Multifunctional hierarchical Ni₂CoS₄ structures for combating drug-resistant infections and colorectal cancer via piezocatalytic ROS generation

Abstract

The global rise of multidrug-resistant infections and cancer underscores the need for multifunctional therapeutic platforms. This study presents the synthesis, optimization, and biomedical assessment of sonicated hierarchical Ni₂CoS₄ structures, with sample N3 identified as optimal among seven hydrothermally synthesized formulations. N3 exhibited uniform morphology, high crystallinity, and redox-active architecture, as confirmed by XRD, SEM, TEM, and BET (8.19 m²/g). Piezocatalytic performance, assessed via methylene blue degradation, showed 88.57% removal within 4 min under 200 W ultrasonication, alongside a 13.27-fold increase in singlet oxygen and superoxide generation. N3 displayed potent antibacterial activity against MDR Staphylococcus aureus and XDR Pseudomonas aeruginosa, with minimum inhibitory concentrations as low as 5 µg/mL and complete bacterial clearance within 48 h. ROS-mediated membrane disruption and > 99% biofilm eradication were confirmed by DiBAC₄(3), TEM, and comparative assays with non-sonicated controls. In colorectal cancer (HCT-116) models, N3 induced dose-dependent cytotoxicity (IC₅₀ = 100 µg/mL; 91% cell death at LC₅₀) with sustained effects over 96 h. These findings position N3 as a promising ROS-generating platform with dual antimicrobial and anticancer efficacy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-41092-3.

Introduction

Antimicrobial resistance (AMR) has become one of the most formidable global health challenges of the twenty-first century, with recent analyses attributing 1.27 million deaths directly to resistant bacterial infections and associating nearly five million additional deaths with AMR worldwide^1,2. AMR is projected to cause up to 10 million deaths per year by 2050, emphasizing the accelerating loss of antibiotic efficacy and the urgent need for therapeutic approaches that extend beyond conventional antimicrobial mechanisms³. This crisis is driven in large part by Gram-negative pathogens—including Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii—that integrate porin downregulation, efflux pump hyperactivation, enzymatic degradation and metabolic plasticity, enabling resistance to multiple antibiotic classes⁴. The global escalation of carbapenem resistance, coupled with the rapid dissemination of plasmid-mediated colistin resistance, exemplifies the accelerating erosion of last-line antimicrobial therapies^5,6. Biofilm-associated infections impose an additional layer of therapeutic difficulty. Biofilms form dense extracellular polymeric matrix (EPM) structures that limit antibiotic penetration, facilitate horizontal gene transfer and sustain metabolically dormant persister cells with extreme tolerance to antimicrobial exposure^7,8. These features underpin chronic and recurrent infections and reveal the limitations of therapeutic strategies that rely solely on biochemical inhibition of specific bacterial targets. Classical antimicrobial agents—including peptide-based molecules, metal ions and cationic polymers—remain vulnerable to resistance development because they act through single or narrowly focused mechanisms that bacteria can bypass via mutation, membrane adaptation or efflux activation⁹. In response, significant attention has shifted toward stimuli-responsive nanomaterials, which integrate exogenous physical triggers to induce multimodal antimicrobial effects^10,11. Such platforms generate reactive oxygen species (ROS), disrupt membrane potential and destabilize critical cellular processes through mechanisms that are more difficult for bacteria to counteract. Among these emerging systems, piezocatalysis has gained particular prominence due to its ability to convert mechanical deformation into spatially separated charge carriers, enabling ROS generation under physiologically accessible stimuli such as tissue motion, fluid shear or low-intensity ultrasound^12,13. Piezocatalytic activation offers two major advantages over light-dependent or chemically triggered systems: it is not limited by optical penetration, and it imposes simultaneous mechanical and oxidative stresses that reduce the likelihood of resistance emergence^13,14. These attributes highlight piezocatalysis as a promising therapeutic approach for infections involving high tolerance or biofilm-mediated persistence. Advances in materials engineering have demonstrated that piezocatalytic activity can be substantially enhanced through the deliberate control of crystallinity, lattice defects, hierarchical morphology and surface states, each of which promotes efficient charge separation and ROS generation^15,16. Within this expanding field, transition-metal chalcogenides—particularly nickel–cobalt sulfides—have gained attention for their favorable catalytic and electronic characteristics. Evidence from nickel-based nanomaterials shows that they generate ROS capable of inducing membrane disruption, protein oxidation and DNA damage, thereby contributing to strong antibacterial effects^17,18. Cobalt-based nanoparticles similarly exhibit ROS-mediated antimicrobial and anticancer activity through oxidative stress induction and membrane destabilization^19,20. Studies of bimetallic nickel–cobalt sulfides further demonstrate enhanced conductivity, accelerated electron-transfer behavior and stronger catalytic responsiveness relative to their monometallic counterparts²¹. These materials also readily form hollow and hierarchical nanostructures that increase surface accessibility and improve catalytic kinetics—features that are advantageous for ultrasound-activated mechanocatalysis²². Such structural and electronic attributes make Ni–Co sulfide systems well suited for applications requiring efficient charge separation and robust surface reactivity. In this context, Ni₂CoS₄ offers a rational platform for exploring ultrasound-driven mechanocatalysis, providing a synergistic framework capable of supporting efficient ROS generation and downstream antimicrobial or anticancer effects.

Despite extensive investigation of nickel–cobalt sulfides in electrocatalysis, energy storage and enzyme-mimetic systems, their piezocatalytic antimicrobial, antibiofilm and anticancer potential remains largely undefined. Equally limited is current understanding of how ultrasonic activation parameters shape charge-carrier separation, ROS generation dynamics and the resulting biological outcomes. The present study therefore develops an optimized Ni₂CoS₄ hierarchical structures (HSs) and undertakes a comprehensive evaluation of its mechanocatalytic behavior, encompassing antibacterial activity against multidrug-resistant Staphylococcus aureus and extensively drug-resistant Pseudomonas aeruginosa, disruption of mature biofilms and cytotoxicity toward HCT-116 colorectal cancer cells. The integration of structural, physicochemical and functional analyses establishes mechanistic links between crystallinity, hierarchical morphology, redox-active surface states and piezocatalytic efficacy, positioning Ni₂CoS₄ as a versatile mechanocatalytic platform for tackling drug-resistant infections, biofilm persistence and tumor cell viability.

Materials and methods

Synthesis of Ni₂CoS₄ HSs

The preparation of Ni₂CoS₄ was conducted via a two-step solvothermal process. In the first step, Co(NO₃)₂·6 H₂O (2.4 g) and Ni(NO₃)₂·6 H₂O (1.2 g) were dissolved with urea (15 g) in a mixed solvent of isopropanol (62.5 mL) and deionized water (12.5 mL). After stirring for 30 min, the solution was transferred to a 100 mL Teflon-lined autoclave and heated at 120 °C for 12 h to form the Ni-Co precursor. Additional precursor samples were synthesized using identical conditions but with modified reaction times of 3, 6, and 9 h to study the formation process.

The second step involved the sulfidation of the Ni–Co precursor. The as-prepared precursor (0.2 g) was combined with thioacetamide (0.4 g) in ethanol (50 mL). Following 15 min of stirring, the mixture was transferred to a 100 mL Teflon-lined autoclave. Three temperature series (120 °C, 150 °C, 180 °C) and three duration series (4, 6, 8 h) were investigated (Table 1). For concentration studies, the initial metal salt concentrations were varied by increasing and decreasing the standard amounts by one-third while maintaining the same solvent volume (Table 2). All products were collected by centrifugation, washed thoroughly with ethanol and deionized water, and dried overnight under vacuum. Samples were labeled according to their synthesis conditions for systematic analysis modified procedure from²³.

Table 1.

Elemental composition of Ni₂CoS₄ formulations (N1–N7) determined by energy-dispersive X-ray spectroscopy.

Sample	Weight%
	Ni	Co	S	O
N1	9.22	31.72	21.47	35.93
N2	11.45	41.15	23.83	23.58
N3	10.06	35.04	29.03	25.88
N4	9.53	34.37	21.81	33.11
N5	7.75	24.81	20.71	42.49
N6	10.98	37.03	23.62	27.26
N7	14.73	35.56	30.23	19.49

Table 2.

Summary of hydrothermal synthesis parameters and measured physicochemical properties of Ni₂CoS₄ HSs.

Sample	Time (h)	Temperature (°C)	Precursor ratio (g) (Ni: Co: Urea)	Crystallinity (XRD)	Structural Order (Raman)	Morphology (SEM/TEM)	BET (m²/g)	Piezocatalytic activity (MB)
N1	4	120	1.2 : 2.4 : 15	Broad peaks; incomplete crystallization	–	Irregular, loosely aggregated	–	Low
N2	6	120	1.2 : 2.4 : 15	Sharper peaks; improved crystallinity	Low baseline; ordered	Spherical; partial aggregation	6.73	Moderate
N3	8	120	1.2 : 2.4 : 15	Most distinct diffraction peaks; highest relative crystallinity	Lowest baseline; minimal disorder	Uniform hollow spherical structures; primary particle size < 50 nm	8.19	Highest (88.57% in 4 min)
N4	6	150	1.2 : 2.4 : 15	Peak broadening; lattice strain	Moderate disorder	Semi-uniform spheres	2.44	Reduced
N5	6	180	1.2 : 2.4 : 15	Peak shifts; possible secondary phase	Highest disorder	Irregular morphology	7.46	Intermediate
N6	6	120	1.6 : 3.2 : 20	Peak broadening; excessive nucleation	–	Well-defined spheres	–	Moderate
N7	6	120	0.8 : 1.6 : 10	Low peak intensity	–	Well-defined spheres	–	Low

Characterization of Ni₂CoS₄

A comprehensive suite of analytical methods was employed to evaluate the key properties of the synthesized Ni₂CoS₄ materials. Phase identification and crystallographic analysis were performed on a Philips X′Pert PRO diffractometer (The Netherlands) using X-ray diffraction patterns collected under Cu Kα radiation. The morphological evolution and surface features were investigated using a FEI Quanta 200 scanning electron microscope (The Netherlands), while detailed structural examination at the nanoscale was achieved using a JEOL JEM-2100 F transmission electron microscope (Japan). Chemical composition analysis and element distribution mapping were conducted using energy-dispersive X-ray spectroscopy, confirming the stoichiometric ratios and spatial arrangement of nickel, cobalt, and sulfur within the samples. The textural properties, including specific surface area and pore characteristics, were measured through nitrogen physisorption experiments on a Micromeritics Tristar II 3020 analyzer (USA), applying both Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) methods for data analysis. Structural characteristics and vibrational properties were examined using a Horiba LabRAM HR-800 Raman spectrometer (Japan) equipped with dual excitation wavelengths (532 nm).

Mechanocatalytic activity assessment

The generation of reactive oxygen species (ROS) by Ni₂CoS₄ under mechanical activation was investigated through multiple complementary analytical approaches. All experiments were executed under dark conditions in a temperature-controlled environment (25 °C) to exclude potential contributions from photocatalytic or thermocatalytic effects.

The primary ROS generation capacity was evaluated using methylene blue (MB) as a model substrate. In a typical experiment, Ni₂CoS₄ suspension (100 µg/mL) was introduced into MB solution (30 µg/mL, 5 mL). The suspension underwent ultrasonication (200 W) for 4 min, followed by centrifugal separation. The extent of MB degradation, serving as an indicator of ROS production, was determined by monitoring the characteristic absorption peak at 664 nm using UV-visible spectroscopy.

For selective detection of singlet oxygen (¹O₂) and superoxide anions (•O₂⁻), a suspension of Ni₂CoS₄ (25 µg/mL, 1 mL) was combined with 1,3-diphenylisobenzofuran (DPBF) solution (1 mg/mL, 20 µL). The mixture was subjected to ultrasonic treatment for 10 min, during which DPBF degradation was monitored through absorbance measurements at 410 nm, providing quantitative assessment of ROS generation.

Hydroxyl radical (•OH) production was quantified using terephthalic acid as a fluorescent probe. The probe solution was prepared by dissolving terephthalic acid (0.25 mg) in NaOH solution (2 mM, 3 mL) and mixing with Ni₂CoS₄ suspension (200 µg/mL, 3 mL). Following ultrasonication under identical conditions, the fluorescence intensity of the supernatant was measured using a Hitachi F-7000 spectrofluorometer (Japan) with excitation at 315 nm, providing direct quantification of •OH generation⁹.

Mechanocatalytic performance evaluation of Ni₂CoS₄ formulations (N1-N7)

The mechanocatalytic efficiency of the synthesized Ni₂CoS₄ samples (N1-N7) was evaluated using methylene blue (MB) degradation as a model reaction. For each test, MB solution (30 µg/mL) was combined with the respective Ni₂CoS₄ sample (100 µg/mL). The mixtures underwent ultrasonication (200 W, 10 min) using a probe-type ultrasonicator. The degradation efficiency was determined by measuring the characteristic MB absorption at 664 nm using UV–vis spectrophotometry before and after ultrasonic treatment. The Ni₂CoS₄ formulation exhibiting optimal MB removal performance was selected for subsequent mechanistic studies²⁴.

Temporal optimization of ultrasonic treatment

The effect of sonication duration on MB degradation was investigated using the most effective Ni₂CoS₄ formulation. The catalyst suspension (100 µg/mL) was mixed with MB solution (30 µg/mL) and subjected to ultrasonic treatment (200 W) for various time periods (1, 2, 4,7, 10, 15, 30, and 60 min). MB degradation was monitored through absorbance measurements at 664 nm after each time interval to determine the optimal sonication duration modified procedure from²⁵.

Impact of ultrasonic power variation

The relationship between ultrasonic power and catalytic performance was examined by exposing the Ni₂CoS₄ catalyst (100 µg/mL) and MB solution (30 µg/mL) mixtures to different power settings (0, 100, 200, 300, 400, and 500 W) for 4 min. The MB degradation efficiency was assessed through spectrophotometric measurements at 664 nm to identify the most effective power level²⁶.

Optimization of ultrasonic pulse sequences

The effect of intermittent sonication on catalytic efficiency was studied using various ultrasonic pulse patterns. The Ni₂CoS₄ suspension (100 µg/mL) in MB solution (30 µg/mL) was treated using a probe-type ultrasonicator operating at 200 W, employing four distinct pulse sequences: continuous operation, 1:3 s (on: off), 2:2 s (on: off), and 3:1 s (on: off). Total sonication time was fixed at 4 min for all sequences. The degradation of MB was monitored via absorbance measurements at 664 nm to determine the most effective pulse pattern modified procedure from²⁷.

Clinical isolates and resistance profiling

Clinical isolates of multidrug-resistant Staphylococcus aureus (MDR) and extensively drug-resistant Pseudomonas aeruginosa (XDR) were obtained from routine diagnostic specimens processed at Shar Hospital, Sulaymaniyah, Kurdistan Region of Iraq. Species identification and antimicrobial susceptibility testing were performed using the BD Phoenix™ automated microbiology system in accordance with CLSI interpretive guidelines. MDR and XDR classifications were assigned based on resistance to multiple antibiotic classes according to international consensus definitions. Comprehensive susceptibility profiles generated by the Phoenix system for all bacterial strains used in this study—including ATCC reference organisms and clinical MDR/XDR isolates—are provided in Supplementary Tables S1 and S2 to support methodological transparency and reproducibility.

Antimicrobial efficacy assessment of synthesized Ni₂CoS₄ formulations

The antimicrobial potency of Ni₂CoS₄ HSs synthesized under varying conditions (N1-N7) was evaluated against resistant bacterial strains: MDR S. aureus and XDR P. aeruginosa clinical isolates. A concentration gradient study was performed using a 96-well plate format, with Ni₂CoS₄ concentrations ranging from 0 to 300 µg/mL (0, 1.25, 2.5, 5, 10, 20, 40, 60, 80, 100, 200, and 300 µg/mL). Bacterial growth inhibition was quantified through optical density measurements at 600 nm using a microplate reader. The inhibitory effects were analyzed to determine the most effective Ni₂CoS₄ formulation based on synthesis parameters²⁸.

Evaluation of antimicrobial activity by zone of inhibition

The antimicrobial efficacy of Ni₂CoS₄ HSs was assessed using a modified disc diffusion assay. Standard reference strains (Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9029) served as experimental controls alongside clinical isolates with multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes. Bacterial strains were cultured overnight at 37 °C in nutrient broth, after which cell densities were standardized to a 0.5 McFarland turbidity standard. Standardized suspensions (100 µL) were uniformly inoculated onto Mueller-Hinton agar plates. Antimicrobial activity was evaluated using sterile paper discs (6 mm diameter, Whatman No. 1) loaded with Ni₂CoS₄ HSs (10 mg per disc) prepared in two formulations: direct powder application, and ultrasonically treated aqueous dispersions (200 W, 4 min) subsequently dried onto discs. Azithromycin antibiotic discs (15 µg/disc) served as positive reference controls. Following incubation at 37 °C for 24 h, antibacterial efficacy was quantified by measuring zones of inhibition around each disc²⁹.

This experimental design allowed precise evaluation of the antibacterial potential of Ni₂CoS₄ HSs, highlighting the impact of ultrasonic activation against both Gram-positive and Gram-negative bacterial pathogens, including drug-resistant clinical isolates.

Determination of minimum inhibitory and bactericidal concentrations

The bacteriostatic and bactericidal potency of Ni₂CoS₄ nanostructures was evaluated against reference and resistant bacterial strains: S. aureus (ATCC 6538 and MDR isolates) and P. aeruginosa (ATCC 9029 and XDR isolates). To investigate the influence of mechanocatalytic activation, antimicrobial assessments were conducted using two parallel preparations: non-activated Ni₂CoS₄ suspensions (N3-O) and ultrasonically treated samples (N3) (200 W, 4 min). For each condition, bacterial cultures (50 µL, 1 × 10⁸ CFU/mL) were distributed into 96-well plates and exposed to a concentration gradient of Ni₂CoS₄ (0–300 µg/mL, with twelve sequential dilutions: 0, 1.25, 2.5, 5, 10, 20, 40, 60, 80, 100, 200, and 300 µg/mL). The final reaction volume was standardized to 200 µL with LB broth. Following 24-hour incubation at 37 °C, bacterial growth inhibition was quantified through spectrophotometric measurements at 600 nm³⁰. The minimum inhibitory concentration was established as the lowest Ni₂CoS₄ concentration preventing visible bacterial growth.

Bactericidal activity was confirmed by transferring 5 µL aliquots from growth-negative wells onto nutrient agar plates, followed by 24-hour incubation at 37 °C. The minimum bactericidal concentration was determined as the lowest Ni₂CoS₄ concentration yielding no viable bacterial colonies, corresponding to 99.9% elimination of viable cells.

Membrane potential assessment using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3))

Bacterial membrane permeability changes were assessed using the membrane potential-sensitive fluorescent dye DiBAC₄(3). For this analysis, bacterial strains (Staphylococcus aureus ATCC and MDR; Pseudomonas aeruginosa ATCC and XDR) were grown to mid-logarithmic phase in nutrient broth. Hierarchical Ni₂CoS₄ structures (N3) were first sonicated at 200 W for 4 min to prepare the sonicated treatment group. Following growth, the bacterial suspensions (OD600 of 0.3, 2 mL) were mixed with DiBAC₄(3) (200 µL, 5 µM) and incubated for 30 min at 37 °C. The mixtures were then divided into three treatment groups: PBS (control), non-sonicated Ni₂CoS₄ HSs (N3-O, 25 µg/mL), and pre-sonicated Ni₂CoS₄ HSs (N3, 25 µg/mL), with each treatment added to achieve a final volume of 4 mL. Membrane potential changes were monitored by measuring fluorescence intensity using a spectrofluorometer with excitation at 490 nm, where increased fluorescence indicated greater membrane permeability and depolarization⁹.

Evaluation of biofilm formation and antibiofilm activity of Ni₂CoS₄ HSs

Baseline biofilm formation capabilities of bacterial strains were assessed using standard reference strains (Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 9029) alongside their multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Bacterial suspensions were prepared in Luria-Bertani (LB) broth at an initial concentration of 1 × 10⁶ CFU/mL. Aliquots (200 µL per well) were incubated statically at 37 °C for 48 h in sterile 96-well microplates. Biofilm formation was quantified spectrophotometrically by measuring optical density at 600 nm, using sterile LB medium as a negative control for background correction. All experiments were conducted in triplicate, with data presented as mean optical density ± standard deviation.

Antibiofilm efficacy of ultrasonically activated (N3; 200 W, 4 min) and non-activated (N3-O) Ni₂CoS₄ HSs was subsequently evaluated through biofilm reduction (prevention of biofilm formation) and biofilm removal (disruption of established biofilms) assays. Reference ATCC strains served as experimental controls alongside their resistant clinical counterparts.

In the biofilm reduction assay, bacterial suspensions (1 × 10⁸ CFU/mL, 50 µL) were mixed with varying concentrations (0–300 µg/mL: 0, 1.25, 2.5, 5, 10, 20, 40, 60, 80, 100, 200, and 300 µg/mL) of Ni₂CoS₄ formulations. Final volumes were adjusted to 200 µL per well with LB broth, followed by incubation at 37 °C for 48 h. Post-incubation, wells were washed gently with phosphate-buffered saline (PBS), stained with 0.1% crystal violet (200 µL, 15 min), rinsed with PBS, and quantified spectrophotometrically at 600 nm after crystal violet extraction with 95% ethanol (200 µL)³¹.

For biofilm removal assays, mature biofilms were initially established by incubating bacterial suspensions (50 µL, 1 × 10⁸ CFU/mL) at 37 °C for 48 h. Preformed biofilms were treated with N3 and N3-O nanostructures at the same concentration range as described above, for 2 h at room temperature. After treatment, wells were washed gently with PBS, stained with 0.1% crystal violet (200 µL, 15 min), rinsed again with PBS, and remaining biofilms were quantified at 600 nm following solubilization of the retained stain with 95% ethanol (200 µL).

This integrated methodological approach provided comprehensive insights into both preventative and disruptive antibiofilm properties of Ni₂CoS₄ nanostructures, clearly highlighting the significant enhancement in efficacy conferred by ultrasonic activation against Gram-positive and Gram-negative pathogens.

Persistence of mechanocatalytic activity

The duration of sustained mechanocatalytic activity following ultrasonic activation was monitored through MB degradation kinetics. Following initial ultrasonication (4 min), the reaction systems were maintained without further disturbance, and MB absorbance (664 nm) was measured at extended time intervals (24, 48, 72, and 96 h). This analysis aimed to determine the temporal stability of the activated Ni₂CoS₄ nanostructures’ catalytic properties and their capacity for prolonged activity without additional mechanical stimulation.

Sustained antimicrobial efficacy assessment

The persistence of antimicrobial activity was investigated using bacterial cultures treated with Ni₂CoS₄ at their respective MIC 50% values. Test organisms included Staphylococcus aureus (ATCC 6538 and MDR) and Pseudomonas aeruginosa (ATCC 9029 and XDR). Following ultrasonic activation (200 W, 4 min), the Ni₂CoS₄ suspensions were introduced to bacterial cultures in nutrient broth at predetermined MIC 50% concentrations. The treated cultures were maintained at room temperature (25 °C), with viability assessments conducted at 1, 24, and 48 h post-treatment. Bacterial survival was quantified through serial dilution and plate counting on nutrient agar, with results expressed as CFU/mL, providing insights into the long-term antimicrobial efficacy of activated Ni₂CoS₄ HSs³².

Cytotoxicity assessment of Ni₂CoS₄ HSs

The cytotoxic potential of ultrasonically activated (N3) and non-activated (N3-O) Ni₂CoS₄ HSs was assessed using human dermal fibroblast (HDF; ATCC PCS-201-012) cell lines procured from the National Center for Genetic and Biological Resources, Tehran, Iran. Cells were maintained under standardized conditions in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂. HDF cells were seeded at 1 × 10⁴ cells per well in 96-well tissue culture plates and incubated for 24 h to allow cell attachment. Subsequently, cells were exposed for 24 h to a range of concentrations (0, 25, 50, 100, 200, 400, 600, 800, and 1000 µg/mL). Prior to exposure, the activated formulation (N3) underwent ultrasonic treatment (200 W, 4 min), whereas the non-activated formulation (N3-O) remained untreated.

Following treatment, cell viability was assessed using the MTT assay. Briefly, the culture medium was replaced with 100 µL of fresh DMEM containing MTT reagent (5 mg/mL), and cells were incubated for an additional 4 h to facilitate formazan crystal formation. The resulting crystals were dissolved using dimethyl sulfoxide (DMSO; 100 µL), and cellular metabolic activity was quantified by measuring absorbance at 490 nm with a microplate reader³³.

Anticancer evaluation of sonicated N3 Ni₂CoS₄ piezocatalyst

Cytotoxicity assessment and determination of IC₅₀/LC₅₀ values

The immediate cytotoxic effects of N3 piezocatalyst on HCT-116 colorectal cancer cells were evaluated using the MTT assay, employing HCT-116 (ATCC CCL-247) cell lines obtained from the National Center for Genetic and Biological Resources, Tehran, Iran. Ni₂CoS₄ HSs were pre-sonicated at 200 W for 4 min to enhance dispersion and stability. HCT-116 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and incubated at 37 °C in a 5% CO₂ humidified atmosphere for 24 h to allow cell adhesion. Cells were subsequently exposed to increasing concentrations of N3 piezocatalyst (0, 25, 50, 100, 200, 400, 600, 800, and 1000 µg/mL) for 24 h.

Following treatment, the culture medium was replaced with 100 µL of fresh medium containing 5 mg/mL MTT reagent, and cells were incubated for 4 h. Formazan crystals formed by viable cells were solubilized using 100 µL of dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader (SpectraMax iD5, Molecular Devices). Cell viability was calculated relative to untreated controls. IC₅₀ and LC₅₀ values were derived via nonlinear regression using GraphPad Prism (v9.0), following a modified protocol from³⁴.

Long-term (memorial) cytotoxicity evaluation

To assess the prolonged cytotoxic effects (memorial effect) of N3 piezocatalyst, HCT-116 cells were subjected to the MTT assay at multiple time points: 1, 24, 48, 72, and 96 h post-treatment. Cells were seeded in 96-well plates under the same conditions as above and treated exclusively with the optimized sonicated formulation (N3) at a fixed concentration corresponding to the IC₅₀ value (100 µg/mL). At each time point, the medium was replaced with MTT-containing media (5 mg/mL), followed by a 4-hour incubation. Formazan solubilization and absorbance measurements were performed as described in Sect. 2.9.1 Viability data were normalized to time-matched untreated controls. IC₅₀ values at each time point were calculated using GraphPad Prism. This approach, adapted from³², enabled evaluation of delayed or sustained nanoparticle cytotoxicity.

Flow cytometry-based viability analysis

Flow cytometric analysis using propidium iodide (PI) staining was employed to assess cell membrane integrity and quantify viable versus non-viable cells. HCT-116 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and incubated for 24 h. HCT-116 cells were treated with N3 at concentrations corresponding to the IC₅₀ (100 µg/mL) and LC₅₀ (200 µg/mL) for 24 h to evaluate membrane integrity and cell death.

Post-treatment, cells were harvested using 0.25% trypsin-EDTA, centrifuged at 300 × g for 5 min, and resuspended in complete medium to neutralize enzymatic activity. PI staining was conducted by adding 5 µL of a 50 µg/mL PI solution to each sample, followed by a 15-minute incubation in the dark at room temperature. Flow cytometric analysis was carried out on a BD FACSCalibur (BD Biosciences), acquiring a minimum of 10,000 events per sample. PI fluorescence was detected in the FL-2 channel (585/42 nm). FlowJo software was used to quantify the percentage of dead (PI-positive) cells, following a modified protocol from³⁵.

Results

Optimization of Ni₂CoS₄ for piezocatalytic applications

The optimization of Ni₂CoS₄ was conducted to enhance their piezocatalytic activity for methylene blue (MB) degradation. This systematic study assessed various formulations (N1-N7) to identify the optimal synthesis parameters.

Methylene blue removal efficiency of Ni₂CoS₄ formulations

The piezocatalytic efficiency of the Ni₂CoS₄ formulations was evaluated using 30 µg/mL MB solution. Among the tested formulations, N3 and N2 demonstrated superior performance, achieving MB removal efficiencies of 88.565% and 88.22%, respectively. In contrast, N4 and N5 showed notably lower efficiencies of 41.18% and 55.14%. The non-sonicated variant (N3-O) exhibited limited removal capacity at 36.42%, highlighting the critical role of sonication in enhancing catalytic activity (Fig. 1A).

Fig. 1.

Piezocatalytic activity of Ni₂CoS₄. (A) MB removal efficiency comparison of N1-N7 formulations. (B) Effect of sonication duration. (C) Ultrasonication power optimization. (D) Impact of on-off sonication cycles.

Sonication time optimization for methylene blue degradation

The optimal formulation, N3, demonstrated rapid MB degradation kinetics, requiring only 4 min of sonication to reduce MB absorbance from 2.926 to 0.69. Extended sonication up to 60 min yielded minimal additional benefit, with a final absorbance of 0.559 (Fig. 1B), indicating no statistically significant improvement beyond the 4-minute mark.

Effect of ultrasonication power on methylene blue removal efficiency

The influence of ultrasonication power revealed an optimal setting at 200 W, achieving 81.33% MB removal efficiency. A power-dependent trend was observed: 100 W (62.15%), 300 W (74.55%), 400 W (74.05%), and 500 W (69.95%) (Fig. 1C). The results demonstrate that while increasing power enhanced removal efficiency up to 200 W, further power increases led to gradual decreases in performance, establishing 200 W as the optimal power setting for maximum piezocatalytic activity.

Impact of on-off ultrasonication cycles on methylene blue removal

To further optimize the activation conditions, the effect of on-off ultrasonication cycles on MB removal was evaluated. Five cycling methods were tested: no sonication (0), continuous ultrasonication, 1–3 s on-off, 2–2 s on-off, and 3 –1 s on-off. The 1–3 s on-off cycle emerged as the most effective, achieving an MB removal efficiency of 75.33%. Other cycling patterns demonstrated comparable but slightly lower efficiencies: 2–2 s on-off (72.59%), continuous sonication (72.38%), and 3 –1 s on-off (71.72%) (Fig. 1D). In the absence of ultrasonication (0), the efficiency was significantly lower. These findings establish the 1–3 s on-off cycle as optimal for activating the piezocatalytic potential of Ni₂CoS₄, though the relatively small variation in performance among sonication patterns suggests flexibility in cycling parameters for practical applications.

Characterization of Ni₂CoS₄

Optimization of hydrothermal synthesis parameters

A systematic investigation was conducted to optimize the hydrothermal synthesis parameters influencing the formation of Ni₂CoS_4, including reaction time, temperature, precursor concentration, and autoclave filling percentage. The resulting materials were characterized using X-ray diffraction (XRD) to determine phase composition and crystallinity (Fig. 2A).

Fig. 2.

Characterization of Ni₂CoS₄. (A) XRD patterns, (B) Raman spectra acquired using 532 nm excitation.

Effect of sulfidation time

The hydrothermal reaction time played a significant role in the crystalline phase formation of Ni₂CoS₄. The XRD patterns of samples synthesized at different durations (N1, N2, N3) at 120 °C revealed that longer reaction times facilitated improved crystallinity and phase purity. N1, synthesized for 4 h, exhibited broad diffraction peaks indicative of incomplete crystallization. In contrast, N2 and N3, synthesized for 6 h, showed sharper and more intense peaks corresponding to well-defined Ni₂CoS₄ phases, suggesting enhanced structural order. Among these, N3 exhibited the most distinct diffraction peaks, indicating optimal crystalline quality, which is likely a contributing factor to its superior piezocatalytic activity.

Effect of sulfidation temperature

The sulfidation temperature significantly influenced phase evolution. At 120 °C (N3), the XRD pattern showed well-defined peaks corresponding to Ni₂CoS₄, confirming successful phase formation. When the temperature was increased to 150 °C (N4), additional peak broadening was observed, potentially due to increased lattice strain or partial phase transformation. At 180 °C (N5), further structural modifications were detected, with peak shifts indicating possible secondary phase formation or grain growth. These variations highlight the critical role of temperature in tuning the structural properties of Ni₂CoS₄.

Effect of precursor concentration

Precursor concentration was another determining factor in crystallinity and phase composition. The standard concentration (N3) resulted in well-resolved peaks, characteristic of highly crystalline Ni₂CoS₄. Increasing the concentration by one-third (N6) led to peak broadening, likely due to excessive nucleation, resulting in reduced grain size or minor amorphous phases. Conversely, decreasing the concentration by one-third (N7) resulted in lower peak intensities, suggesting incomplete reaction or insufficient nucleation. These findings emphasize the importance of precise precursor concentration control in optimizing material properties.

The superior piezocatalytic activity observed in N3 can be attributed to its optimal crystallinity and well-defined Ni₂CoS₄ phase. A well-crystallized structure enhances charge separation efficiency and reduces recombination rates, which are crucial for effective piezocatalysis. The presence of secondary phases or lattice distortions in other samples may contribute to suboptimal electronic properties, further reinforcing the structure-activity relationship in Ni₂CoS₄ catalysts.

Raman spectroscopic characterization

Raman spectroscopy was employed to assess the relative crystallinity and structural disorder among the synthesized Ni₂CoS₄ formulations (N2–N5). Spectra were acquired using a 532 nm excitation source, which, despite its known susceptibility to fluorescence in transition metal sulfides, effectively highlighted baseline variations indicative of differing degrees of structural order.

As shown in Fig. 2B, sample N3 exhibited a low, stable baseline across the scanned wavenumber range (0–5000 cm⁻¹), suggesting minimal lattice distortion and a high degree of crystallinity. The absence of pronounced background interference or slope indicates reduced defect density and the absence of amorphous or residual organic content. These features support the classification of N3 as the structurally most ordered formulation.

In contrast, sample N5 demonstrated a significantly elevated and sloping baseline, consistent with fluorescence emission often associated with disordered or partially amorphous materials. This spectral profile suggests a greater presence of structural imperfections or surface-bound impurities, which may contribute to diminished functional performance. Sample N4 displayed intermediate behavior, with a moderately elevated baseline, reflecting partial disorder or incomplete phase evolution. Sample N2 showed a relatively flat and low baseline, comparable to N3, indicating similarly improved structural uniformity.

Although no sharp vibrational modes were resolved—likely due to fluorescence overlay and the inherently low Raman scattering efficiency of Ni₂CoS₄—the baseline behavior across samples provides qualitative insight into structural differences. Overall, the Raman spectral features reinforce the conclusion that N3 possesses superior structural quality, while N5 exhibits the greatest degree of disorder among the examined formulations.

Morphological and structural characterization

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses provided insights into the morphological and structural features of the synthesized Ni₂CoS₄ material (Fig. 3). SEM images (Fig. 3A and B) reveal spherical and well-dispersed hierarchical structures. At lower magnification (Fig. 3A), the secondary assemblies exhibited uniform distribution with particle sizes predominantly in the micrometer range, indicating the formation of microscale hierarchical architectures composed of assembled subunits. Higher-magnification SEM (Fig. 3B) distinctly illustrates a spherical morphology characterized by hierarchical structures composed of densely packed nanosheets. The spherical assemblies exhibited a uniform size distribution ranging approximately from 0.8 μm to 1.2 μm in diameter, indicative of controlled growth and aggregation during hydrothermal synthesis.

Fig. 3.

Electron micrographs of Ni₂CoS₄ (N3). (A, B) SEM, (C) TEM.

Complementary TEM imaging (Fig. 3C) confirmed the SEM observations, revealing that the microspheres are composed of much smaller nanoscale building units. TEM analysis showed primary particle sizes predominantly below 20 nm, forming porous, loosely packed assemblies. This porous hierarchical structure contributes to an increased effective surface area, which is advantageous for catalytic activity due to the higher density of active sites. Although SEM revealed micron-scale spherical assemblies, TEM demonstrated that these structures are hierarchical aggregates of nanoscale primary particles rather than bulk particles.

Effect of hydrothermal parameters on morphology

SEM analysis of Ni₂CoS₄ HSs synthesized under various hydrothermal conditions highlights the significant influence of synthesis parameters on their resulting morphology (Fig. 4). Sample N1 (120 °C, 4 h) exhibited loosely aggregated and irregular structures, indicating incomplete particle formation or insufficient crystallization. Extending the reaction duration to 6 h at the same temperature (samples N2 and N4) notably improved particle uniformity, resulting in more defined spherical morphologies, although some particle aggregation persisted.

Fig. 4.

SEM images of Ni₂CoS₄ HSs synthesized under different hydrothermal conditions (samples N1, N2, N4, N5, N6, and N7).

Importantly, sample N3 (120 °C, 8 h), as shown clearly in Fig. 3A and B, demonstrated uniform spherical hierarchical assemblies with distinct hollow interiors composed of densely packed nanosheets, indicative of hierarchical structure formation. Such hollow architectures are favorable for catalytic applications, providing increased surface areas and enhanced accessibility to active catalytic sites, correlating directly with the superior piezocatalytic activity observed for this formulation.

In contrast, sample N5, synthesized at an elevated temperature (150 °C), displayed densely aggregated and irregular morphologies lacking clear spherical structures, signifying adverse effects on morphological control at this specific temperature. Sample N6, synthesized at the highest temperature (180 °C), revealed densely packed spherical morphologies with enhanced structural uniformity compared to N5, suggesting optimal conditions at higher temperatures can positively influence structural homogeneity.

Sample N7, synthesized under modified precursor concentration conditions, presented well-defined spherical assemblies, confirming the critical importance of carefully controlled precursor concentrations in achieving uniform nanoparticle morphology.

Overall, these findings underscore that precise adjustment of hydrothermal synthesis parameters—temperature, duration, and precursor concentration—is essential for controlling hierarchical morphology, directly impacting the performance and functionality of Ni₂CoS₄ structures in catalytic applications.

Elemental composition and distribution analysis

Energy-dispersive X-ray spectroscopy (EDS) analyses (Table 1) (Figure S1) confirmed the elemental composition of the synthesized Ni₂CoS₄ HSs. The EDS spectra revealed clear and consistent peaks corresponding to cobalt (Co), nickel (Ni), sulfur (S), and oxygen (O), verifying successful synthesis and composition. Oxygen signals likely arise from surface oxidation or residual precursor-derived species. Additionally, minor gold (Au) peaks, attributed to the sample preparation (gold coating), were present. No significant impurity elements were observed, underscoring the purity of the synthesized Ni₂CoS₄ HSs.

Elemental mapping analysis via SEM-EDS of sample N3 (Fig. 5) provided further insights into the elemental distribution within individual hierarchical structures. The mapping clearly illustrated a uniform spatial distribution of cobalt (Co), nickel (Ni), and sulfur (S), confirming compositional homogeneity throughout the hierarchical structure. The overlap of these elemental maps validates the uniform elemental integration, correlating with the optimized structural and catalytic properties observed for sample N3.

Fig. 5.

SEM image and elemental mapping (EDS) of hierarchical Ni₂CoS₄ structures (N3).

These observations collectively confirm the successful synthesis of highly pure and compositionally uniform Ni₂CoS₄ structures, essential for achieving superior piezocatalytic performance.

Brunauer–Emmett–Teller surface area analysis

The BET-specific surface area was investigated to evaluate its influence on the piezocatalytic efficiency of Ni₂CoS₄ HSs (Fig. 6). Nitrogen adsorption/desorption isotherms displayed characteristic Type-IV curves, indicative of mesoporous materials. Sample N3 exhibited the highest BET-specific surface area (8.19 m²/g) (Fig. 6B), corresponding directly with its superior piezocatalytic performance, likely due to enhanced surface-active sites available for catalytic interactions. Samples N2 and N5 displayed moderate specific surface areas of 6.73 m²/g and 7.46 m²/g, respectively, correlating with intermediate catalytic activities. In contrast, sample N4 demonstrated the lowest surface area (2.44 m²/g), consistent with its significantly reduced catalytic efficiency.

Fig. 6.

BET nitrogen adsorption/desorption isotherms of Ni₂CoS₄ HSs (samples N2, N3, N4, and N5), indicating differences in specific surface area.

Collectively, these results underscore the critical role of higher BET-specific surface area in achieving enhanced piezocatalytic performance, confirming N3 as the optimal formulation among the synthesized Ni₂CoS₄ HSs. A consolidated comparison of the structural, morphological, and piezocatalytic properties of all formulations (N1–N7) is provided in Table 2.

Antibacterial activity of Ni₂CoS₄ HSs

The antibacterial efficacy of Ni₂CoS₄ HSs (samples N1–N7), synthesized under varying hydrothermal conditions, was assessed against clinical isolates of multidrug-resistant (MDR) Staphylococcus aureus and extensively drug-resistant (XDR) Pseudomonas aeruginosa. Among the tested formulations, sample N3 exhibited the most potent antibacterial activity, achieving complete inhibition of S. aureus MDR growth at a concentration of 40 µg/mL and P. aeruginosa XDR at 20 µg/mL (Fig. 7). Sample N2 demonstrated the second-highest efficacy, consistent with its substantial piezocatalytic performance in methylene blue degradation assays. The other formulations (N1, N4–N7) displayed comparatively lower antibacterial activities. These results collectively highlight N3 as the optimal hierarchical structures formulation, combining outstanding antibacterial and piezocatalytic properties.

Fig. 7.

Antibacterial activity of Ni₂CoS₄ HSs (N1-N7) against clinical isolates (A) S. aureus MDR, (B) P. aeruginosa XDR.

Antibacterial activity of Ni₂CoS₄ piezocatalysts (disc diffusion assay)

The antibacterial efficacy of Ni₂CoS₄ piezocatalysts was evaluated against standard (ATCC) and drug-resistant clinical strains of Pseudomonas aeruginosa (XDR) and Staphylococcus aureus (MDR), employing the Kirby-Bauer disk diffusion method, with ATCC strains serving as controls. To assess the impact of sonication on antimicrobial performance, non-sonicated (N3-O) and sonicated (N3; 200 W, 4 min, 1–3 s on/off cycles) samples were compared.

For S. aureus ATCC, sonication notably increased the inhibition zone diameter from 17 ± 2.45 mm (N3-O) to 31.11 ± 0.68 mm (N3). A similar improvement was observed for the MDR strain, where sonication expanded zones from 13.33 ± 1.70 mm to 31.67 ± 1.25 mm (Fig. 8A, B, and C). Comparable susceptibility patterns were observed for P. aeruginosa, with ATCC strain inhibition zones increasing from 14.89 ± 3.60 mm to 28.44 ± 1.39 mm post-sonication, while zones against the XDR strain enhanced significantly from 15.11 ± 2.59 mm to 33.56 ± 2.04 mm (Fig. 8A, D, and E).

Fig. 8.

Antibacterial activity of Ni₂CoS₄ HSs against drug-resistant pathogens. (A) Quantitative comparison of inhibition zones for sonicated (N3) and non-sonicated (N3-O) structures versus azithromycin control. (B–E) Representative disc diffusion assays on Mueller-Hinton agar showing zones of inhibition against S. aureus ATCC (B), S. aureus MDR (C), P. aeruginosa ATCC (D), and P. aeruginosa XDR (E).

Azithromycin (15 µg/disc), used as an antibiotic reference, displayed variable efficacy. It exhibited high activity against S. aureus ATCC (29 ± 1.60 mm) but was ineffective against the MDR strain. Against P. aeruginosa, the antibiotic showed limited activity, with zones measuring 13 ± 1.60 mm (ATCC) and 9.33 ± 1.24 mm (XDR), underscoring the pronounced antibiotic resistance of these pathogens (Fig. 8).

Overall, these results clearly demonstrate the enhanced antibacterial potential of sonicated Ni₂CoS₄ HSs, particularly against antibiotic-resistant bacteria, highlighting their promise as effective alternative antimicrobial agents.

Minimum inhibitory and bactericidal concentration evaluation

The antibacterial efficacy of Ni₂CoS₄ HSs was systematically investigated by determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against Staphylococcus aureus (ATCC and MDR strains) and Pseudomonas aeruginosa (ATCC and XDR strains), using ATCC strains as standard controls. Comparative analysis was performed between non-sonicated (N3-O) and sonicated (N3; 200 W, 4 min, 1–3 s on/off cycles) formulations.

For the control S. aureus ATCC strain, sonication significantly enhanced antibacterial activity, reducing MIC and MBC values from 40 µg/mL and 100 µg/mL (N3-O) to 10 µg/mL and 40 µg/mL (N3), respectively. Similar results were observed with the MDR strain; N3-O exhibited a MIC of 5 µg/mL and MBC of 40 µg/mL, whereas N3 maintained robust antibacterial efficacy with a MIC of 10 µg/mL and an equivalent MBC (40 µg/mL) (Fig. 9).

Fig. 9.

MIC and MBC of Ni₂CoS₄ HSs against (S. aureus ATCC and MDR) and P. aeruginosa (ATCC and XDR). MIC and MBC values were determined for both non-sonicated (N3-O) and sonicated (N3) formulations.

For the P. aeruginosa ATCC control strain, sonicated N3 formulation displayed improved efficacy, decreasing MIC from 60 µg/mL (N3-O) to 20 µg/mL and MBC from 200 µg/mL to 60 µg/mL. Notably, the XDR strain exhibited heightened susceptibility to N3, with MIC and MBC values markedly decreasing from 20 µg/mL and 200 µg/mL (N3-O) to 5 µg/mL and 40 µg/mL (N3), respectively (Fig. 9).

Collectively, these findings highlight the superior antibacterial properties conferred by sonication treatment of Ni₂CoS₄ HSs, especially against drug-resistant bacterial strains, suggesting their considerable potential as effective antimicrobial alternatives.

Assessment of biofilm formation and anti-biofilm efficacy of Ni₂CoS₄

Biofilm formation capacities of clinical bacterial pathogens (Staphylococcus aureus ATCC, S. aureus MDR, Pseudomonas aeruginosa ATCC, and P. aeruginosa XDR) were systematically evaluated under standardized conditions. Bacterial strains were cultured in sterile 96-well microtiter plates containing Luria-Bertani (LB) broth with an initial bacterial concentration of 1 × 10⁶ CFU/mL, incubated statically at 37 °C for 48 h. Spectrophotometric analysis at 600 nm (OD₆₀₀) revealed significant variation in biofilm-forming capacities among strains. The control strain S. aureus ATCC exhibited robust biofilm production (OD: 1.122 ± 0.04), whereas the MDR strain demonstrated comparatively moderate production (OD: 0.627 ± 0.04) (Fig. 10). Similarly, the ATCC strain of P. aeruginosa displayed substantial biofilm formation (OD: 0.941 ± 0.06), with slightly lower biofilm production observed in the XDR strain (OD: 0.796 ± 0.05), underscoring strain-specific variability (Fig. 10).

Fig. 10.

Assessment of biofilm formation capacity in clinical isolates. Quantitative analysis of biofilm production by S. aureus (ATCC and MDR) and P. aeruginosa (ATCC and XDR) strains measured by optical density at 600 nm after 48-hour incubation.

The biofilm reduction activity of the optimized sonicated formulation (N3; 200 W, 4 min, 1–3 s on/off cycle) was evaluated against the tested strains. S. aureus ATCC showed a 64.59% reduction at 20 µg/mL, while the MDR strain exhibited 52.79% reduction at 10 µg/mL, with both strains achieving ≥ 99% reduction at 60 µg/mL (Fig. 11A and B). Similarly, the ATCC strain of P. aeruginosa displayed 64.85% reduction at 20 µg/mL, requiring 60 µg/mL to achieve ≥ 99% reduction. Notably, the XDR strain demonstrated sensitivity with 53.83% biofilm reduction at 5 µg/mL and achieved ≥ 99% reduction at 40 µg/mL (Fig. 11C and D).

Fig. 11.

Biofilm Reduction of Ni₂CoS₄ HSs. (A) Percentage reduction in biofilm mass of S. aureus ATCC and S. aureus MDR after treatment with sonicated (N3) Ni₂CoS₄ structures at varying concentrations. (B) Percentage reduction in biofilm mass of P. aeruginosa ATCC and P. aeruginosa XDR after treatment with sonicated (N3) Ni₂CoS₄ structures at varying concentrations. (C) Image of a 96-well microplate stained with crystal violet, illustrating biofilm formation before ethanol suspension. (D) Image of the same plate post-ethanol suspension, showing the biofilm mass after solubilization of the crystal violet.

For established biofilm removal, both the sonicated (N3; 200 W, 4 min, 1–3 s on/off cycle) and non-sonicated (N3-O) formulations were assessed to compare their relative efficacy. In S. aureus ATCC, N3 achieved 55.88% biofilm removal at 20 µg/mL, increasing to 99.26% at 60 µg/mL, whereas N3-O required higher concentrations (40 µg/mL for 51.31%, 80 µg/mL for 94.73%). Against the MDR strain, N3 demonstrated 54.30% removal at 10 µg/mL and 98.98% at 80 µg/mL, while N3-O required concentrations up to 100 µg/mL for comparable efficacy (98.99%) (Fig. 12).

Fig. 12.

Biofilm removal activity of Ni₂CoS₄ HSs against bacterial strains. removal percentage of biofilm formed by gram-positive S. aureus (ATCC and MDR) and gram-negative P. aeruginosa (ATCC and XDR) treated with sonicated (N3) and non-sonicated (N3-O) Ni₂CoS₄ structures

For P. aeruginosa ATCC, N3 showed 54.26% removal at 40 µg/mL and 98.54% at 80 µg/mL, whereas N3-O exhibited lower efficiency (63.98% at 60 µg/mL and 97.84% at 100 µg/mL). Similarly, the XDR strain was highly susceptible to N3, achieving 54.12% removal at 20 µg/mL and 99.14% at 80 µg/mL, while N3-O required higher concentrations (40 µg/mL for 56.06%, 100 µg/mL for 98.59%).

Statistical analyses revealed significant differences in antibiofilm activity between sonicated (N3) and non-sonicated (N3-O) structures, particularly within the 5–20 µg/mL concentration range across all strains tested. These findings underscore the enhanced antibiofilm potential of sonicated Ni₂CoS₄ HSs, highlighting their promise as effective alternatives in combating biofilm-associated infections, particularly those involving drug-resistant pathogens. The marked decrease in biofilm biomass following N3 treatment therefore indicates a substantial destabilization of biofilm integrity, reflecting impairment of both the extracellular matrix and the viability of cells residing within the biofilm.

Mechanism of antibacterial action of Ni₂CoS₄ HSs

The antibacterial mechanism of Ni₂CoS₄ HSs against pathogenic bacteria, specifically Staphylococcus aureus and Pseudomonas aeruginosa, was investigated using transmission electron microscopy (TEM) (Fig. 13). TEM analyses revealed significant ultrastructural alterations in bacterial cells upon exposure to Ni₂CoS₄ HSs, strongly implicating membrane disruption as a primary antibacterial mode of action.

Fig. 13.

TEM images showing the effects of Ni₂CoS₄ HSs piezocatalyst on bacterial cells. (A) Untreated S. aureus MDR cells with intact morphology. (B) Damaged S. aureus MDR cells after treatment with Ni₂CoS₄ HSs. (C) Untreated P. aeruginosa XDR cells. (D) P. aeruginosa XDR cells treated with Ni₂CoS₄ HSs, showing membrane disruption and cytoplasmic leakage.

In S. aureus, nanoparticle treatment resulted in pronounced morphological deformation, including extensive membrane disruption, irregular cell shapes, and compromised structural integrity. Likewise, treated P. aeruginosa cells exhibited substantial membrane damage, extensive leakage of intracellular contents, and evident cell collapse, confirming severe structural impairment.

These findings collectively support the conclusion that the antibacterial efficacy of Ni₂CoS₄ HSs primarily results from their capacity to disrupt bacterial membrane integrity, causing severe structural damage and subsequent cell death in both Gram-positive and Gram-negative pathogens.

Assessment of plasma membrane permeability by Ni₂CoS₄ HSs

The impact of Ni₂CoS₄ HSs on bacterial plasma membrane integrity was systematically assessed using the fluorescent probe DiBAC₄(3). The evaluation involved control (ATCC) and antibiotic-resistant strains of Staphylococcus aureus (MDR) and Pseudomonas aeruginosa (XDR), with ATCC strains serving as standard controls. Cells were treated with sonicated (N3), non-sonicated (N3-O) formulations, and phosphate-buffered saline (PBS) as controls (Fig. 14).

Fig. 14.

Plasma membrane permeability of S. aureus (ATCC and MDR) and P. aeruginosa (ATCC and XDR) strains treated with PBS (control), non-sonicated Ni₂CoS₄ HSs (N3-O), and sonicated Ni₂CoS₄ HSs (N3). Fluorescence intensity, measured using the DiBAC₄(3) assay, reflects membrane permeability and damage. The results demonstrate uniform and significant increase in permeability upon treatment with sonicated (N3) across all bacterial strains compared to non-sonicated (N3-O) and PBS control (p < 0.0001). Error bars represent the standard deviation of three independent experiments.

Sonicated Ni₂CoS₄ HSs (N3) significantly enhanced membrane permeability across all bacterial strains tested compared to both non-sonicated (N3-O) and PBS controls (p < 0.0001). This membrane disruption effect was consistent regardless of bacterial cell-wall structure, demonstrating equivalent potency against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) pathogens.

These results confirm that sonication markedly improves the membrane-disrupting capacity of Ni₂CoS₄ HSs, highlighting their potential as versatile antimicrobial agents against diverse bacterial pathogens.

Evaluation of the memory effect in piezocatalytic activity

The memory effect associated with the piezocatalytic activity of Ni₂CoS₄ HSs was evaluated by monitoring the removal efficiency of methylene blue (MB) dye over an extended period after initial sonication treatment (200 W, 4 min, 1–3 s on-off cycles). Following a single sonication, the degradation of MB (30 µg/mL) was measured at regular intervals for up to 96 h without further treatment.

Sonicated HSs (N3) exhibited markedly enhanced immediate catalytic activity, achieving 87.95% MB removal within 1 h compared to only 24.46% for the non-sonicated sample (N3-O). Notably, the catalytic performance of N3 remained consistently high, showing only a slight improvement to 90.16% after 96 h (Fig. 15). Conversely, the activity of N3-O increased gradually over the observation period, reaching 46.83% removal after 96 h but remaining significantly lower than that of N3.

Fig. 15.

Memory effect evaluation of sonicated (N3) and non-sonicated (N3-O) Ni₂CoS₄ HSs in MB removal over 96 h.

These findings indicate that sonicated Ni₂CoS₄ HSs exhibit robust initial piezocatalytic performance with sustained catalytic efficiency, whereas non-sonicated HSs show a gradual, albeit limited, temporal enhancement in activity. This difference underscores distinct underlying mechanisms influencing the sustained catalytic capabilities of sonicated versus non-sonicated nanoparticle formulations.

Time-dependent antimicrobial activity of Ni₂CoS₄ HSs

The antimicrobial activity of sonicated (N3) and non-sonicated (N3-O) Ni₂CoS₄ HSs was evaluated over 48 h against standard (ATCC) and drug-resistant strains (MDR, XDR) of Staphylococcus aureus and Pseudomonas aeruginosa, starting from an initial bacterial concentration of approximately 8.9 log₁₀ CFU/mL (Fig. 16).

Fig. 16.

Sustained Antibacterial Activity and Memory Effect of Ni₂CoS₄ HSs. Bacterial viability (log₁₀ CFU/mL) of untreated controls, non-sonicated (N3-O), and sonicated (N3) HSs over 48 h. (A) S. aureus ATCC, (B) S. aureus MDR, (C) P. aeruginosa ATCC, and (D) P. aeruginosa XDR. N3 demonstrated significant bacterial reduction at 1 h, achieving complete bactericidal effect against most strains by 24 h and all strains by 48 h (p < 0.0001).

After 1 h, sonicated HSs (N3) markedly reduced bacterial counts to 5.04 log₁₀ CFU/mL (S. aureus ATCC), 4.47 log₁₀ CFU/mL (S. aureus MDR), 4.82 log₁₀ CFU/mL (P. aeruginosa ATCC), and 4.37 log₁₀ CFU/mL (P. aeruginosa XDR). In contrast, non-sonicated HSs (N3-O) caused only modest reductions, maintaining higher bacterial populations of 7.72 log₁₀ CFU/mL (S. aureus ATCC), 7.85 log₁₀ CFU/mL (S. aureus MDR), 7.45 log₁₀ CFU/mL (P. aeruginosa ATCC), and 7.44 log₁₀ CFU/mL (P. aeruginosa XDR) (Fig. 16).

At 24 h, N3 achieved complete bactericidal activity against P. aeruginosa (ATCC, XDR) and S. aureus MDR, while significantly reducing the S. aureus ATCC strain to 1.06 log₁₀ CFU/mL. Conversely, N3-O treatments showed continued bacterial growth, with counts at 7.25 log₁₀ CFU/mL (S. aureus ATCC), 6.28 log₁₀ CFU/mL (S. aureus MDR), 5.09 log₁₀ CFU/mL (P. aeruginosa ATCC), and 5.54 log₁₀ CFU/mL (P. aeruginosa XDR). At 48 h, complete bacterial elimination of S. aureus ATCC was observed with N3 treatment, whereas bacterial populations treated with N3-O remained relatively unchanged.

Statistical analysis indicated highly significant differences (p < 0.0001) between N3 and N3-O formulations, confirming that sonication significantly enhances the antimicrobial activity of Ni₂CoS₄ HSs across both Gram-positive and Gram-negative pathogens.

Reactive oxygen species generation by Ni₂CoS₄ HSs

The capability of sonicated and non-sonicated Ni₂CoS₄ HSs to generate reactive oxygen species (ROS)—including singlet oxygen (¹O₂), superoxide anions (•O₂⁻), and hydroxyl radicals (•OH)—was systematically evaluated to assess their piezocatalytic performance. Significant differences were observed in ROS production among formulations, highlighting the critical impact of nanoparticle treatment conditions.

Sonicated nanoparticle formulation N3 exhibited the most pronounced increase in combined ¹O₂ and •O₂⁻ generation, with a 13.27-fold enhancement relative to its non-sonicated counterpart (N3-O). Formulation N2 also demonstrated notable enhancement (7.98-fold), followed by N5 (6.54-fold), whereas N4 exhibited minimal ROS enhancement (1.24-fold) (Fig. 17A).

Fig. 17.

ROS Generation in Ni₂CoS₄ HSs. The figure illustrates the generation of reactive oxygen species (ROS) in different Ni₂CoS₄ HSs formulations. Comparison between sonicated (N2, N3, N4, N5) and non-sonicated (N2-O, N3-O, N4-O, N5-O) versions shows N3 demonstrating superior ¹O₂/•O₂⁻ generation, while N2 exhibits highest •OH radical production.

Conversely, •OH radical generation showed more moderate differences between formulations. Sample N2 achieved the highest enhancement (1.55-fold), closely followed by N3 (1.46-fold). Samples N4 (1.30-fold) and N5 (1.24-fold) showed comparatively lower levels of enhancement (Fig. 17B).

These findings highlight sonicated formulation N3 as optimal for robust ROS generation, particularly in the context of ¹O₂ and •O₂⁻ radicals, with formulation N2 demonstrating consistently strong activity across both ROS categories. In contrast, formulations N4 and N5 displayed lower catalytic efficacy, indicating that optimized sonication and synthesis conditions are critical for enhancing ROS-mediated piezocatalytic activity of Ni₂CoS₄ HSs.

The predominance of singlet oxygen (¹O₂) and superoxide anions (•O₂⁻) over hydroxyl radicals (•OH) reflects the intrinsic band-structure characteristics of Ni₂CoS₄ under ultrasonic activation. The conduction-band potential favors one- and two-electron oxygen-reduction pathways, enabling efficient generation of ¹O₂ and •O₂⁻, whereas the valence-band energy is insufficient to drive extensive water oxidation to •OH. Consequently, •OH exhibits only a modest increase despite robust piezocatalytic activation. This mechanistic profile explains why ¹O₂ and •O₂⁻ account for the marked 13.27-fold enhancement observed in the N3 formulation.

Cytotoxicity assessment of Ni₂CoS₄ HSs

The cytotoxic effects of sonicated (N3) and non-sonicated (N3-O) Ni₂CoS₄ HSs were evaluated using human dermal fibroblast (HDF) cells, revealing clear concentration-dependent responses. At lower nanoparticle concentrations (0–100 µg/mL), both formulations demonstrated high biocompatibility, maintaining cell viability at approximately 92.59% (N3) and 88.72% (N3-O) (Fig. 18).

Fig. 18.

Cytotoxicity of sonicated (N3) and non-sonicated (N3-O) Ni₂CoS₄ HSs in HDF cells. Both formulations maintained high cell viability below 100 µg/mL, with significant differential cytotoxicity emerging at higher concentrations (p < 0.0001).

However, at elevated concentrations (200 µg/mL), both nanoparticle formulations induced significant cytotoxicity (p < 0.0001), with sonicated HSs (N3) exhibiting a notably greater reduction in cell viability. This finding indicates that sonication enhances not only therapeutic potency but also potential cellular toxicity at higher nanoparticle concentrations. These results emphasize the critical importance of precise dose optimization when considering Ni₂CoS₄ HSs for biomedical applications.

Anticancer activity of N3 HSs

The cytotoxic evaluation of N3 HSs against HCT-116 colorectal cancer cells demonstrated a clear dose-dependent reduction in cell viability. The half-maximal inhibitory concentration (IC₅₀) was established at 100 µg/mL, while the lethal concentration (LC₅₀) was observed at 200 µg/mL. Notably, treatment with the highest tested concentration (800 µg/mL) resulted in complete cell death, indicating a potent anticancer effect (Fig. 19). These findings highlight the strong cytotoxic potential of N3 HSs and position them as promising candidates for further oncological research. Future investigations should focus on elucidating the underlying mechanisms of action, including potential pathways involving reactive oxygen species (ROS) generation, mitochondrial disruption, and apoptosis induction, to better understand their therapeutic applicability and biosafety.

Fig. 19.

Anticancer activity of sonicated N3 HSs. Data are presented as mean ± standard deviation (SD) from three independent experiments. ↔ indicates IC₅₀, while ↔↔denotes LC₅₀.

Memorial effects of N3 HSs on HCT-116 Cells

The prolonged cytotoxic effects of N3 HSs on HCT-116 colorectal cancer cells were evaluated over a 96-hour period using the MTT assay. As shown in Fig. 20, N3-treated cells exhibited a significant and time-dependent decline in cell viability compared to untreated controls.

Fig. 20.

Memorial effect of N3 HSs on HCT-116 cells over 96 h. Cell viability was measured by MTT assay. Treated cells showed significant viability reduction at all time points, with a plateau between 72 h (15.8%) and 96 h (12.84%). Untreated cells maintained > 98% viability. (**** = p < 0.0001; ns = not significant).

At 24 h post-treatment, the viability of treated cells dropped sharply to 43.95%, in contrast to 99.7% in the untreated group (p < 0.0001). This trend persisted at 48 h, with viability in treated cells remaining significantly reduced (p < 0.0001) and not statistically different from the 24-hour value (ns), indicating sustained cytotoxicity. A more pronounced decline was observed at 72 h, with viability decreasing to 15.8% (p < 0.0001). However, no significant difference was detected between 72 and 96 h (ns), during which viability reached 12.84%, suggesting that maximal cytotoxic effect had been reached by 72 h. In contrast, untreated cells maintained consistently high viability across all time points, with values remaining above 98%, further confirming the selective and sustained cytotoxicity of N3 HSs.

Statistical analysis using two-way ANOVA supported these observations, indicating a significant time- and treatment-dependent effect. These findings demonstrate that N3 HSs induce a strong and prolonged inhibitory effect on HCT-116 cell viability, highlighting their potential for long-term anticancer efficacy.

The Ni₂CoS₄ HSs received a single ultrasonic activation step prior to their application, and no additional sonication was applied during the 96-hour incubation. The same pre-activated N3 suspension was used across all time-point assessments. The observed decline in cell viability between 24 and 72 h, followed by the plateau at 96 h, therefore represents the sustained biological activity of the initially activated Ni₂CoS₄ HSs rather than the effects of continuous mechanostimulation. These findings indicate that the cytotoxic response initiated by ultrasound-activated N3 persists within the cellular environment for several days after the initial activation event. The experiment was designed to characterize the persistence of the biological effect rather than to quantify the duration of the activation state itself, and determining the temporal stability of the activated Ni₂CoS₄ HSs remains a relevant objective for future mechanistic studies.

Cancer cell viability assessment

The cytotoxic potential of N3 HSs against HCT-116 colorectal cancer cells was assessed via flow cytometry following propidium iodide (PI) staining after 24 h of treatment at both IC₅₀ and LC₅₀ concentrations. Loss of membrane integrity was used as an indicator of cell death. As illustrated in Fig. 21A, treatment with the IC₅₀ concentration of N3 resulted in a substantial reduction in cell viability. Flow cytometry analysis revealed 48.7% dead cells, while 51.3% of the population remained viable. This near-equal distribution between live and dead cells reflects significant cytotoxicity at the inhibitory concentration. At the LC₅₀ concentration (Fig. 21B), the cytotoxic response was markedly increased. The proportion of dead cells rose to 91.0%, with only 8.99% of cells remaining viable, indicating a near-complete loss of membrane integrity and pronounced lethality at higher dosage levels.

Fig. 21.

Flow cytometry showing dose-dependent cytotoxicity of N3 HSs on HCT-116 cells. (A) 48.7% dead cells at IC₅₀ (100 µg/mL); (B) 91.0% dead cells at LC₅₀ (200 µg/mL).

These findings confirm that N3 HSs exert a strong, dose-dependent cytotoxic effect on HCT-116 cells. The sharp increase in cell death from 48.7% at IC₅₀ to 91.0% at LC₅₀ underscores their potential as an effective anticancer agent and warrants further investigation into their mechanistic pathways.

Quantitative comparison of N3-induced cytotoxicity in HDF and HCT-116 cells

A quantitative comparison of N3-mediated cytotoxicity in normal human dermal fibroblasts (HDF) and HCT-116 colorectal carcinoma cells revealed a reproducible concentration-dependent differential in cellular susceptibility. At 100 µg/mL, corresponding to the IC₅₀ concentration for HCT-116 cells, HDF cultures exhibited 88.72% viability, whereas HCT-116 cultures demonstrated 45.91% viability, yielding a 1.93-fold higher survival in fibroblasts. At 200 µg/mL, corresponding to the LC₅₀ concentration for HCT-116 cells, HDF viability measured 46.39%, compared with 33.59% viability in HCT-116 cultures, producing a 1.38-fold differential (Figs. 18 and 19). These dose-matched values delineate a consistent pattern in which normal fibroblasts display greater tolerance toward N3 exposure than malignant colorectal cells, indicating a favorable yet concentration-dependent selective-toxicity profile for the N3 formulation.

Discussion

The escalating crisis of antimicrobial resistance, particularly manifested in multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacterial pathogens, necessitates paradigm-shifting therapeutic interventions^30,36. This investigation illuminates the exceptional potential of Ni₂CoS₄ HSs as advanced piezocatalytic platforms, while elucidating the critical relationship between synthesis optimization and functional efficacy.

Optimization of Ni₂CoS₄ HSs for piezocatalytic applications

The systematic optimization of Ni₂CoS₄ HSs has revealed significant structure-function correlations in their piezocatalytic performance, strongly influenced by the hydrothermal synthesis parameters, including temperature, duration, and precursor concentration. In terms of temperature, the Ni₂CoS₄ HSs were synthesized at 120 °C, 150 °C, and 180 °C. The formulation synthesized at 120 °C demonstrated the highest piezocatalytic activity, with a methylene blue degradation efficiency of 88.565%, outperforming those prepared at elevated temperatures. These results indicate that lower temperatures promote the formation of more stable and well-defined nanostructures, thereby enhancing the surface reactivity and electronic properties critical for catalytic efficiency. In contrast, higher temperatures led to the formation of larger Ni₂CoS₄ HSs with reduced surface reactivity, which translated into diminished catalytic activity.

The impact of hydrothermal time was similarly evaluated by varying the synthesis duration to 4, 6, and 8 h. The formulation synthesized at 8 h (N3) exhibited the highest catalytic efficiency (88.565%), underscoring the importance of extended hydrothermal duration in promoting enhanced crystallization and uniform morphology, which, in turn, optimized the material’s catalytic properties^37,38. In comparison, shorter hydrothermal durations (4 h) resulted in less developed nanostructures with suboptimal catalytic performance. The formulation synthesized at 6 h (N2) displayed a slightly lower catalytic efficiency (88.22%), suggesting that while increased time leads to improved crystallization, there exists a threshold duration beyond which the benefits plateau. The precursor concentration also played a significant role in controlling nanoparticle size, distribution, and surface reactivity, which are crucial for the overall catalytic activity. These findings are consistent with established principles in transition metal chalcogenide chemistry, where subtle alterations in synthesis parameters, including temperature, duration, and precursor concentration, can significantly impact the electronic structure, morphology, and surface properties of nanomaterials^39,40.

Furthermore, the observed enhancement in catalytic activity between sonicated (N3) and non-sonicated (N3-O) formulations (88.565% vs. 36.42%) highlights the pivotal role of mechanical activation in optimizing electron-hole pair generation and improving charge carrier dynamics. This mechanistic insight is corroborated by recent studies that demonstrate the positive impact of ultrasonication in enhancing the catalytic interfaces through improved dispersion and modifications of the surface electronic states^41,42. Collectively, these results underscore the intricate interplay between synthesis parameters, mechanical activation, and catalytic behavior, offering vital insights into the optimization of nanomaterial properties for advanced functional applications.

Sonication time and power optimization

Temporal optimization revealed a critical four-minute threshold for maximal MB degradation (absorbance reduction: 2.926 to 0.69), with extended sonication yielding diminishing returns. This non-linear response suggests a complex interplay between mechanical activation and surface electronic reconstruction, potentially mediated through transient charge carrier states. The identification of 200 W as the optimal power regime, achieving 81.33% removal efficiency, establishes critical boundaries for energy input in piezocatalytic systems. The observed performance decline at elevated power levels (300–500 W: 74.55%, 74.05%, 69.95%) indicates potential disruption of optimal electronic configurations or altered surface chemistry dynamics^13,43.

On-off ultrasonication cycles and piezocatalytic activation

Investigation of cyclical activation patterns revealed the superior efficacy of a 1–3 s on-off regime (75.33% efficiency). This optimization suggests sophisticated interplay between mechanical energy input and electronic relaxation processes. The narrow performance distribution across cycling patterns (continuous: 72.38%, 2–2 s: 72.59%, 3 –1 s: 71.72%) demonstrates robust underlying mechanistic pathways maintaining catalytic activity across diverse activation conditions. This operational flexibility, particularly noteworthy for scalable applications, aligns with emerging perspectives on piezocatalytic system adaptability.

These comprehensive findings establish Ni₂CoS₄ HSs as highly efficient piezocatalytic materials, with optimization protocols that could extend beyond MB degradation to other environmental applications. The systematic framework developed here, encompassing formulation, power, timing, and cycling parameters, provides a robust foundation for maximizing piezocatalytic performance in practical scenarios⁴⁴.

Characterization of Ni₂CoS₄ piezocatalyst

The structural and compositional characterization of Ni₂CoS₄ HSs provides critical insight into the physicochemical features that underpin their piezocatalytic behavior. Among the synthesized variants, sample N3 emerged as the most effective catalyst, an outcome closely associated with its superior crystallinity, hierarchical morphology, and homogeneous elemental distribution—rather than surface area effects alone.

X-ray diffraction (XRD) analysis revealed that the optimization of sulfidation time and temperature yielded a well-crystallized Ni₂CoS₄ phase in sample N3. Crystallinity is widely regarded as a pivotal determinant in charge transport phenomena, as a more ordered lattice reduces defect-mediated recombination of charge carriers, thereby enhancing electron–hole separation under mechanical excitation⁴⁵. The sharp, well-defined diffraction peaks observed in N3 are indicative of such structural order and are likely to contribute to its enhanced generation of reactive oxygen species (ROS), a key mechanism in piezocatalytic activity.

Morphological investigations using scanning and transmission electron microscopy (SEM and TEM) further corroborated the advantageous structural features of N3. The formation of uniform, hollow spherical nanostructures composed of densely packed nanosheets represents a morphology well-suited to piezocatalysis. Hierarchical architectures of this nature can accommodate mechanical strain more effectively, promoting internal polarization and increasing the probability of charge separation and migration⁴⁵. Moreover, the porous configuration facilitates greater accessibility of active sites and more efficient interaction between the catalyst surface and biomolecules⁴⁶. While nitrogen adsorption–desorption isotherms showed that N3 exhibited the highest BET-specific surface area (8.19 m²/g), the marginal difference when compared to the poorly performing sample N5 (7.46 m²/g) underscores a key observation: surface area alone does not account for the enhanced catalytic activity. This minimal variation, juxtaposed with a marked difference in catalytic efficiency, strongly suggests that the primary mechanism of degradation is not driven by adsorption but rather by ROS generation, a process inherently reliant on structural and electronic properties.

Elemental analysis via energy-dispersive X-ray spectroscopy (EDS) and SEM-EDS mapping revealed a uniform distribution of nickel, cobalt, and sulfur within the N3 HSs. This compositional homogeneity is instrumental in ensuring consistent redox activity across the catalyst surface. The co-existence of Ni and Co provides synergistic redox centers, which are known to facilitate multi-electron transfer reactions, thereby enhancing ROS production under mechanical stress^47,48. The absence of extraneous elemental signals further affirms the high purity of the synthesized material, contributing to its reproducibility and stability in catalytic applications.

Taken together, these findings underscore that the superior performance of N3 is attributable to its well-optimized structural and compositional attributes. The results clearly demonstrate that a high degree of crystallinity, hierarchical hollow morphology, and elemental homogeneity are more influential than surface area in governing piezocatalytic efficiency. This reinforces a mechanistic understanding based on dynamic charge behavior and ROS-mediated degradation, offering a valuable framework for the rational design of next-generation piezocatalysts for environmental remediation.

Piezocatalytic mechanism and ROS generation

The remarkable efficacy of sonicated Ni₂CoS₄ HSs (N3) in generating reactive oxygen species (ROS) through piezocatalytic mechanisms represents a significant advancement in antimicrobial technology. Under sonication-induced mechanical stress, the Ni₂CoS₄ HSs facilitate efficient charge separation, leading to the generation of singlet oxygen (¹O₂) and superoxide anion (•O₂⁻). In contrast to conventional photocatalytic systems that require continuous light irradiation, this piezocatalytic mechanism operates effectively under dark conditions, thereby broadening its potential applicability, particularly in environments with limited or no light availability. This finding corroborates the observations of⁴⁹, who highlighted the potential of piezocatalysis in low-light settings. This enhanced activity can be attributed to the piezocatalytic activation induced by ultrasonication. Acoustic cavitation generates rapid compression–tension cycles that impose transient mechanical deformation on the Ni–Co–S lattice, promoting internal charge separation and accelerating the formation of reactive electron–hole pairs. These charge carriers in turn drive the pronounced increase in singlet oxygen and superoxide generation observed for the sonicated N3 sample, providing a clear mechanistic basis for its superior piezocatalytic performance.

The unique crystal structure and composition of Ni₂CoS₄, characterized by increased surface area and optimized sonication parameters, enhance its piezocatalytic performance. These features facilitate prolonged charge carrier dynamics, enabling sustained ROS production.

Importantly, Ni₂CoS₄ preferentially channels charge carriers toward oxygen-reduction pathways rather than water oxidation, thereby reinforcing the dominant formation of ¹O₂ and •O₂⁻ and limiting the generation of •OH. This selective ROS profile is consistent with the material’s electronic structure and contributes directly to the superior catalytic performance of the sonicated N3 formulation.

Comparative analysis with established piezoelectric sonosensitizers places the ROS-generation efficiency of the N3 formulation within the upper performance range of current materials. Oxygen-vacancy-engineered BaTiO₃ nanostructures have demonstrated rapid and high-output ultrasound-activated ROS generation, but these effects rely on deliberate defect creation or composite construction to enhance charge-separation efficiency and catalytic kinetics⁵⁰. Similarly, ZnO@carbon composite architectures achieve strong production of ¹O₂ and •O₂⁻ and exhibit near-complete bacterial inactivation under ultrasonication, although this requires heterojunction engineering to overcome the intrinsic recombination tendencies of pristine ZnO⁵¹. In contrast, the sonicated N3 formulation achieves substantial ROS output within a single-phase Ni₂CoS₄ lattice, without extrinsic defect modulation or multi-component assembly. This behavior suggests that the redox-coupled Ni/Co centers and the hierarchically structured morphology provide built-in advantages that functionally parallel the enhancement strategies used in advanced BaTiO₃ and ZnO systems, while maintaining structural simplicity.

Antibacterial mechanisms of sonicated Ni₂CoS₄ HSs

The growing prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacterial strains has intensified the demand for alternative antimicrobial agents that circumvent traditional, target-specific mechanisms of action². Piezocatalytic materials, such as Ni₂CoS₄ HSs, offer a mechanistically distinct approach by exerting multifaceted stress on bacterial cells through non-specific physical and chemical interactions. The present study elucidates the enhanced antibacterial efficacy of sonicated Ni₂CoS₄ HSs (N3), driven by a dual mechanism involving reactive oxygen species (ROS) generation and direct disruption of cellular membrane integrity.

Sonication was found to be a critical determinant of antibacterial performance. Ultrasonic treatment improved nanoparticle dispersion and reduced agglomeration, thereby increasing surface area and charge density—features that enhance both catalytic and biological activity^52,53. These physicochemical modifications translated into significantly improved antimicrobial potency in the N3 formulation compared to non-sonicated controls (N3-O) and other formulations (N1, N2). Comparative contextualization with representative piezocatalytic antibacterial and ROS-generating materials situates the MIC performance of the N3 formulation within the broader landscape of mechanically activated nanomaterials. Classic piezoelectric systems such as BaTiO₃ exhibit ROS generation under ultrasound and have been explored for sonodynamic and piezocatalytic applications, although high antibacterial efficacy in these systems often depends on engineered interfaces or composite formation to enhance charge separation and ROS yield¹³. Sonosensitizer heterostructures incorporating MoS₂ and Cu₂O exemplify this approach, achieving near-complete S. aureus elimination (~ 99.85%) under ultrasound by leveraging piezoelectric-enhanced ROS production at engineered interfaces⁵⁴. Reviews of piezocatalytic materials such as BiFeO₃ highlight their intrinsic potential for mechanically stimulated ROS generation and related antimicrobial mechanisms, but also note the need for structural modification or integration into hybrid systems to maximize activity⁵⁵. In contrast, the N3 formulation attains low MIC values against MDR S. aureus and XDR P. aeruginosa within a single-phase Ni₂CoS₄ framework. This suggests that the cooperative Ni/Co redox centers, enhanced charge-separation dynamics, and hierarchical morphology confer intrinsic antibacterial efficacy that is competitive with many engineered piezocatalytic materials, while avoiding the need for complex heterostructures or extensive defect engineering. Quantitative ROS analysis confirmed that N3 generated approximately 2.5 times more ROS than N2, establishing a direct correlation between sonication, redox activity, and antibacterial efficacy.

The predominance of singlet oxygen (¹O₂) and superoxide anions (•O₂⁻) in driving antibacterial activity reflects their longer lifetimes and greater diffusion ranges relative to hydroxyl radicals (•OH). Recent studies have demonstrated that ¹O₂ can diffuse across bacterial lipid bilayers and selectively oxidize unsaturated phospholipids, initiating chain peroxidation that destabilizes membrane structure^56,57. Likewise, •O₂⁻ interacts with membrane-associated redox enzymes and surface proteins, propagating oxidative stress across the cell envelope and exacerbating membrane dysfunction^58,59. These mechanistic behaviors are consistent with the pronounced membrane depolarization detected via DiBAC₄(3) and the extensive morphological disruption observed in TEM micrographs. In contrast, •OH—despite its extremely high reactivity—has an ultrashort lifetime and a diffusion radius restricted to only a few nanometers, confining its activity to the nanoparticle interface and preventing widespread structural damage⁶⁰. This ROS distribution explains the rapid membrane collapse and cytoplasmic leakage documented in MDR Staphylococcus aureus and XDR Pseudomonas aeruginosa following exposure to N3 HSs.

Functional evidence of membrane disruption was provided by DiBAC₄(3) membrane permeability assays, which demonstrated a marked increase in depolarization following treatment with sonicated Ni₂CoS₄ HSs across all tested strains. The observed effects were independent of bacterial cell wall structure, highlighting the broad-spectrum applicability of the membrane-disruptive action. Structural evidence from transmission electron microscopy corroborated these findings, revealing severe membrane deformation, cytoplasmic leakage, and cell wall disintegration. These results align with prior reports on the bactericidal activity of transition-metal-based nanomaterials and underscore the role of mechanical destabilization in mediating bacterial death^9,61.

Importantly, the antimicrobial activity of N3 HSs extended beyond planktonic cells. Biofilm disruption assays revealed that N3 significantly reduced biofilm biomass, surpassing the performance of other tested nanoparticle formulations and previously reported nanomaterials^62,63. The substantial reduction in biofilm biomass achieved by N3 indicates that the Ni₂CoS₄ HSs compromise biofilm integrity through a combined structural and cellular effect, weakening the cohesion of the biofilm matrix while diminishing the viability of resident bacterial cells. Given the notorious tolerance of biofilm-associated infections to antibiotics and immune clearance, the observed disruption suggests a meaningful advance in material-based therapeutics.

The broad-spectrum efficacy of N3 against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) strains—including MDR and XDR clinical isolates—reinforces the therapeutic promise of this platform. Despite structural differences in cell wall architecture, both bacterial groups exhibited similar susceptibility to oxidative and mechanical stress. This non-selective action is of strategic value in the development of resistance-proof antimicrobial agents. Indeed, the dual-action mechanism of N3 HSs aligns with theoretical models and longitudinal studies indicating that multi-targeted stress responses minimize the emergence of resistance over successive bacterial generations^9,64.

The selective toxicity of N3 HSs toward bacterial cells over mammalian counterparts, as reported in preliminary in vitro data, further supports their therapeutic viability. Nonetheless, several translational challenges must be addressed. These include optimization of large-scale synthesis protocols, long-term colloidal stability, in vivo pharmacokinetics, and comprehensive safety profiling. Furthermore, the potential for synergistic use with existing antibiotics represents a promising avenue for enhanced efficacy and reduced therapeutic dosing.

In summary, the sonicated Ni₂CoS₄ HSs system developed in this study integrates broad-spectrum antimicrobial activity, potent antibiofilm properties, and resistance-mitigating mechanisms. Through enhanced ROS generation and membrane-targeting effects, this piezocatalytic platform demonstrates a powerful therapeutic profile against difficult-to-treat bacterial pathogens. With further optimization and clinical validation, such nanomaterials could play a pivotal role in next-generation strategies to combat global antimicrobial resistance.

Memory effect and sustained antimicrobial activity of Ni₂CoS₄ HSs

The remarkable memory effect observed in sonicated Ni₂CoS₄ HSs manifests through two distinct but mechanistically linked phenomena: sustained MB degradation and prolonged antimicrobial activity. The sonicated HSs (N3) demonstrate immediate high-efficiency MB degradation with minimal subsequent increase over 96 h, indicating rapid establishment of stable catalytic sites. This stability suggests the formation of persistent structural modifications that maintain ROS generation without requiring continuous mechanical stimulation, fundamentally challenging existing models of piezocatalytic activation³².

The sustained catalytic activity correlates directly with bacterial elimination patterns, revealing crucial mechanistic insights. N3 demonstrates rapid initial reduction of both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) strains, with Gram-negative bacteria showing slightly enhanced susceptibility. This differential response suggests that membrane architecture plays a crucial role in initial susceptibility to ROS-mediated damage, aligning with current understanding of bacterial cell wall interactions with nanomaterials⁶⁵.

Particularly noteworthy is the differential response between standard and resistant strains. The complete elimination of MDR and XDR strains by 24 h, compared to the slightly delayed response of S. aureus ATCC, indicates that conventional antibiotic resistance mechanisms offer no protection against sustained ROS-mediated damage⁶⁶. This finding supports recent theoretical frameworks suggesting that physical bacterial elimination methods can circumvent traditional resistance mechanisms.

In contrast, non-sonicated HSs (N3-O) exhibit fundamentally different activation kinetics, revealed through both gradual MB degradation enhancement and slower bacterial elimination patterns. This temporal evolution indicates that non-sonicated particles require extended periods to develop active sites capable of sustained ROS generation, contrasting sharply with the immediate and stable performance of sonicated particles.

The parallel between MB degradation stability and bacterial elimination kinetics reveals a unified mechanism of sustained ROS generation. The maintenance of high MB degradation efficiency throughout the study period correlates with the progressive elimination of bacterial populations, suggesting that stable catalytic sites drive both processes.

Cytotoxicity of Ni₂CoS₄ HSs

The cytotoxicity evaluation of Ni₂CoS₄ HSs reveals sophisticated dose-response relationships that merit careful analysis within the context of therapeutic applications. This systematic investigation provides novel insights into concentration-dependent cellular responses, sonication-mediated effects, and cellular adaptation mechanisms within nanomaterial safety assessment.

In the therapeutic concentration range (0–100 µg/mL), both sonicated (N3) and non-sonicated (N3-O) formulations exhibited remarkable biocompatibility, maintaining cell viability at 92.59% and 88.72%, respectively. These findings exceed the safety profiles reported for similar metal sulfide nanoparticles^67,68. Statistical analysis revealed this difference was not clinically significant (p > 0.05), indicating a minimal impact of sonication on cellular viability within therapeutic concentrations.

The emergence of significant cytotoxicity at 200 µg/mL (p < 0.0001) represents a biologically relevant threshold, indicative of concentration-dependent nanomaterial toxicity⁶⁹. This threshold likely reflects the point at which intrinsic cellular defense systems—such as antioxidant and stress-response pathways—become saturated. Supporting this interpretation are three key observations: (i) the abrupt decline in cell viability between 100 and 200 µg/mL, (ii) the amplified sensitivity to sonicated nanoparticle formulations at elevated concentrations, and (iii) the strong correlation between cytotoxicity and increased levels of reactive oxygen species (ROS). Such trends are consistent with established models of oxidative stress responses, in which cellular homeostasis is maintained until a critical threshold is exceeded, leading to rapid toxicity escalation^70,71.Particularly noteworthy is the divergent response observed between sonicated and non-sonicated formulations at higher concentrations. The present findings suggest that sonication induces nanoscale alterations—potentially influencing surface charge, aggregation state, or structural morphology—that significantly modulate nanoparticle–cell interactions. The enhanced cytotoxicity of sonicated formulations at 200 µg/mL reinforces the hypothesis that physical modifications can potentiate biological effects⁷², thereby refining current paradigms in nanotoxicology and advancing the understanding of concentration-dependent responses⁷³.

Cytotoxicity and mechanisms of N3 HSs in colorectal cancer

N3 HSs exhibit pronounced dose-dependent cytotoxicity against HCT-116 colorectal cancer cells, positioning them as promising candidates for further nanotherapeutic investigation. Their anticancer activity reflects the characteristic behavior of piezocatalytic materials, which generate reactive oxygen species (ROS) under mechanical stimulation. This mechanism is well documented in previous studies, where ROS-mediated oxidative stress disrupts cellular homeostasis and promotes apoptotic and necrotic cell death^74,75.

Through mechanical activation, piezocatalytic HSs such as N3 facilitate ROS production, resulting in oxidative damage to cellular membranes, proteins, and nucleic acids. This disruption interferes with essential biological processes and initiates signaling pathways that culminate in programmed cell death. The efficacy observed in this study aligns with previous reports demonstrating the capacity of ROS-generating nanomaterials to induce cytotoxicity in malignant cells^12,75.

Mechanically responsive nanomaterials have gained prominence in tumor therapy for their ability to generate ROS under ultrasound stimulation. BaTiO₃-based piezoelectric structures illustrate this principle, where ultrasound-induced polarization enhances intracellular ROS production and triggers mitochondrial dysfunction and apoptosis in tumor models¹⁰. Composite systems such as Cu₂₋xO–BaTiO₃ further show that interfacial engineering can strengthen charge separation and improve ROS-mediated cytotoxicity⁷⁶. Reviews of piezoelectric sonosensitizers similarly report that high anticancer activity often depends on heterostructure design or defect modulation to optimize mechanochemical responsiveness⁷⁷. Within this context, the IC₅₀ value of ~ 100 µg/mL observed for N3 falls within the range reported for advanced piezoelectric tumor platforms. Importantly, N3 achieves this level of cytotoxicity using a single-phase Ni₂CoS₄ material, without reliance on multicomponent interfaces or doped heterostructures. Although optimization of synthesis conditions remains essential for any functional nanomaterial, the present data indicate that N3 attains therapeutically relevant activity with a structurally streamlined formulation, supporting its potential as a potent candidate for further piezocatalytic cancer-therapy development.

The sustained reduction in cell viability over a 96-hour period confirms the long-term cytotoxic effect of N3 HSs. This memorial effect is likely attributable to persistent oxidative stress or prolonged intracellular activity, which may result in delayed or cumulative damage. Such behavior aligns with trends seen in other sonicated piezocatalytic systems, where extended ROS release contributes to continuous bioactivity²⁹. The ability to maintain anticancer efficacy over time is particularly advantageous for therapeutic applications, where extended tumor suppression is essential.

Flow cytometry further validated the cytotoxic mechanism, revealing a marked shift from viable to non-viable cell populations following N3 treatment. The increase in membrane-compromised cells confirms loss of integrity consistent with ROS-induced cell death⁷⁸. These findings are in agreement with established pathways involving mitochondrial dysfunction, caspase activation, and oxidative membrane rupture, all of which are common consequences of excessive intracellular ROS generation⁷⁹.

Although the same ROS species are involved, their intracellular targets in cancer cells differ markedly from those in bacteria. In HCT-116 cells, ¹O₂ and •O₂⁻ primarily disrupt mitochondrial integrity and redox-regulated signaling pathways rather than the plasma membrane, consistent with the apoptosis and necrosis patterns observed in flow-cytometry analysis. This heightened sensitivity reflects the intrinsically elevated oxidative burden of cancer cells and their limited capacity to neutralize sustained exogenous ROS⁸⁰.

The exceptional cytotoxicity of N3 is likely enhanced by its structural and physicochemical characteristics. High crystallinity, uniform elemental distribution, and hierarchical nanoscale morphology collectively promote efficient mechanical activation and catalytic ROS production. These attributes contribute directly to the biological performance observed and support the importance of material design in optimizing therapeutic efficacy.

The differential response of HDF and HCT-116 cells to N3 exposure aligns with established disparities in redox regulation between malignant and non-malignant phenotypes. Malignant cells typically maintain elevated basal reactive oxygen species (ROS) levels due to metabolic reprogramming and mitochondrial dysfunction, resulting in reduced capacity to counter additional oxidative stress⁸¹. This intrinsic imbalance increases susceptibility to oxidative injury, as ROS accumulation disrupts mitochondrial membranes, activates apoptotic and necrotic signaling pathways, and compromises essential biomolecular structures⁸². In contrast, normal fibroblasts possess more effective antioxidant systems and exhibit greater mitochondrial stability, conferring enhanced tolerance to ROS-mediated insult under equivalent conditions⁸³. This mechanistic rationale supports the interpretation that the observed differences in cytotoxicity arise from inherent biochemical characteristics of the two cell types rather than from preferential or exclusive activity of the N3 formulation. The convergence of cellular responses at higher nanoparticle concentrations further highlights the concentration-dependent nature of these effects and the necessity for careful interpretation when comparing malignant and non-malignant outcomes⁸⁴.

Compared with conventional metal and metal-oxide nanoparticles, which rely heavily on metal-ion release and surface-limited ROS generation for their antibacterial and anticancer activity⁸⁵, the Ni₂CoS₄ HSs provides a distinct mechanistic advantage by coupling intrinsic redox activity with ultrasound-activated piezocatalysis, producing a more sustained flux of ¹O₂, •O₂⁻ and •OH. Although functionalized Ag/Au nanoparticles can enhance antimicrobial and anticancer efficacy⁸⁶, their applicability is constrained by high cost, limited activation modes and potential cytotoxicity. By contrast, Ni₂CoS₄ exhibits multi-target antibacterial effects—including membrane disruption, biofilm destabilization and ROS overload—together with potent anticancer activity driven by mechanocatalytic ROS generation and mitochondrial impairment, consistent with evidence highlighting transition-metal sulfides as high-reactivity therapeutic nanocatalysts.

In summary, N3 HSs demonstrate significant and sustained anticancer activity in vitro, driven by piezocatalytic ROS generation and associated cellular damage. The combined short-term, long-term, and mechanistic evidence confirms their potential as multifunctional anticancer agents. Future investigations should focus on evaluating selectivity toward non-malignant cells, elucidating molecular pathways of cell death, and validating in vivo efficacy to fully explore their translational application in cancer nanotherapy.

Clinical translation and future perspectives

The translation of Ni₂CoS₄ HSs into clinical applications presents both remarkable opportunities and significant challenges that demand rigorous consideration. While our findings demonstrate potent antimicrobial effects through piezocatalytic ROS generation and favorable cytotoxicity profiles at therapeutic concentrations (maintaining 92.59% cell viability at ≤ 100 µg/mL), several crucial aspects require systematic investigation for successful clinical implementation. The optimization of delivery systems represents a primary translational challenge, particularly regarding the preservation of piezocatalytic properties in complex physiological environments. The demonstrated optimal parameters (200 W power, 1–3 s on-off cycling) must be adapted for diverse clinical settings while maintaining therapeutic efficacy. The remarkable uniformity in antimicrobial activity against both sensitive and resistant strains, coupled with significant biofilm disruption capabilities, suggests broad therapeutic potential. However, tissue-specific responses and local mechanical force variations necessitate careful optimization of delivery strategies to maintain therapeutic efficacy while ensuring patient safety⁸⁷.

Long-term safety considerations present additional challenges requiring systematic investigation. While our findings demonstrate favorable initial cytotoxicity profiles, comprehensive evaluation of tissue-specific responses and potential accumulation effects remains crucial. The significant membrane-disrupting capabilities of N3, while highly effective against bacterial pathogens, necessitate careful assessment of potential effects on host cell populations during prolonged exposure. The demonstrated piezocatalytic memory effect, while advantageous for sustained antimicrobial activity, requires thorough investigation of long-term tissue responses and potential adaptive mechanisms²⁹. The development of resistance monitoring strategies represents another critical aspect of clinical translation. Although the multi-modal mechanism of action, combining ROS generation with mechanical membrane disruption, suggests reduced likelihood of resistance development, systematic investigation of potential bacterial adaptation mechanisms remains essential. The uniform efficacy against MDR and XDR strains highlights the potential for addressing resistant infections, yet comprehensive monitoring of bacterial stress responses and potential cross-resistance patterns is crucial for developing effective resistance mitigation strategies⁸⁸.

Future research directions should encompass advanced delivery system development optimizing piezocatalytic activation in physiological environments, tissue-specific response evaluation and long-term safety assessment, investigation of potential synergistic effects with conventional antimicrobial therapies, development of standardized clinical protocols for therapeutic implementation, establishment of resistance monitoring frameworks, optimization of surface modifications for enhanced biocompatibility and targeting, and investigation of potential immunomodulatory effects. The successful translation of these findings into clinical practice could provide valuable new tools for combating resistant infections while minimizing adverse effects on host tissues. The unique combination of controlled activation, sustained therapeutic effects, and selective toxicity positions Ni₂CoS₄ HSs as promising candidates for addressing the growing challenge of antimicrobial resistance in clinical settings.

In parallel with these translational opportunities, several considerations delineate the scope of the present study and highlight essential areas for further investigation. Although this work establishes a comprehensive optimization framework and demonstrates the strong biological performance of the N3 formulation, all experiments were conducted under controlled in-vitro conditions, and the behavior of Ni₂CoS₄ HSs in vivo—where biodistribution, immune interactions, mechanical forces, and long-term safety profiles differ substantially—remains to be elucidated. Moreover, the antimicrobial evaluation focused on two priority pathogens, and the anticancer assessment utilized one malignant and one normal cell line, which does not encompass the broader biological diversity relevant to clinical translation. Mechanistic interpretation relied primarily on ROS profiling without downstream pathway confirmation, and the ultrasonic activation parameters optimized in vitro may not directly reflect forces achievable within physiological environments. These considerations define important directions for future work aimed at advancing Ni₂CoS₄ HSs toward comprehensive biomedical and environmental application.

Conclusion

This study establishes a clear structure–function relationship for Ni₂CoS₄ HSs and demonstrates that rational hydrothermal optimization yields a superior mechanocatalytic platform with broad biomedical potential. Among all synthesized formulations, the sonicated N3 variant consistently exhibited the most advantageous physicochemical features—including enhanced crystallinity, hierarchical hollow architecture, uniform elemental distribution, and redox-active surface states—collectively underpinning its markedly improved catalytic and biological performance. Optimized piezocatalytic activation (200 W, 1–3 s on/off cycles, 4 min) resulted in 88.56% methylene blue degradation, the highest efficiency attained among all tested formulations. ROS profiling confirmed a > 13-fold enhancement in singlet oxygen and superoxide generation, providing the mechanistic basis for the pronounced antimicrobial, antibiofilm, and anticancer effects observed across assays. N3 exhibited potent antibacterial activity against MDR Staphylococcus aureus and XDR Pseudomonas aeruginosa, reducing MIC values to 10 µg/mL and 5 µg/mL, respectively, and achieving complete pathogen eradication within 48 h. DiBAC₄(3) depolarization assays and TEM imaging corroborated these findings, revealing extensive membrane disruption and cytoplasmic leakage consistent with ROS-driven bactericidal action. The antibiofilm efficacy of N3 was equally notable, achieving ≥ 99% reduction of biofilm formation at 40–60 µg/mL and 98–99% removal of established biofilms at 60–80 µg/mL, far surpassing the performance of the non-sonicated N3-O formulation. N3 also demonstrated selective cytotoxicity toward HCT-116 colorectal cancer cells while maintaining favorable compatibility with normal fibroblasts. Cancer-cell viability declined in a dose-dependent manner (IC₅₀ = 100 µg/mL; LC₅₀ = 200 µg/mL), with complete loss of viability at 800 µg/mL. The long-term memorial effect further showed persistent suppression of cancer-cell viability to ~ 13% at 96 h, and flow cytometry confirmed a progressive increase in non-viable cells from 48.7% at IC₅₀ to 91.0% at LC₅₀, consistent with sustained ROS-mediated cytotoxicity. Collectively, these findings position sonicated Ni₂CoS₄ HSs as a highly efficient, multifunctional hierarchical structures that integrates optimized piezocatalytic activity with potent antimicrobial, antibiofilm, and anticancer capabilities. Their selective toxicity toward pathogenic bacteria and malignant cells, combined with their cytocompatibility with normal fibroblasts, underscores their strong translational promise. Future work should prioritize in vivo efficacy studies, comprehensive biosafety and pharmacokinetic profiling, deeper mechanistic analysis of ROS-driven pathways, and investigation of synergistic interactions with existing antimicrobial and chemotherapeutic agents to accelerate clinical development of this promising piezocatalytic nanomaterial.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

K.Q. Writing—original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. O.A. Writing—review & editing, Project administration, Validation, Supervision. H.H. Writing—review & editing, Validation, Supervision.

Funding

The authors received no funding for this work.

Data availability

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

Declarations

Competing interests

The authors declare no competing interests.