rectangular shapes can be distorted by various factors, including faradaic reactions and pseudocapacitive behavior.⁶⁹ The CV curve also indicates that current changes instantaneously with voltage changes, with constant capacitance within the voltage range.

The Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode showed an oxidation peak at a voltage of 0.335 V, indicating Faradaic behavior of pseudocapacitance electrode. Furthermore, the CV curve has a rectangular shape, thus, Ti₃C₂T_x–SnO₂ NPs/PPy is a pseudocapacitance-type supercapacitor electrode. The CV results for PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy are shown in Fig. S(3). Furthermore, the GCD comparison profile of PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy, is displayed in Fig. 4(D), and the detailed GCD curves are presented in Fig. S(4). From the GCD curves, it was observed that a rectangular shape was obtained with no IR drop. Therefore, the CV and GCD revealed that Ti₃C₂T_x–SnO₂ NPs/PPy is a supercapacitor-type electrode. The CV specific capacitance comparison is shown in Fig. 4(E), where Ti₃C₂T_x–SnO₂ NPs/PPy show higher than PPy, Ti₃C₂T_x–PPy, and Ti₃C₂T_x–SnO₂ NPs, which was calculated from eqn (5). The specific capacitance of Ti₃C₂T_x–SnO₂ NPs/PPy was found to be 795.0 F g⁻¹, 271.85 F g⁻¹, 444.0, and 468 F g⁻¹ for PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs at a scan rate of 0.005 V s⁻¹. Similarly, the specific capacitance of the GCD curve is shown in Fig. 4(F) and was calculated using eqn (6). The PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy, nanocomposites show specific capacitances of 290.4, 447.5, 471.32, and 806.67 F g⁻¹ at a 2 A g⁻¹ current density. In Fig. 4(F), Ti₃C₂T_x–SnO₂ NPs/PPy shows a higher specific capacitance curve than that of PPy, Ti₃C₂T_x–PPy, and Ti₃C₂T_x–SnO₂ NPs. eqn (5) and (6) were used to calculate specific capacitance (C_sp) from the CV and GCD curves, respectively:⁷⁰

where ∫I(V)dV is the constrained area under the CV curve under the current in (A), ΔV is the potential window or cut-off voltage in (V), I is the instant discharge current value in (A), v is the scan rate in (mV s⁻¹), m represents the mass of the electrode materials in grams, and Δt denotes the discharge time in seconds.

This highlights the fact that Ti₃C₂T_x–SnO₂ NPs/PPy modified is a highly capacitive supercapacitor electrode. The Nyquist plot for Ti₃C₂T_x–SnO₂ NPs/PPy and the Bode plot for the phage angle are displayed in Fig. 4(G and H). The Nyquist plot displayed in Fig. 4(G), shows an upper trending linear diagram without a semicircle. The Bode plot of the phage angle versus log(frequency) shown in Fig. 4(H) presents the maximum phage angle of 83.37°, which is near the ideal capacitive (−85°) nature of Ti₃C₂T_x–SnO₂ NPs/PPy electrode. Furthermore, the Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode shows 98.3% stable performance after 10 000 consecutive cycles as presented in Fig. 4(I). The GCD stability graph at the end of 10 000 cycles is shown in Fig. S(5). The stability of GCD was driven at the current density of 4.0 A g⁻¹. A comparison of the supercapacitor performance of this work and previously published Ti₃C₂T_x/PPy-based electrodes are summarized in Table 1.

Table 1 Supercapacitor performance comparison of this work with previously published Ti₃C₂T_x/PPy-based electrodes

Electrode material	Synthesis method of PPy	Specific capacitance F g^−1	Current density or scan rate	Potential range (V)	Electrolyte	Capacitance retention (%)	Ref.
Ti3C2Tx/PPy	Chemical oxidation	416	5 mV s^−1	−0.2–0.35	H2SO4	92% over 25 × 10^3 cycles	71
Ti3C2Tx/PPy-PVA	Chemical oxidation	614	1 A g^−1	−0.5–0.6	H2SO4	100% over 10^3 cycles	72
Ti3C2Tx@PPY NW	Chemical oxidation	610	25 A g^−1	0–0.6	KOH	100% over 14 000 cycles	73
Ti3C2Tx/PPy	Chemical oxidation	437	5 mV s^−1	−0.8–0.2	H2SO4	76% 10^3 cycles	74
Ti3C2Tx/PPy@Cotton	Chemical oxidation	506.6	1 A g^−1	0–0.5	H2SO4	83.3% over 2000 cycles	75
Ti3C2Tx–SnO2	Chemical oxidation	471.32	2.0 A g^−1	−0.3–0.4	H2SO4	N/A	This work
Ti3C2Tx/PPy	Chemical oxidation	448.4	2.0 A g^−1	−0.5–0.5	H2SO4	N/A	This work
Ti3C2Tx–SnO2 NPs/PPy	Chemical oxidation	806.67	2.0 A g^−1	−0.5–0.5	H2SO4	98.3% over 10^4 GCD cycles	This work

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3.5. Electrochemical mechanism of K. pneumoniae detection

The link between the structural and compositional characterization of the nanocomposite with its functional performance, particularly in biosensing and electrochemical energy storage, is explained through Scheme 2(A and B). In Scheme 2A, the arrangement of Ti₃C₂T_x, SnO₂ NPs, and PPy is presented, where SnO₂ NPs are inserted in Ti₃C₂T_x and PPy is covered on Ti₃C₂T_x–SnO₂ NPs. Thus, the successful formation of Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite was characterized via electrochemical testing for biosensors and their capacitive nature through supercapacitor properties studies. Additionally, in Scheme 2B, electrochemical charging alters the electrical properties of the modified surface. These changes influence how effectively bacteriophages can attach and bind to the surface. Thereafter, the phage can attack the K. pneumoniae, and the binding bacterial concentration is calculated by using electrochemical EIS and DPV techniques. This work explores the nanocomposite of Ti₃C₂T_x, SnO₂ NPs, and PPy for dual applications in biosensing and electrochemical energy storage.⁷⁶

Scheme 2 (A) electrochemical mechanism of Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite formation towards biosensor and electrochemical capacitor, (B) mechanism of PPy in biosensor and electrochemical capacitor.

The composite leverages the individual properties of each component: Ti₃C₂T_x for conductivity and surface area, SnO₂ NPs for enhanced electrochemical interaction, and PPy for electroactivity and charge transport, leading to a material capable of both biosensing and electrochemical capacitive functionality.⁷⁷ The study investigates the electrochemical mechanisms of negative ion attachment and phage immobilization on the electrode surface as capacitive phenomena to optimize the dual functionality of the nanocomposite.

3.6. Electrochemical detection of K. pneumoniae

The analytical performance of Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposites depends on their building block materials, like SnO₂ NPs, Ti₃C₂T_x, and PPy. Here, the analytical performance is tested in standard [Fe(CN)₆]^3−/4− solution by DPV and EIS techniques. The DPV experiment was conducted at potential window of 0–0.5 V. EIS was operated at a wide frequency range, from as low as 10⁻¹ Hz to as high as 10⁶ Hz. With the optimized immobilization protocol, prepared SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage was infected with multiple concentrations of K. pneumoniae. Host bacterial concentrations were 10¹, 10², 10³, 10⁴, 10⁵, and 10⁶ CFU mL⁻¹. After bacterial immobilization on SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage, the electrode was used for DPV anodic oxidation and EIS non-Faradic impedance measurement in [Fe(CN)₆]^3−/4−. The respective data are plotted in Fig. 5(A–E). In the DPV plot, a linear dynamic range of 10–10⁶ CFU mL⁻¹ was maintained with the corresponding regression equation of y = −7.845x + 65.452, and R² value of 0.98594, as shown in Fig. 5(A and B). The bacteria-infected electrode surface impedance was measured using EIS in the same [Fe(CN)₆]^3−/4−.

Fig. 5 (A) DPV and (B) calibration plot; (C) Nyquist, (D) Bode, and (E) calibration plot of SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage biosensor-based detection of 10¹, 10², 10³, 10⁴, 10⁵, and 10⁶ CFU mL⁻¹ of K. pneumoniae.

The corresponding Nyquist plot and Bode plot of impedance spectra are shown in Fig. 5(C–E). The R_ct value shows a linearity with the regression equation. In both DPV and EIS, it appears that the peak current is decreased with increasing bacterial concentration in DPV; in contrast, the diameter of a semicircle of the Nyquist plot grew with increasing K. pneumoniae concentrations 10–10⁶ CFU m L⁻¹. A comparison of phage-modified electrochemical biosensors between this work and previously published research for K. pneumoniae quantification is summarized in Table 2.

Table 2 Comparison of phage-modified electrochemical biosensors for the detection of K. pneumoniae^a

Electrodes	Modification type	Target bacteria	Assay type	Detection range (CFU mL^−1)	LOD (CFU mL^−1)	Sample	Ref.
a Abbreviations: BC: bacterial cellulose, c-MWCNT: carboxylated multiwalled carbon nanotube, PEI: polyethyleneimine, PAN: poly-acetonitrile; CNF: carbon nanofibre, 11-MUA: 11-mercaptoundecanoic acid, 3-MPA: 3-mercaptopropionic acid, NHS: N-hydroxy-succinimide, EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
BC/c-MWCNT/PEI	CL	Staphylococcus aureus	DPV	3.0–3.0 × 10^7	3.0	PBS	78
				5.0–5.0 × 10^6	5.0	Milk	78
PEI/c-CNT	EI	Staphylococcus aureus	EIS	10^2–10^7	1.23 × 10^2	Water	13
					1.23 × 10^2	Blood plasma
PAN-derived CNF	EI	Escherichia coli	EIS	10^2–10^6	36	PBS, apple juice	18
AuE/11-MUA-3MPA/NHS-EDC	EI	Escherichia coli	DPV	1.9 × 10^1–1.9 × 10^8	14 ± 5	SM buffer	14
Ti3C2Tx–SnO2 NPs/PPy	EI	K. pneumoniae	DPV, EIS	10–10^6	1.0	PBS	This work

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3.7. Selectivity, repeatability, stability, and practical application

To evaluate the performance of the Ti₃C₂T_x–SnO₂ NPs/PPy electrode-based biosensor, a series of experiments were conducted to assess the selectivity, repeatability, stability, and real sample analysis. DPV measurements were carried out after attacking several bacteria (S. typhi, E. coli DH5α, P. aeruginosa, E. coli 0157:H7, S. flexneri, V. cholerae, S. enterica, L. monocytogenes) onto the SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage to study the biosensor's selectivity, and the results are presented in Fig. S6(A). The targeted host K. pneumoniae (10³ CFU mL⁻¹) attacked the SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage modified electrode, and in DPV response, a 34 μA oxidation signal is recorded. On the contrary, the non-target host is not attacked on SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage, and the DPV response remains around 100 μA oxidation signal. So, the results can reveal that the biosensor can quantify K. pneumoniae with superior selectivity in the presence of many other interfering bacterial cells. Reproducibility is a key parameter for assessing the precision of electrochemical biosensors, primarily reflecting the stability of the biosensor itself. The precision of the biosensor was assessed by measuring its response five times at a K. pneumoniae concentration of 10⁴ CFU mL⁻¹, as shown in Fig. S6(B).

To evaluate the stability of the SPE/Ti₃C₂T_x–SnO₂ NPs/PPy biosensor, the electrodes were stored at 4 °C. Every seven days, the functionalized electrodes were refreshed by introducing K. pneumoniae (10³ CFU mL⁻¹) and immobilizing the phage. DPV measurements were then performed, and the findings are displayed in Fig. S6(C). These findings demonstrate the impact of storage duration on the stability of the developed biosensor. The stability reaches 98.1% after eleven consecutive weeks with a gradual decrease in the activity of Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite modified electrode. This indicates that, compared to previously reported phage-modified electrochemical biosensors, the biosensors developed in this study demonstrate superior stability.^13,79 To confirm the practical application of the developed biosensors as an effective diagnostic tool for monitoring K. pneumoniae, a recovery test was conducted in environmental samples (soil, agricultural water, hospital effluent, tap water, and urine). The collected data is presented in Table 3. To prepare a real sample, the bacteria were combined with an environmental sample and extracted using the procedure described in Subsection 2.4.1. The whole protocol was similarly maintained for the quantification of K. pneumoniae. The recovery efficiency of the designed SPE/Ti₃C₂T_x–SnO₂ NPs/PPy/Phage biosensor was outstanding, ranging from 99.75 to 99.90%. Despite the presence of background bacterial interference in the environmental sample, K. pneumoniae detection remained highly reliable and consistent.

Table 3 Recovery test of K. pneumoniae in soil, agricultural water, hospital effluent, tap water, and urine using the proposed biosensor

Sample name	Add (CFU mL^−1)	Detected (CFU mL^−1)	Recovery (%)
Soil	10^4	9975	99.75
Agricultural water	10^4	9990	99.90
Hospital effluent	10^4	9980	99.80
Tap water	10^4	9993	99.93
Urine	10^4	9988	99.88

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4 Conclusion

Herein, a capacitive electrochemical biosensor electrode based on a Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite for bacteriophage immobilization and the selective detection of K. pneumoniae was successfully engineered. The fabricated Ti₃C₂T_x–SnO₂ NPs/PPy electrode exhibited capacitive behavior, with a b-value of 0.830 derived from the power law equation and a 90% capacitive contribution at a scan rate of 10 mV s⁻¹, as determined using the Dunn method. Comparative evaluation of individual and binary components PPy, Ti₃C₂T_x–PPy, Ti₃C₂T_x–SnO₂ NPs, and Ti₃C₂T_x–SnO₂ NPs/PPy revealed b-values of 0.656, 0.456, and 0.962, and corresponding specific capacitances of 290.4, 447.5, and 471.32 F g⁻¹, respectively. Remarkably, the Ti₃C₂T_x–SnO₂ NPs/PPy nanocomposite achieved an elevated specific capacitance of 806.67 F g⁻¹ and a phase angle of −83.37°, closely approximating the ideal capacitive response (−85°). These findings confirm the capacitive nature and superior electrochemical performance of the Ti₃C₂T_x–SnO₂ NPs/PPy modified electrode. Upon immobilization of bacteriophages, the resulting Ti₃C₂T_x–SnO₂ NPs/PPy/Phage biosensor demonstrated efficient detection of K. pneumoniae using DPV and EIS techniques, covering a dynamic linear detection range of 10–10⁶ CFU mL⁻¹. The practical applicability of the developed sensor was validated using complex real-world samples, including soil, agricultural water, hospital effluent, and urine matrices. Overall, the Ti₃C₂T_x–SnO₂ NPs/PPy/Phage biosensor offers a promising platform for both environmental surveillance and clinical diagnostics, owing to its high sensitivity, stability, and adaptability to diverse sample types. This is the first to report on PPy-based functionalized capacitive electrochemical detection of K. pneumoniae.

Ethics approval

Healthy urine samples were collected from healthy donors, who provided written informed consent for this study according to the Declaration of Medical Services. The blood drawing and experimental procedures were performed in accordance with the Guidelines for Care and Use of Human Samples of Jashore University of Science and Technology (JUST) and approved by the Medical Ethics Committee of Jashore-7408, Bangladesh.

Author contributions

Md. Abdul Khaleque: electrode fabrication, data collection, analysis, writing original draft, review and editing; Moumita Rahman: bacteriophage and bacteria culture, and writing original draft; Selina Akter: methodology, data analysis, and writing original draft; Md. Romzan Ali: data analysis, review and editing; Syed Imdadul Hossain: methodology, investigation, and review and editing; Rahman Saidur: Ti₃C₂T_x synthesis, reviewing and editing; Mohamed Aly Saad Aly: conceptualization, methodology, analysis and discussion, supervision, project management, and reviewing and editing; Md. Zaved H. Khan: conceptualization, methodology, review and editing, and supervision.

Conflicts of interest

The authors declare that there is no conflict of interest.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5ra03642a.

Acknowledgements

This work received specific funding from Georgia Tech Shenzhen Institute (GTSI) and Jashore University of Science and Technology.

References

1. C. L. Gorrie, M. Mirčeta, R. R. Wick, L. M. Judd, M. M. C. Lam, R. Gomi, I. J. Abbott, N. R. Thomson, R. A. Strugnell, N. F. Pratt, J. S. Garlick, K. M. Watson, P. C. Hunter, D. V. Pilcher, S. A. McGloughlin, D. W. Spelman, K. L. Wyres, A. W. J. Jenney and K. E. Holt, Nat. Commun., 2022, 13, 3017,  DOI:10.1038/s41467-022-30717-6.
2. A. G. Elliott, D. Ganesamoorthy, L. Coin, M. A. Cooper and M. D. Cao, Genome Announc., 2016, 4(3), e00438,  DOI:10.1128/genomeA.00438-16.
3. D. Chang, L. Sharma, C. S. Dela Cruz and D. Zhang, Front. Microbiol., 2021, 12, 750662,  DOI:10.3389/fmicb.2021.750662.
4. Z. Zhang, H. W. Yu, G. C. Wan, J. H. Jiang, N. Wang, Z. Y. Liu, D. Chang and H. Z. Pan, J. AOAC Int., 2017, 100, 548–552.
5. N. Florien, S. Sharma, D. Kala, S. Gupta, A. Kaushal and D. Kumar, Discov. Life, 2025, 55(1), 1–12.
6. M. R. Ali, M. S. Bacchu, M. A. A. Setu, S. Akter, M. N. Hasan, F. T. Chowdhury, M. M. Rahman, M. S. Ahommed and M. Z. H. Khan, Biosens. Bioelectron., 2021, 188, 113338,  DOI:10.1016/j.bios.2021.113338.
7. M. S. Bacchu, M. R. Ali, S. Das, S. Akter, H. Sakamoto, S. I. Suye, M. M. Rahman, K. Campbell and M. Z. H. Khan, Anal. Chim. Acta, 2022, 1192, 339332,  DOI:10.1016/j.aca.2021.339332.
8. H. A. Hussein, A. Kandeil, M. Gomaa and R. Y. A. Hassan, Microsyst. Nanoeng., 2023, 9, 105,  DOI:10.1038/s41378-023-00578-0.
9. N. Gupta, in Journal of Cytology, Wolters Kluwer Medknow Publications, 2019, vol. 36, pp. 116–117.
10. Y. Ding, Y. Zhang, C. Huang, J. Wang, H. Li and X. Wang, Talanta, 2024, 270, 125561.
11. Y. Zhou, A. Marar, P. Kner and R. P. Ramasamy, Anal. Chem., 2017, 89, 5734–5741.
12. N. Bhardwaj, S. K. Bhardwaj, J. Mehta, G. C. Mohanta and A. Deep, Anal. Biochem., 2016, 505, 18–25.
13. D. Patel, Y. Zhou and R. P. Ramasamy, J. Electrochem. Soc., 2021, 168, 057523.
14. J. Xu, C. Zhao, Y. Chau and Y. K. Lee, Biosens. Bioelectron., 2020, 151, 111914,  DOI:10.1016/j.bios.2019.111914.
15. Å. Richter, K. Matuła, A. Les̈niewski, K. Kwas̈nicka, J. Łos̈, M. Łos̈, J. Paczesny and R. Hołyst, Sens. Actuators, B, 2016, 224, 233–240.
16. I. U. Haq, K. Rahim, S. Maryam and N. P. Paker, Biotech. Rep., 2025, 45, e00872,  DOI:10.1016/j.btre.2024.e00872.
17. U. Farooq, M. W. Ullah, Q. Yang, A. Aziz, J. Xu, L. Zhou and S. Wang, U. Farooq, M. W. Ullah, Q. Yang, A. Aziz, J. Xu, L. Zhou and S. Wang, Biosens. Bioelectron., 2020, 157, 112163,  DOI:10.1016/j.bios.2020.112163.
18. Y. L. Wang, L. Q. Fan, S. J. Sun, J. J. Chen, Z. X. Wu, T. T. Zhu, Y. F. Huang and J. H. Wu, Chem. Eng. J., 2022, 428, 131993.
19. I. B. Qader, H. K. Ismail, H. F. Alesary, J. H. Kareem, Y. T. Maaroof and S. Barton, J. Electroanal. Chem., 2023, 951, 117943.
20. L. Lan, Y. Li, J. Zhu, Q. Zhang, S. Wang, Z. Zhang, L. Wang and J. Mao, Chem. Eng. J., 2023, 455, 140675.
21. Ö. Yavuz, M. K. Ram, M. Aldissi, P. Poddar and H. Srikanth, Synth. Met., 2005, 151, 211–217.
22. W. A. El-Said, O. Nasr, A. I. A. Soliman, E. A. Elshehy, Z. A. Khan and F. K. Abdel-Wadood, Appl. Surf. Sci. Adv., 2021, 4, 100065,  DOI:10.1016/j.apsadv.2021.100065.
23. F. Liu, Y. Yuan, L. Li, S. Shang, X. Yu, Q. Zhang, S. Jiang and Y. Wu, Composites, Part B, 2015, 69, 232–236.
24. M. A. Khaleque, M. Aly Saad Aly and M. Z. H. Khan, Chem. Eng. J., 2025, 507, 160444,  DOI:10.1016/j.cej.2025.160444.
25. H. Ding, M. Zhang, Y. Liu, Y. Yao, Z. Mai, H. Zheng, B. Song, B. Fan, H. Wang and H. Lu, Ceram. Int., 2024, 50, 46334–46346.
26. K. Tian, X. Hui, H. Wang, Z. Zhang, L. Zhang, C. Wang and L. Yin, Electrochim. Acta, 2022, 415, 140242.
27. R. Shafique, M. Rani, K. Batool, A. A. Alothman, M. S. Saleh Mushab, A. A. Shah, A. Kanwal, S. Ali and M. Arshad, J. Mater. Res. Technol., 2023, 24, 2668–2677.
28. X. Liu, Y. Chen, H. Zhang, L. Zhuo, Q. Huang, W. Zhang, H. Chen and Q. Ling, J. Colloid Interface Sci., 2024, 660, 735–745.
29. E. Azizi, J. Arjomandi, H. Shi and M. A. Kiani, J. Energy Storage, 2024, 75, 109665,  DOI:10.1016/j.est.2023.109665.
30. D. Li, H. Liang and Y. Zhang, Carbon, 2024, 226, 119205.
31. T. K. Paul, M. A. Khaleque, M. R. Ali, M. Aly Saad Aly, M. S. Bacchu, S. Rahman and M. Z. H. Khan, RSC Adv., 2025, 15(12), 8948–8976,  10.1039/d5ra00271k.
32. W. Zhao, Y. Lei, Y. Zhu, Q. Wang, F. Zhang, X. Dong and H. N. Alshareef, Nano Energy, 2021, 86, 106120,  DOI:10.1016/j.nanoen.2021.106120.
33. N. Zhao, Y. Yang, D. Yi, Y. Xiao, K. Wang, W. Cui and X. Wang, Chem. Eng. J., 2021, 422, 130018,  DOI:10.1016/j.cej.2021.130018.
34. Q. Lu, C. Liu, Y. Zhao, W. Pan, K. Xie, P. Yue, G. Zhang, A. Omar, L. Liu, M. Yu and D. Mikhailova, SusMat, 2023, 3, 471–497.
35. D. Liang, P. Song, M. Liu and Q. Wang, Ceram. Int., 2022, 48, 9059–9066.
36. M. Cao, S. Liu, S. Liu, Z. Tong, X. Wang and X. Xu, Microchem. J., 2022, 175, 107068.
37. A. P. S. GAHLOT, R. Pandey, S. Singhania, A. choudhary, A. Garg and P. M. Gupta, Evolution of tin oxide (SnO2) nanostructures synthesized by hydrothermal method, 2021,  DOI:10.21203/rs.3.rs-1097943/v1.
38. B. Dharmasiri, K. A. S. Usman, S. A. Qin, J. M. Razal, N. T. Tran, P. Coia, T. Harte and L. C. Henderson, Chem. Eng. J., 2023, 476, 146739,  DOI:10.1016/j.cej.2023.146739.
39. M. R. Lukatskaya, O. Mashtalir, C. E. Ren, Y. Dall'Agnese, P. Rozier, P. L. Taberna, M. Naguib, P. Simon, M. W. Barsoum and Y. Gogotsi, Science, 2013, 341, 1502–1505.
40. M. Hu, Z. Li, T. Hu, S. Zhu, C. Zhang and X. Wang, ACS Nano, 2016, 10(12), 11344–11350,  DOI:10.1021/acsnano.6b06597.
41. L. Tong, C. Jiang, K. Cai and P. Wei, J. Power Sources, 2020, 465, 228267,  DOI:10.1016/j.jpowsour.2020.228267.
42. C. Zhao, Z. Wei, J. Zhang, P. He, X. Huang, X. Duan, D. Jia and Y. Zhou, J. Alloys Compd., 2022, 907, 164428,  DOI:10.1016/j.jallcom.2022.164428.
43. M. A. Khaleque, M. R. Ali, M. Aly Saad Aly, M. I. Hossain, K. H. Tan, M. A. Zaed, R. Saidur, M. M. Rahman, N. M. Mubarak and M. Z. H. Khan, Diamond Relat. Mater., 2024, 15, 111749,  DOI:10.1016/j.diamond.2024.111749.
44. Y. Kang, Z. Li, K. Xu, X. He, S. Wei and Y. Cao, J. Alloys Compd., 2019, 779, 728–734.
45. L. Tan, L. Wang and Y. Wang, Hydrothermal synthesis of SnO 2 nanostructures with different morphologies and their optical properties, J. Nanomater., 2011, 2011(1), 529874,  DOI:10.1155/2011/529874.
46. Y. Zhao, J. Liu, Q. Liu, Y. Sun, D. Song, W. Yang, J. Wang and L. Liu, Mater. Lett., 2014, 136, 286–288.
47. M. Sravanthi and K. G. Manjunatha, in Materials Today: Proceedings, Elsevier Ltd, 2020, vol. 46, pp. 5964–5968.
48. A. Yussuf, M. Al-Saleh, S. Al-Enezi and G. Abraham, Int. J. Polym. Sci., 2018, 2018, 4191747,  DOI:10.1155/2018/4191747.
49. J. Parayangattil Jyothibasu, M. Z. Chen and R. H. Lee, ACS Omega, 2020, 5, 6441–6451.
50. M. R. Ali, M. S. Bacchu, D. D. Ridoy, P. L. Mozumder, M. N. Hasan, S. Das, M. F. H. Palash, S. Akter, N. Sakib, A. Khaleque, D. Chakrobortty and M. Z. H. Khan, RSC Adv., 2022, 12, 31497–31505.
51. F. Zhang, W. Liu, S. Wang, C. Liu, H. Shi, L. Liang and K. Pi, Composites, Part B, 2021, 217, 108900,  DOI:10.1016/j.compositesb.2021.108900.
52. S. Z. Shahanshahi and S. Mosivand, Appl. Phys. A:Mater. Sci. Process., 2019, 125, 652,  DOI:10.1007/s00339-019-2949-2.
53. Y. Fu and A. Manthiram, RSC Adv., 2012, 2, 5927–5929.
54. G. Kulkarni, P. Kandesar, N. Velhal, H. Kim and V. Puri, J. Appl. Polym. Sci., 2021, 138(22), 50447,  DOI:10.1002/app.50447.
55. R. Turczyn, K. Krukiewicz, A. Katunin, J. Sroka and P. Sul, Compos. Struct., 2020, 232, 111498,  DOI:10.1016/j.compstruct.2019.111498.
56. E. Smidt and K. Meissl, Waste Manage., 2007, 27, 268–276.
57. V. A. Lorenz-Fonfria, Chem. Rev., 2020, 120(7), 3466–3576,  DOI:10.1021/acs.chemrev.9b00449.
58. M. Naguib, M. Kurtoglu, V. Presser, J. Lu, J. Niu, M. Heon, L. Hultman, Y. Gogotsi and M. W. Barsoum, Adv. Mater., 2011, 23, 4248–4253.
59. A. Debataraja, D. W. Zulhendri, B. Yuliarto, H. Nugraha and B. Sunendar, in Procedia Engineering, Elsevier Ltd, 2017, vol. 170, pp. 60–64.
60. T. Preethi, K. Senthil, S. Ashokan, R. Balakrishnaraja, B. Sundaravel and P. Saravanan, J. Nano- Electron. Phys., 2021, 13, 1–5.
61. C. Z. Brusamarello, L. M. Santos, A. P. Romio, M. Di Domenico, A. F. Santos, P. H. H. de Araújo and C. Sayer, Mater. Today Commun., 2021, 26, 101740,  DOI:10.1016/j.mtcomm.2020.101740.
62. N. Khan, R. Ullah, M. Khan and Almas, Electrochim. Acta, 2024, 505, 144988,  DOI:10.1016/j.electacta.2024.144988.
63. S. Dhineshkumar, E. Elanthamilan, S.-F. Wang, S. Rajkumar and J. P. Merlin, Electrochim. Acta, 2025, 512, 145525.
64. D. Liang, P. Song, M. Liu and Q. Wang, Ceram. Int., 2022, 48, 9059–9066.
65. S. F. Tseng, S. J. Cheng, W. T. Hsiao, S. H. Hsu and C. C. Kuo, Ceram. Int., 2024, 50, 43728–43737.
66. P. Sharma and V. Kumar, Mater. Today: Proc., 2020, 33, 1573–1578.
67. K. Krishnamoorthy, P. Pazhamalai, V. K. Mariappan, S. Manoharan, D. Kesavan and S. J. Kim, Adv. Funct. Mater., 2021, 3(10), 2008422,  DOI:10.1002/adfm.202008422.
68. R. Anjana, P. M. Anjana, J. Alex, R. Isaac, R. S. S. Hussain and D. Sajan, Diamond Relat. Mater., 2024, 146, 111241,  DOI:10.1016/j.diamond.2024.111241.
69. A. A. Mohamad, Inorg. Chem. Commun., 2025, 172, 113677.
70. B. Karuppaiah, R. Sukanya, S. M. Chen, P. R. Chavan, R. Karthik, M. Hasan, A. Milton and J. J. Shim, J. Energy Storage, 2024, 79, 110145.
71. M. Boota, B. Anasori, C. Voigt, M. Q. Zhao, M. W. Barsoum and Y. Gogotsi, Adv. Mater., 2016, 28, 1517–1522.
72. W. Zhang, J. Ma, W. Zhang, P. Zhang, W. He, J. Chen and Z. M. Sun, Nanoscale, 2020, 12, 6637–6643.
73. T. A. Le, N. Q. Tran, Y. Hong and H. Lee, Chem.—Eur. J., 2019, 25, 1037–1043.
74. J. Cao, Y. Han, X. Zheng and Q. Wang, J. Appl. Polym. Sci., 2019, 136(4), 47003,  DOI:10.1002/app.47003.
75. L. Yang, F. Lin, F. Zabihi, S. Yang and M. Zhu, Int. J. Biol. Macromol., 2021, 181, 1063–1071.
76. A. Al Mahmud, A. H. Alshatteri, H. S. Alhasan, W. Al Zoubi, K. M. Omer and M. R. Thalji, Electrochim. Acta, 2024, 503, 144857.
77. N. Neha and A. Dalvi, RSC Adv., 2025, 15, 6518–6530.
78. U. Farooq, M. W. Ullah, Q. Yang, A. Aziz, J. Xu, L. Zhou and S. Wang, Biosens. Bioelectron., 2020, 157, 112163,  DOI:10.1016/j.bios.2020.112163.
79. R. F. Wang and R. Wang, Bioelectrochemistry, 2022, 148, 108229,  DOI:10.1016/j.bioelechem.2022.108229.

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