Introduction

As a new kind of nanomaterial, zero-dimensional quantum dots (QDs) have attracted much interest recently due to their unique and superior optical and electrical properties. These nanomaterials have been widely explored in bioimaging, medical diagnosis, and chemical sensing, which makes them excellent candidates to be effectively used in various industrial and technical applications. The most important feature of nanosized materials was the increased surface area-to-volume ratio (large aspect ratio) and enhanced targeting action of nanomaterials, even in low concentrations1,2. Pulsed Laser Ablation in Liquids (LAL) is a versatile technique for synthesizing nanoparticles, involving the use of a pulsed laser focused onto a target material immersed in a liquid3,4. The process begins with the laser pulse interacting with the target, leading to rapid heating, melting, and vaporization of the material5. This results in the formation of a plasma plume containing ablated species6. The plasma then rapidly cools due to the surrounding liquid, leading to nucleation and growth of nanoparticles5. A cavitation bubble is generated by the energy exchange between the plasma and the liquid, and nanoparticles are released from the bubble into the solution5. The size, shape, and composition of the nanoparticles can be controlled by adjusting laser parameters such as wavelength, pulse duration, and fluence, as well as the properties of the liquid medium5,7. Transparent conductive oxide (TCO) nanomaterials such as zinc oxide (ZnO) draw significant attention because of their excellent electrical and optical characteristics, which are used widely in optoelectronic devices, gas sensing, medical applications, and photocatalysis8,9. ZnO nanoparticles have been found to have high antibacterial activity against spores, which have high resistance against high-temperature pressure conditions, as well as against both Gram-negative and Gram-positive bacteria9,10.

Their size and morphology play a vital role in their toxic nature; whenever the particles are smaller, they will have higher antibacterial activity against bacteria and spores. Based on reported information, the toxicity mechanism of ZnO nanoparticles is not well understood yet. Some researchers propose that these nanoparticles are connected to the surface of bacteria via electrostatic force, bind it, and cause death; others suggest that the main reason for the antibacterial activity is the production of hydrogen peroxide in the cell. Quantum dots (QDs) in general can easily cause damage to DNA and oxidative stress when attached to bacteria cells with targeting action, even at low concentrations, making them good candidates for bioimaging and drug delivery9,10,11. As reported, the ZnO nanoparticles can be synthesized via numerous chemical and physical routes, and their physical and chemical properties depend strongly on the size, surface chemistry, morphology, and chemical composition. Laser ablation in liquid (LAL) is one of these routes used efficiently to synthesize colloidal ZnO nanoparticles11. LAL is a simple process that does not require any catalysts, produces high-purity nanoparticles quickly and at low cost, maintains product stoichiometry, and eliminates the need for a cooling process after laser action, as the surrounding liquid serves as both a cooling and confinement factor12,13.

Basically, the efficiency of the LAL method depends mainly on two factors: the physical features of the metal (target) and the surrounding medium properties. The size, morphology, and concentration of synthesized nanoparticles depend on the laser energy, spot size, wavelength, pulse duration, and pulse repetition frequency14,15. In this regard, we report on the synthesis of zinc oxide quantum dots (ZnO QDs) by means of the LAL technique. The effect of structural and optical properties was studied as a function of laser energy. The antibacterial activities of ZnO QDs against two kinds of bacteria, one gram-negative Escherichia coli (E. coli) and one gram-positive Streptococcus pyogenes (S. pyogenes), were investigated. The biocompatible effect of ZnO QDs using normal rat embryonic fibroblast (REF) cells was also assessed.

Although there is extensive research on ZnO nanoparticles, a significant gap remains in understanding how variations in laser energy during the synthesis process affect their antibacterial efficacy and structural properties. This study presents a novel approach to synthesizing zinc oxide quantum dots (ZnO QDs) through Nd: YAG laser ablation in water, revealing how varying laser energy impacts their structural and antibacterial properties. Unlike traditional methods, our investigation demonstrates that smaller ZnO QDs (3–6 nm) exhibit significantly higher antibacterial activity compared to larger nanoparticles (16–22 nm) at reduced concentrations. Therefore, this study fills a gap by systematically investigating the relationship between laser energy and the resulting properties of ZnO quantum dots, highlighting their potential as improved antibacterial agents.

Materials and methods

Preparation of ZnO QDs

The preparation of colloidal ZnO NPs and ZnO QDs was performed by laser ablation of high-purity zinc pellets positioned in a quartz vessel filled with 2 ml of deionized water as a medium. The laser used for the ablation was a pulsed Q-switched Nd: YAG laser of 1064 nm wavelength. A pulse width of 7 ns and a pulse repetition frequency (PRF) of 1 Hz were applied. As shown in Fig. 1, the laser beam was focused on the zinc pellet by a converging lens with a focal length of 12 cm. In addition, the spot size of the laser beam was measured and found to be 1.3 mm. The laser energy used for the ablation was 60 and 120 mJ, and the number of pulses was 120 for all experiments.

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Schematic diagram of LAL system.

Characterization of ZnO QDs

The structural properties of ZnO were examined by X-ray diffractometer (XRD Shimadzu-6000) pattern that measured with Cu-Kα X-ray source at diffraction angle range of 2θ = 20–80º. Fourier transform infrared spectroscopy (FTIR) (Bruker-7619) was used to range between 400 and 4000 cm⁻¹ to investigate their crystallographic structure and their vibrational bonds, respectively. The morphological characterization was investigated by high-resolution transmission electron microscopy (TEM Titan 80–300 HRTEM). The optical absorption of the ZnO colloid QDs was measured using a double-beam UV-VIS spectrophotometer (Shimadzu-1800).

Antibacterial activity

The antibacterial activity of the ZnO QDs was investigated against two pathogens: one gram-negative, Escherichia coli (E. coli), and one gram-positive, Streptococcus pyogenes (S. pyogenes), using nutrient broth and nutrient agar. For nutrient broth, the bacteria suspensions were set by adjusting with 0.5 McFarland turbidity basis (5 × 107 cell/mL) tubes. A UV spectrophotometer was employed to determine the optical density (OD) at a 600 nm wavelength. The cell viability (the number of bacteria cells in one milliliter of broth medium) is determined from the equation:

$$CellViability=\frac{\text{C}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\text{o}\text{l}\text{O}\text{D}}{\text{T}\text{e}\text{s}\text{t}\text{O}\text{D}}$$
(1)

A lawn of bacterial culture was placed on solid nutrient agar plates and allowed to rest for 15 min. The tip of a sterile micropipette was used to punch 5 mm-sized wells into the agar. Colloid concentrations ranged from 110 µg/mL at 60 mJ laser energy to 430 µg/mL at 120 mJ.

Bacterial biofilm formation assay

Bacterial strains were cultured in 24-well plates at a concentration of 1 × 10⁶ CFU/mL. They were then treated with ZnO NPs and QDs at concentrations of 430 µg/mL and 110 µg/mL, respectively, for 24 h. After treatment, the samples were washed with PBS. The adhered microbial cells were stained with 0.1% crystal violet (CV) and then rinsed with PBS. To quantify biofilm formation, 0.2 ml of 95% ethanol was added to the CV-stained wells, followed by incubation with shaking for 2 h. The optical density (OD) was measured at 595 nm.

Thiazolyl blue tetrazolium bromide (MTT) assay

The cytotoxic activity of the ZnO NPs and ZnO QDs was assessed against normal rat cell line (REF) cells. In brief, 1 × 10⁴ cells were seeded into 96-well plates and incubated overnight at 37 °C to allow for cell attachment. Following this, ZnO NPs and ZnO QDs were added to the REF cells, and the cells were incubated for 48 h. After treatment, MTT solution (Sigma–Aldrich, USA) was introduced to each well. Upon completion of the incubation, the medium was removed, and DMSO (Sigma–Aldrich, USA) was added to dissolve the formazan crystals. Absorbance was then recorded at 492 nm using a microplate reader.

Statistical analysis

For statistical analysis of the data obtained, the unpaired t-test with GraphPad Prism 6 was used. The values were presented as the mean ± SD of triplicate measurements. A significance level of p < 0.05 was utilized.

Results and discussion

Synthesis of ZnO QDs

When the laser irradiates the Zn pellet located inside the deionized water, it results in the production of charged ions and atoms of Zn due to rapid vaporization and plasma formation16. The Zn plasma disperses into the surrounding water, and the ablated Zn atoms react with the oxygen and hydroxyl radicals (OH⁻) that are already there to make ZnO nuclei. Finally, these ZnO nuclei combine to form nanoparticles, and their final size and shape are affected by the laser settings and the surrounding water based on the following chemical reaction:

$$Zn+{H}_{2}O{\to}ZnO+{H}_{2}\left(g\right)$$
(2)

Figure 2 shows the effect of laser energy (E) on the mass concentrations (N) of ZnO NPs.

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Mass concentration of ZnO QDs as a function of laser energy.

The mass concentration of the nanoparticles was found to increase with higher laser energy because the penetration depth in the zinc target leads to the formation of more ablated atoms that enter the surrounding water17,18.

Characterization of ZnO QDs

Figure 3 illustrates the X-ray diffraction (XRD) patterns of ZnO NPs synthesized at different laser energies. Seven peaks were observed at 2θ = 31.7º, 34.4º, 35.3º, 47.6º, 56.4º, 62.6º, and 67.3º, corresponding to the (100), (002), (101), (102), (110), (103), and (112) phases, respectively19,20. These peaks belong to the ZnO hexagonal structure according to JCPDs # 036–1451.

There is no shift in the 2θ of the peaks for ZnO as laser energy increases. When the laser energy is increased from 60 to 120 mJ, the peaks’ full width at half maximum decreases while their intensity increases. Decreasing the FWHM indicates an increase in crystallite size as laser energy increases, and the main reason for the increase in peak intensity is the formation of higher mass concentrations. All of the observed peaks correspond to crystalline hexagonal ZnO, according to JCPDs # 036–1451. There were no Zn-related XRD peaks detected, indicating that Zn had completely oxidized and formed ZnO.

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X-ray diffraction patterns of ZnO QDs prepared at laser energy of 60 and 120 mJ.

The average crystallite size of the ZnO QDs was calculated using Scherrer formula

$${D}_{ave}=\frac{K\lambda}{{\beta}cos\theta}$$
(3)

The dislocation density (δ) and lattice strain (ε) produced in ZnO were estimated using the following equations:

$$\epsilon=\frac{{\beta}cos\theta}{4}$$
(4)

Table 1 lists the values of crystallite size, stress, and dislocation density of the ZnO QDs prepared with two laser energies.

Table 1 Crystallite size, strain, and dislocation density of ZnO QDs.
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As shown in Table 1, the crystallite size of ZnO increases with increasing laser energy. When the laser energy increased, the ablation process generated a larger concentration of Zn and O species in the plume, which increased the supersaturation level in the solution/plasma. This high energy favors crystal growth over new nucleation, resulting in fewer but larger crystallites. Additionally, the strain and dislocation density in ZnO were found to be decreased with increasing laser energy due to the formation of larger crystallites, which reduce grain boundary density and allow lattice defects to relax. As crystallites grow, the ratio of surface atoms to bulk atoms reduces, minimizing internal stress and lowering the probability of dislocation formation.

FTIR spectra of ZnO QDs prepared at 60 and 120 mJ in the wavenumber range of 400–4000 cm− 1 are depicted in Fig. 4. FTIR spectra show a broad absorption peak at 3400 cm− 1 was related to the stretching vibration of the O–H band18. There are two peaks that relate to the Zn-O bond that appear at ~ 510 cm⁻¹ and ~ 470 cm⁻¹. The absorption peaks at ~ 1570 cm− 1 and ~ 1400 cm− 1 are indexed to C-O and C = O bonds, respectively19. When the laser energy increases, the ZnO’s FTIR shows no significant variation. Table 2 shows the main FTIR assignments of ZnO QDs.

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FTIR spectra of ZnO QDs synthesized at different laser energies of 60 and 120 mJ.

Table 2 FTIR assignments of ZnO QDs.
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Figure 5 demonstrates the TEM images of ZnO prepared at 60 and 120 mJ. Figure 5a shows that the ZnO nanoparticles prepared at 60 mJ contain quantum dots (QDs) with a particle size ranging from 3 to 6 nm. The TEM image confirmed the formation of spherical QD morphology. The inset of Fig. 4a is the particle size distribution of the ZnO prepared at 60 and 120 mJ. The average particle size of ZnO prepared at 60 and 120 mJ was 4 and 14 nm, respectively. Some agglomerated ZnO NPs were observed, as shown in Fig. 5a, due to the high surface energy and Van der Waals attraction force. While the sample prepared at 120 mJ shows the formation of spherical nanoparticles with a size in the range of 12–22 nm, as depicted in Fig. 5b. We think this result can be ascribed to the fact that lower laser energy produces a lower concentration of material being ablated from the zinc target, which generates a less dense plasma plume17,21.

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TEM images of ZnO nanoparticles prepared at (a) 60 mJ and (b) 120 mJ. Insets are the particle size distribution of ZnO QDs.

Figure 6 shows the effect of laser energy on the optical absorption of colloidal ZnO NPs. The absorbance decreases sharply after a wavelength of 200 nm due to the semiconducting behavior of ZnO. A shoulder was observed at 333 and 336 nm for samples prepared at 60 and 120 mJ, respectively. This result is due to the quantum confinement that comes from the nanosized effect. The optical absorption of the ZnO prepared at 60 mJ is higher than that prepared at 120 mJ, particularly in the UV-Vis region, due to the higher concentration of ZnO and smaller particle size. In the inset of Fig. 6 are the photographs of the ZnO colloids; the colloid prepared with 10 mJ is darker than that prepared with 60 mJ due to the presence of a high concentration of the ZnO nanoparticles. The stability of the ZnO nanoparticles synthesized without stabilizers was examined by repeating the UV–Vis absorption spectra after two weeks of storage under laboratory conditions. The absorption edge position and spectral shape remained unchanged, and no color variation of the colloidal solution was observed, indicating good colloidal stability in the absence of stabilizing agents.

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UV-Vis absorption of colloidal ZnO QDs. Inset is the photograph of ZnO colloids, which reveal the colors of ZnO colloids prepare at different values of laser energy.

Figure 7 shows the variation of (αhν)² against the photon energy (hν) plot for the ZnO NPs. The optical energy gap (Eg) of ZnO QDs was estimated by plotting the variation of (αhν)² with photon energy (hν), as shown in Fig. 7. The absorption coefficient (α) of the CdS nanoparticles was calculated using the Beer–Lambert relation:

$$\alpha=\frac{2.303A}{d}$$
(5)

where A is the measured absorbance of the ZnO nanoparticles and d is the optical path length of the CdS nanostructured sample.

The extrapolation of the linear part on the photon energy axis to (αhν)2 = 0 points provided the optical energy gaps according to Tauc plot22,23.

$$({\alpha{h}}{\nu}{)}^{2}=\left(h\nu-{E}_{g}\right)$$
(6)

As shown in Fig. 7, the optical energy gap of ZnO QDs and NPs was 4.15 and 3.35 eV, respectively. In addition, the linear part observed at higher photon energies belongs to strong absorption and confirms that ZnO shows a direct allowed electronic transition. The low-energy region is associated with weak absorption and does not represent the fundamental band gap transition24,25.

The decrease in the energy gap as the laser energy increases is due to the increase in particle size. When the particle size of ZnO nanoparticles decreases (as in the sample prepared at 60 mJ) and becomes comparable to the exciton Bohr radius (~ 2.34 nm for ZnO), the motion of electrons and holes becomes confined within the ZnO particle. This quantum confinement effect increases the energy separation between the valence and conduction bands, resulting in a wider optical energy gap (a blue shift in absorption). In contrast, when the particle size increases (as in the sample prepared at 120 mJ) beyond the quantum confinement regime, the confinement effect weakens, and the energy levels approach those of bulk ZnO. Consequently, the energy gap decreases (a red shift). Therefore, the observed reduction in the energy gap with increasing particle size indicates a transition from quantum-confined nanoparticles to bulk-like behavior26,27.

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(αhν)2 versus photon energy plot for the colloidal ZnO NPs prepared at 60 and 120 mJ. The extrapolation of the high absorption regions to photon energy axis gives the optical energy gap.

Antibacterial activity of ZnO QDs

We tested the antibacterial activity of ZnO QDs and ZnO NPs prepared at 60 and 120 mJ, respectively, against E. coli and S. pyogenes using nutrient broth and nutrient agar. At 60 mJ laser energy, ZnO QDs had a mass concentration of 110 µg/mL, while ZnO NPs at 120 mJ had a mass concentration of 430 µg/mL. Figures 8 and 9 show the OD and cell viability (calculated from Eq. (1)) of bacteria culture growth (E. coli and S. pyogenes) in the presence of ZnO QDs and ZnO NPs at their mass concentrations.

Increasing the mass concentration of particles does not necessarily increase their toxicity, because any particle’s toxicity is heavily influenced by its chemical composition, shape, and size, all of which control its ability to penetrate the biological cell28. Figure 5 shows this clearly. The higher concentration (430 mg/mL) was three times higher than the lower concentration (110 µg/mL), but the OD and cell viability were lower. Figure 8 (lower panel) depicts the variation in the inhibition zone against bacterial strains following exposure to ZnO NPs and QDs. Despite the significant difference in mass concentration, the lower concentration has higher antibacterial activity than the higher concentration, which is due to the difference in particle size. ZnONPs at 430 µg/mL were approximately 16–22 nm in size, while ZnO QDs at lower concentrations were approximately 3–6 nm. Both nanosized particles (ZnO NPs and ZnO QDs) have the same nature and chemical composition as zinc oxide, but their toxicity varies with size. There are three primary mechanisms that are thought to be involved in harmful antibacterial mechanism of metallic NPs. First, the production of reactive oxidative species (ROS). Second, the release of ions. Finally, the interaction of NPs with the cell membrane29. The antibacterial mechanism is mostly influenced by the size of NPs30. The first step of metallic NPs in antibacterial activity mechanism involves the attachment of nanoscale metallic ions to the cell through transmembrane proteins. The entire process is dependent on size after adhering to bacterial cells, causing structural alterations in the cell membrane, and obstructing the transport channels. While larger NPs have a larger absolute surface area that allows for better van der Waals force adhesion, smaller NPs are more effective. NPs may then be absorbed, cause ionization inside the cell, and harm intracellular structures, ultimately leading to cell death31. The antibacterial efficacy of metal nanoparticles is largely dependent on their generation of reactive oxidative species (ROS). Superoxide radicals (O− 2), hydrogen peroxide (H₂O₂), hydroxyl radicals (OH− 1), and singlet oxygen (O− 2) are examples of short-lived oxidants that make up ROS. ROS can harm peptidoglycan and cell membranes, DNA, mRNA, ribosomes, and proteins. Additionally, ROS can impede the electron transport chain, enzyme activity, transcription, and translation. ROS production is a major cause of toxicity for certain metal oxide nanoparticles32. As a result of the increasing Zn2 + ions inside the bacteria and the hook-up of ZnO NPs there is harm to the microorganism cytomembrane. The positive surface potential of ZnO-NPs improves the assembly of the reactive element species and imposes mechanical membrane stress while communicating with the negative surface potential of the microorganism membrane, contributing to membrane depolarization25.

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Growth curves of tested materials in broth medium against Escherichia coli and Streptococcus pyogenes in present of at concentration 110 µg/mL ZnO QDs prepared at 60 mJ laser energy and 430 µg/ml ZnO NPs prepared at 120 mJ laser energy.

The results showed that the antibacterial activity of both ZnO NPs and ZnO QDs is higher in gram-positive S. pyogenes than in gram-negative. E. coli, as indicated in Fig. 8, which means that the E. coli have higher resistance than S. pyogenes. This may be due to the variation in membrane nature and the biofilm creation between them33,34,35,36. The cell wall of gram-negative bacteria contains a thin layer of peptidoglycan and a thick layer of LPS. On the other hand, Gram-positive bacteria have thin LPS in their cell walls37. Gram-negative bacteria, in contrast to Gram-positive species, have a highly complex cell envelope with an outer membrane rich in lipopolysaccharides, which reduces membrane permeability and improves protection against antimicrobial agents like nanoparticles38.

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Antibacterial activity in present of 110 µg/mL ZnO QDs concentration prepared at 60 mJ laser energy and 430 µg/mL ZnO NPs prepared at 120 mJ laser energy against (a) Escherichia coli and (b) Streptococcus pyogenes.

The initial attachment of bacterial cells to a surface is a crucial step in biofilm formation and can occur through both specific and nonspecific interactions. Biofilms can be visualized by staining the attached cells with crystal violet. In this study, the inhibitory effects of ZnO NPs and ZnO QDs on biofilm formation were evaluated. As shown in Fig. 10, both ZnO NPs and ZnO QDs demonstrated strong anti-biofilm activity. The results indicate that these ZnO NPs and ZnO QDs significantly inhibit the growth of bacterial strains and reduce biofilm development. The results of the current study indicated that the ZnO NPs and ZnO QDs have fewer cytotoxic effects against the normal cell line (REF cells), as indicated in Fig. 11.

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ZnO NPs and ZnO QDs reduce biofilm formation in bacterial strains (Escherichia coli and Streptococcus pyogenes).

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Biocompatibility of ZnO NPs and ZnO QDs in REF cells of at concentration of 110 µg/mL and 430 µg/ml, respectively.

Nanoparticles are toxic to bacteria because of their high surface area-to-volume ratios; they can attach via electrostatic force and penetrate the cell membrane, increasing penetrability39. As a result, uncontrolled mass transfer and proteolytic enzyme release will occur, leading to a decrease in protein content40.

Nanosized particles inhibit bacterial growth via three biological processes: penetration of the cell membrane, production of reactive oxygen species (ROS), and finally binding to nuclear content (DNA, RNA). Oxidative stress causes the release of ROS (O₂H, O₂⁻, HO•, and O₂²•), which accelerates bacteria structure damage41. ROS binds to bimolecular structures using electrostatic force, promoting the discharge of cytoplasmic components and causing the bacteria cell to die42. The proposed mechanisms for ZnO QDs’ antibacterial activity are limited to their ability to penetrate bacterial cells43 and generate excessive amounts of oxidative materials, such as H₂O₂ and OH-radicals, which cause oxidative stress and cellular damage44. Furthermore, ZnOQDs may cause structural changes in the bacterial cell membrane, resulting in membrane lysis and increased cell permeability. This effect causes bacterial intracellular contents to leak out of the cells. The subsequent increase in ZnOQDs concentrations within bacterial cells can disrupt DNA replication, halt gene expression, and cause condensation and damage to bacterial DNA45. Furthermore, ZnO can dissolve and release zinc ions, disrupting essential cellular processes and contributing to the antibacterial effect46. Finally, ZnOQDs’ positive charge may facilitate their binding to negatively charged bacterial cell walls, increasing their uptake and toxicity47. Figure 12 depicts the proposed mechanisms for ZnOQDs as antibacterial agents.

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Plausible mechanisms of the ZnO QDs as antibacterial agent.

Conclusion

Zinc oxide quantum dots and nanoparticles were successfully prepared by low-cost one-step laser ablation in liquid under different laser energies. The prepared ZnO QDs and NPs were crystalline with a hexagonal structure. TEM analysis showed the formation of spherical particles with average particle sizes of 4 and 14 nm for the ZnO samples prepared at 60 and 120 mJ, respectively. The optical energy gap decreased from 3.35 to 4.15 eV as the laser energy increased from 60 to 120 mJ. The ZnO QDs prepared at 60 mJ exhibited higher antibacterial activity against two types of bacteria, E. coli and S. pyogenes, than those prepared at 120 mJ, since the effective concentration of ZnO QDs was 110 μg/mL, while the concentration for the latter was 430 µg/mL. Our findings indicate that ZnO QDs have the potential to mitigate microbial biofilm growth on both bacterial types with less cytotoxicity against the normal cell line, thus supporting the biocompatibility of ZnO QDs. This difference in antibacterial activity can be attributed to the distinct properties of the ZnO QDs produced at varying laser energies, which affect their surface morphology and reactivity. Consequently, optimizing the laser energy during synthesis may enhance the efficacy of these nanoparticles in antibacterial applications.