Carbon quantum dots (CQDs) with exciting optical properties, low toxicity, and high biocompatibility which are suitable for research in biomedical applications. In this work, the CQDs were prepared through microplasma treatment of orange juice as a carbon source, employing atmospheric pressure and low temperatures. The resulting CQDs exhibited a narrow size distribution, with an average diameter of approximately 4.5 nm. These CQDs displayed strong blue emission characteristics. We evaluated the antibacterial properties of these CQDs against Escherichia coli (E. coli) bacteria strains, using minimum inhibitory concentration assays. Our findings revealed that the minimum inhibitory concentration required for effective inhibition of E. coli was 0.1 ppm, while the minimum bactericidal concentration for this kind of bacteria was 1 ppm with an average antibacterial efficacy of 57%. The antibacterial processes of CQDs could occur without additional light or oxidant.
Research Article
Antibacterial Potential of Carbon Quantum Dots against Escherichia coli Bacteria Strains
https://doi.org/10.21203/rs.3.rs-3609682/v1
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Carbon Quantum Dots
Plasma
Antibacterial
Escherichia coli
Bacteria Strains
Advancements in nanotechnology have significantly impacted various application fields, including drug delivery, imaging, and therapeutic agents [1]. The diversification of biological and chemical properties in nanomaterials has paved the way for their application in nanomedicine, offering reduced immunogenicity, prolonged circulation, improved solubilization, and enhanced safety. Nanoparticles have demonstrated potential antibacterial properties due to their capacity to penetrate and disrupt bacterial cells, presenting promising strategies to combat the growing threat of bacterial resistance [2]. Several types of nanoparticles have been explored for antibacterial applications, including Ag, Cu, Au, and Fe, which have shown considerable effectiveness [2–4], However, their clinical utility poses challenges. Moreover, it is crucial to consider the potential toxicity of these nanoparticles on cells, which cannot be overlooked.
In recent years, carbon quantum dots (CQDs) have emerged as a class of photoluminescent metal-free nanomaterials, constructed by a carbon core enveloped within an amorphous carbon structure. CQDs possess several advantageous properties, including photostability, strong biocompatibility, high water dispersibility, low toxicity, and straightforward synthesis methods [1, 5]. Consequently, CQDs have garnered increasing research interest in recent decades. The CQDs exhibit outstanding fluorescence and minimal cytotoxicity, rendering them suitable for a wide range of applications, such as optoelectronics, sensing, and photocatalysis [6, 7]. Notably, their utilization in the biomedical field has garnered recognition and attention from the scientific community, particularly for their theranostic applications, including cellular imaging, bioimaging, drug delivery, probes, and antibacterial activity [8–10].
Diverse approaches have been employed for the synthesis of CQDs, encompassing both (i) bottom-up methods, such as hydrothermal and solvothermal syntheses, microwave synthesis, and plasma treatments, and (ii) top-down techniques, including electrochemical exfoliation, laser ablation, and chemical oxidation [10–12]. However, it's essential to acknowledge that some of these methods involve the use of strong acids, high processing temperatures, and complex multi-step processes, which can pose disadvantages when scaling up production or considering medical applications, potentially introducing hazardous effects due to the presence of toxic chemicals. As a result, there remains a pressing need for greener chemistry routes in the synthesis of nanoparticles, aiming for eco-friendly and cost-effective production. Notably, recent efforts have explored the use of natural precursors, such as juice from Citrus medica [13], orange juice [14], honey [15], and grape seeds [8] for CQD synthesis. These products derived from natural sources are considered "green" and cost-effective, making them suitable for bulk product development. Researchers have highlighted the advantages of synthesizing CQDs from fruit juices, particularly citrus fruits, which are predominantly acidic and rich in carboxyl groups, resulting in higher yields compared to other fruits [13].
Plasma is a fundamental state of matter that comprises photons, charged species, molecular, atomic, and radical species. Cold plasmas can be generated without the need for expensive vacuum systems or pumps, making them applicable in various fields such as plasma sintering, water desalination, nanomaterial synthesis, surface modification, materials deposition, and bacterial inactivation [16]. Xu et al. utilized cold plasmas for synthesizing CQDs, employing chloroplatinic acid and ethanol as precursors, resulting in a single emission wavelength [17]. Kumar et al. achieved the creation of graphene quantum dots enveloped in gold nanoparticles through DC microplasma [18]. Blue emissive CQDs have been successfully synthesized via plasma treatment using a range of carbon precursors, including citric acid, D-fructose, sodium dodecyl sulfate, chitosan, hydrocarbons, and folic acid [18–22]. Notably, CQDs synthesis has proven successful in both acidic and basic reaction environments. However, it's worth noting that the use of fruit juice as a precursor for CQDs synthesis through the plasma method remains an unexplored area of research.
The excessive use of antibiotics, both in agricultural and clinical settings, has resulted in widespread antibiotic resistance, necessitating the urgent development of new antibacterial agents. According to the World Health Organization (WHO), Gram-negative (GN) bacterial pathogens are particularly resistant to antibacterial treatments [23]. The structure of GN bacteria inherently provides greater resistance compared to Gram-positive (GP) bacteria. GN bacteria feature a slim, peptidoglycan-covered outer layer with multiple thin membrane layers that do not readily absorb external materials, making them more challenging to combat with various antibacterial agents. Escherichia coli (E. coli), a representative GN bacteria, has been studied for its resistance to antimicrobial cationic surfactants [24]. CQDs with positive surface charges act as potent antibacterial agents against negatively charged bacteria, impairing the surfaces of bacterial cells. Consequently, CQDs emerge as promising candidates for photo-activated antibacterials capable of effectively preventing and controlling pathogenic bacteria. Notably, the environmental and health hazards posed by antibacterial chemicals, such as hydrogen peroxide and sodium hypochlorite [25], are mitigated by utilizing CQDs, offering a safer alternative to chemical antimicrobial toxicity.
In this study, eco-friendly CQDs were synthesized from orange juice, serving as a green precursor through a plasma method. The optimal CQD was subjected to antibacterial testing, with the primary goal of investigating and determining the average antibacterial rate of the CQDs against E. coli strains. The results indicated that CQDs exhibit potential as novel antibacterial nanoparticles.
2.1. Synthesis of CQDs
The micro-plasma system, illustrated in Fig. 1, was employed for CQDs synthesis. The setup comprised a graphite electrode, a plasma nozzle, a DC high-voltage source, and a glass beaker containing the precursor solution. Specifically, the plasma electrode consisted of a tungsten rod with a diameter of approximately 1.5 mm, connected to the negative polarity through a 100 kΩ resistor.
To synthesize CQDs, a glass beaker was filled with 30 ml of orange juice solution, as depicted in Fig. 1. The plasma treatment was conducted for 20 minutes, maintaining a constant operating current of 5 mA with a high-voltage source. Following treatment, the solution was filtered through filter paper to remove any residues and then centrifuged to separate the deposit. The resulting redispersed product was mixed with water, yielding a pure CQDs solution with a light yellow color.
2.3 Characterization
The synthesized CQDs underwent various characterization techniques. UV-vis absorption spectroscopy was conducted using the Jasco V-570 instrument. Photoluminescence studies were carried out using fluorescence spectroscopy with the Fluorog 4 from Horiba Jobin–Yvon. Particle size and uniformity were analyzed using a transmission electron microscope (TEM) (JEOL 2100). X-ray powder diffraction (XRD) was conducted on a Siemen D5005 X-ray diffractometer using Cu-Kα1 radiation with a wavelength of 0.15406 nm. The functional groups of CQDs were analyzed by Fourier transform infrared spectroscopy (FTIR) (Bruker Alpha T). All of these measurements were performed at room temperature.
2.6. Antibacterial activity assay
In this current study, the GN antibacterial efficacy of the CQDs was assessed against Escherichia coli (E. coli) using the minimum inhibitory concentration (MICs) assays. Briefly, E. coli strains were streaked and cultured on a nutrient agar medium, followed by incubation for 18 to 24 hours at 37°C (Fig. 1). Subsequently, a bacterial suspension was prepared using 0.9% physiological saline (self-mixed) to achieve a bacterial concentration of 0.5 McFarland, corresponding to 108 Colony Forming Units per milliliter (CFU/ml). Next, 50 µL of CQDs were applied to the agar disk, which had been previously inoculated with E. coli, and incubated for 24 hours at 37°C. Visual inspection was conducted to assess the presence of bacterial growth (turbidity) to determine the MICs of the CQDs sample.
3.1 Structural Characterisation
The morphology of CQDs was examined using TEM, revealing the formation of particles, as depicted in Fig. 2. The CQDs exhibited a quasi-spherical shape with an average size of about 4.4 nm and demonstrated a narrow size distribution, as shown in the inset of Fig. 2.
The crystallinity of the CQDs was analyzed by XRD patterns as shown in Fig. 3. It can be seen that the diffraction pattern of CQDs shows a broad peak centered at 2θ ~ 22° which is assigned to the (002) plane of graphitized carbon [26]. The broad peak exhibited poor crystallinity due to the adequate availability of oxygen-containing groups [26].
Figure 4 presents the Fourier transform infrared (FTIR) spectrum of CQDs. The characteristic peak at 3250 cm⁻¹ in the synthesized CQDs is assigned to the adsorption of O-H stretching vibration [28]. Peaks in the range of 1000–1131 cm⁻¹ are associated with C-H stretching [29]. Peaks at 1641 cm⁻¹ and 1412 cm⁻¹ can be attributed to the C = O and -C-OH groups, indicating plasma treatment that results in the formation of sp2 domains. These groups are derived from small precursor molecules such as sugar and citric acid. Consequently, there is the presence of oxygen, hydroxyl, and carboxyl groups in the sp3 carbon area [30]. These functional groups contribute to the aqueous solubility of CQDs after carbonization by facilitating the formation of bonds with surrounding oxygen groups [31–33].
3.2. Optical Properties
UV–Vis absorption spectrum, a simple evaluation technique for the CQDs product, is presented in Fig. 5. This characterization was conducted within a wavelength range of 200–600 nm. The UV − vis absorption spectrum exhibits strong and sharp absorption bands at 283, which are probably due to the n − π* transition of the C = C bond, meanwhile the weak and broad band at 335 nm, corresponding to the n-π* transition in the C = O bond [27]. This outcome confirms the formation of carbon nanoparticles following the reaction, aligning with reported experimental results [28, 29]. Additionally, the results demonstrate that CQDs exhibit blue fluorescence properties under UV light at 254 nm (inset in Fig. 5, attributed to the quantum confinement effect and edge defects) [27].
The normalized photoluminescent (PL) spectrum of CQDs is presented in Fig. 5. The maximum PL emission wavelength of CQDs was recorded at an excitation wavelength of 300 nm. The PL peak fitting in Fig. 5 illustrates the PL spectrum composed of two peaks: (I) the main peak centered at 430.3 nm (green line curve) with a full-width half maximum (FWHM) of 94 nm, and (II) another shoulder peak at 511 nm (wine line curve) with FWHM of 73 nm, representing the emission centers denoting the surface states of the samples. CQDs exhibit multiple emission centers due to the presence of different surface chemical groups on CQDs [30]. The distribution of emission centers on CQDs can be inferred from their PL spectra.
3.3 Antibacterial Properties
Antibacterial efficacy is a critical criterion for evaluating positively charged CQDs as antibacterial agents in biomedical applications. In this study, E. coli was employed as a model to combat Gram-negative bacteria using antimicrobial agents. The images illustrate the results of the antibacterial abilities of E. coli with the MICs of CQDs as shown in Fig. 6. MICs were visually observed by tube turbidity before and after incubation with bacteria. The MICs of QCQD revealed observed bacterial growth at different concentrations of 0.0001, 0.001, 0.01, 0.1, and 1 ppm CQDs. The results show that with concentrations lower than 0.1 ppm, the solution was still impuring solution, clearly transparent solution at a concentration of 0.1 ppm and 1 ppm. This means no bacterial growth was observed with MICs 0.1 ppm. Therefore, it can be concluded that the MICs of CQDs for E. coli is 1 ppm in our experience.
After determining the MICs, the samples were streaked and cultured on nutrient agar, employing the smoothing method, and incubated for 24 hours at 37°C to investigate the Minimal bacterial concentration (MBC) as shown in Fig. 7. It can be seen that the MBC of CQDs for E. coli is 1 ppm in our experience.
Figure 8 shows the amount of viable E. coli bacterial cells after the treatment evaluated by disk-diffusion test with CQDs. E. coli was diluted to control according to various concentrations of 10− 6, 10− 7, and 10− 8 CFU/mL respectively from bacterial suspensions with a concentration of 106 CFU/mL. At 50 µL CQDs inhibit the growth of E. coli by up to 57%. The physicochemical and functional properties characteristic of CQDs contribute to their antibacterial activity, attributed to the induction of oxidative stress by reactive oxygen species. This induces cell wall disruption and damage the matrix of infective biofilms, increasing the measured inhibition diameter of bacteria.
Our study directly investigated the antibacterial properties of the CQDs sample against the E. coli bacterial strain. CQDs demonstrated the ability to inhibit the growth of E. coli bacterial strains after varying exposure times, consistent with some previous studies [31, 32]. This discrepancy can be explained by the direct testing approach in our CQDs sample without the presence of active ingredients or external influences, such as light or ultraviolet irradiation, resulting in a lower recorded performance. Remarkably, our findings indicate that the contact time between bacteria and the CQD sample can inhibit the growth of E. coli after only 30 seconds. This suggests that the antibacterial capability of the CQD sample is notably rapid compared to research involving hydrophobic carbon quantum dots/polydimethylsiloxane nanocomposites, which effectively kill bacteria after 15 minutes of irradiation [33, 34].
CQDs were produced in a quasi-spherical form with an average size of approximately 4.4 nm using microplasma processing of orange juice under ambient conditions. The photoluminescence (PL) spectrum of the CQDs exhibited a broad luminescence spectrum, with a main peak centered at 449.8 nm and an additional shoulder peak at 509 nm. The synthesized CQDs were evaluated for their bactericidal activity against E. coli bacteria. Notably, CQDs demonstrated the ability to inhibit the growth of the E. coli strain after just 30 seconds of exposure, with a minimum bactericidal concentration of 1 ppm for standard E. coli strains. The antibacterial efficacy of CQDs, measured by the average antibacterial rate against E. coli, was determined to be 57%. These findings suggest that green-synthesized CQDs hold promise for diverse applications, including biological and optoelectronic applications, owing to their tunable optical properties, low cytotoxicity, and excellent dispersibility.
Funding
This research was funded by the project under the Physics Program at the Vietnam Academy of Science and Technology (Project No. KHCBVL.05/21-22) and Project No. NVCC05.03/22-23.
Author information
Authors and Affiliations
1. Institute of Physics, Vietnam Academy of Science and Technology, 100000 Ha Noi, Vietnam
Van Duong PHAM, Hong Minh PHAM, Hoang Tung DO, Thanh Binh NGUYEN
2. Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam
Anh Thi LE
3. Natural Sciences Department, Phu Yen University, Tuy Hoa City, Phu Yen 5600, Vietnam
Duc Toan Le
4. Faculty of Fundamental Sciences, Hue University of Medicine and Pharmacy, Hue University, Hue 530000, Vietnam
Minh Hoa NGUYEN
Contributions
P.V.D and N.M.H conceived the experiment. P.H.M, D.H.T, L.D.T, and L.A.T carried out the experiment and analyzed the data. All authors discussed the results. P.V.D, N.T.B, and N.M. H wrote the original paper draft. N.T.B, P.V.D, L.D.T and N.M.H supervised the project.
Corresponding authors
Correspondence to Minh Hoa NGUYEN
Ethical Approval
Not applicable.
Conflict of interest
There are no potential conflicts of interest relevant to this article.
Availability of Data and Materials
The datasets used in this study are available from the corresponding author upon reasonable request.
Declaration of Interest Statement
We wish to confirm that there are no known conflicts of interest associated with this publication. There has been no significant financial support for this work that could have influenced its outcome.
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Van Duong PHAM Anh Thi LE Hong Minh PHAM Hoang Tung DO
Duc Toan LE Thanh Binh NGUYEN Minh Hoa NGUYEN
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No competing interests reported.