Copper and silver doped in CdTe quantum dots: C. albicans and C. tropicalis antifungal nanomaterials

Quantum dots based on CdTe and Copper or Silver doped CdTe were used for antifungal against C. albicans and C. tropicalis by microdilution method protocol by CLSI. Pure and doped QDs were characterized by UV–vis and fluorescence spectroscopy, x-ray diffraction and transmission electron microscopy which showed sizes between 7.1 and 15.9 nm. Energy dispersive x-ray spectroscopy was carried out to determinate the metal doping in the QDs. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was obtained. The pure QDs fungicidal effect at 500 mg l−1 but 10 mg l−1 of 10% Copper doped QDs show fungicidal for both yeast.


Introduction
Microbial infections have been present around the world for many years, many countries are affected no matter their income levels. These types of infections are very difficult for the public health organizations to deal with [1]. There has been research carried out for fungal diseases by the World Health Organization (WHO). It was shown that microbial resistance is very common due to the high overuse of antibiotics. Most common nosocomial infections are caused by respiratory infections, post-surgery infection, urinary infection and others [2][3][4]. Even though, that the most frequent isolated micro-organisms in nosocomial infections are bacteria like Pseudomonas aeruginosa from ventilator-associated pneumonia, Staphylococcus aureus from bloodstream infections and Enterococcus faecalis from bloodstream and urinary infections, Candida yeast are less common with only 4% of the nosocomial infections. Candida infections have high incidence and cause many problems due to the complication to treat them. These infections have attracted attention due to the 50% mortality in invasive candidiasis [5]. Nevertheless, candidiasis is most common as a superficial disease and so, is very important to develop new and more efficient disinfection systems for superficies.
Tipicall disinfection processes are carried out using special equipment or pre-fabricated chemical mixtures. One of the most common pieces of equipment are UV-light lamps, they are easy to manipulate but are very expensive. Nevertheless, long exposures are required depending on the micro-organism [6,7]. The chemical mixtures commonly used for disinfection are ethanol, chlorine, peracetic acid or ozone [8][9][10] but some of these can be very toxic. One of the most promising methods that can be used to avoid this, is the use of nanoparticles. They can be less toxic that some chemicals and cheaper that instrumental procedures like UV treatments [11,12].
These nanomaterials have been widely studied for antibacterial properties and most of them have shown non-toxic effects to humans and the environment [13,14]. One of these nanomaterials are quantum dots (QDs). Many research has been carried out using them for sensing or biológical applications involving cellular imaging or energy harvesting [14][15][16][17], unlike silver or copper ions [18][19][20][21], QDs have shown great biocompatibility Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. depending on the capping ligands. Nevertheless, QDs have not been used with silver or copper doping for antifungal applications. These nanoparticles can generate reactive oxygen species (ROS) which can be used for antimicrobial studies [22], also silver and copper can generate these ROS [23,24]. CdTe quantum dots have been widely studied for analytical purposes due to their smaller bandgap and to their high optical and chemical stability [25,26].
The synthesis procedures for QDs obtention are basically carried out by water [27] chemical synthesis, precipitation [28], microwave [29], hydrothermal synthesis [30], or using hydrophobic ligands [31] at high temperatures, however, most of them require very specific equipment rising the price of the material or pyrophoric compounds. Chemical synthesis with water as solvent was used in this work and to enhance the antifungal effect, silver and copper were used as dopants to obtain doped CdTe QDs. In this work we report the synthesis of Cu-CdTe and Ag-CdTe doped QDs as antifungal materials against C. albicans and C. tropicalis. Each material was characterized by transmission electron microscopy (TEM) and x-ray diffraction (XRD). Clinical and Laboratory Standards Institute (CLSI) protocols were used to test the antifungal properties of the obtained materials, the minimum inhibition concentration (MIC) and minimum fungicidal concentration (MFC) were calculated.

Doped quantum dots synthesis
Quantum dots were synthesized using a modified version of a previous report methodology [31]. First, 3.2 mmol of CdCl 2 was dissolved in deionized water followed by the addition of the doping agent (Copper or Silver) 0.16 or 0.32 mmol (5% or 10% respectively). After a complete dissolution, 4.8 mmol of ligand (MPA) was added and the solution´s pH was adjusted to 10 with NaOH 0.1 M. This solution was stirred for 30 min the mixture was heated up to 100°C and then a solution containing 0.64 mmol of Te and 300 mg NaBH 4 in 10 ml of water was added drop-wise. The reaction was kept stirring for 1 h, after that the doped QDs were precipitated with acetone and washed with 3 more cycles of acetone to remove unwanted materials. The obtained powder was dried overnight. A control undoped-QDs were obtained without the metal nitrates in the same procedure.

Material characterization
The UV spectra were obtained using a Shimadzu UV 2700 UV-vis spectrophotometer. Fluorescence measurements were carried out using a Horiba Jovin Yvon Nanolog fluorometer. The powder was dried under vacuum before any further characterizations. The morphology and structural properties of the doped QDs were characterized with a JEOL scanning transmission electron microscope (STEM), model JEM2200FS, with spherical aberration correction in a probe mode working at 200 KeV accelerating voltage. The bright field and Z-contrast images were acquired in a scanning transmission mode. The x-ray diffraction spectra (XRD) were carried out with a Bruker Da Vinci model diffractometer.

MIC and MFC assays
The minimum inhibition concentration (MIC) was determined using the CLSI protocol M27-A4. The protocol establishes a microdilution method which allows visual determination of the MIC after 50 h of incubation at 35°C. Afterwards, the minimum fungicidal concentration was determined also visually in agar. Each material and the SGA were sterilized in autoclave before every assay. First, the obtained nanomaterials were dissolved in RPMI-medium and diluted in a range of 20 to 1000 mg l −1 also in RPMI-medium. Additionally, a 0.5 Mc Farland standard with the desired bacteria was obtained in a 0.1% NaCl solution to ensure a 5 × 10 8 CFU ml −1 suspension. This was diluted in RPMI-medium to obtain a 5 × 10 5 CFU ml −1 suspension, this was used as inoculum. Then, 100 μl of the nanomaterial dispersion was mixed with 100 μl of inoculum in a 1 ml vial. For a positive control, the inoculum was mixed with RPMI-medium. On the other hand, for a negative control, 200 μl of RPMI-medium was used. The mixtures were placed in an incubator for 50 h at 30°C. The turbidity indicated the MIC. After this, 20 μl of the suspension in the vials without turbidity were transferred to fresh SGA to determine the MFC by incubating the agar also for 50 h at 30°C.

Results and discussion
3.1. Characterization UV-vis spectra were carried out for each sample(figure 1). It can be seen that pure CdTe have a maximum absorption wavelength at 529 nm. When doping with both silver and copper at 10%, these values decrease to 513 and 516 nm respectively. Which means that the particle sizes are smaller than the pure CdTe QDs. For all samples, the absorption curve have wide peaks which means that the size distribution is poor.
Also fluorescence measurements were carried out. Figure 2 shows all spectra in which the optical property can be seen, in this case, the maximum wavelength also has the same tendency like the UV-vis spectra in which the pure Ag10%CdTe QDs have lower maximum wavelengths in comparison with the pure CdTe QDs. Nevertheless, the fluorescence intensity is observed which confirms the presence of QDs structure in all samples.
Crystalline structures of the obtained materials were studied by x-ray diffraction. The XRD patterns were compared with the reported CdTe structure [32]. Figure 3 shows the XRD patterns for the pure and doped QDs including the standard patterns. The undoped and doped QDs show diffraction peaks that can be seen at intensities at 24.2°(111), 40.5°(220) and 46.9°(311), these peaks are are consistent with the standard XRD data for the zinc blende phase CdTe (JCPDS No. 75-2086).  The CdTe QDs doped with 10% of Ag shows a lower intensity for the (111) and on the other hand a slightly intense peak at 30°appears. This can be attributed to the formation of a Ag 2 Te phase in the material [33].
The average size of the doped and undoped QDs were calculated using Scherrer´s equation [34].

D k Cos
In this equation, D is the average size of the QDs, k is the Scherrer constant (k = 0.9 for QDs), λ is the wavelength of x-ray (1.54059744 Angstroms), β is the full width at half maxima (FWHM) of the XRD pattern peak and θ is the diffraction angle. Using the (1 1 1) plane the average size for each QDs were 2.88, 3.64, 2.56, 3.01, 3.24 nm for the pure CdTe, Ag5%CdTe, Ag10%CdTe, Cu5%CdTe, Cu10%CdTe QDs respectively. These measurements accord with the UV-vis, and fluorescence studies in which the spectra revealed that Ag10%CdTe has smaller particle size in comparison with the pure CdTe. Figure 4 shows the TEM images for each QDs obtained. The shape and size of all QDs were analyzed. The TEM micrographs show that the obtained QDs have a uniform spherical shape. The size for each doped QDs and the undoped QDs were measured and the results were 4.5 ± 0.7, 5.9 ± 0.3, 3.1 ± 0.8, 4.7 ± 0.7, 2.9 ± 1.1 nm for the pure CdTe, Ag5%CdTe, Ag10%CdTe, Cu5%CdTe, Cu10%CdTe QDs respectively.
Figures S1 to S5 shows the EDS analysis for all samples in which it can be seen the effect of the doping in the presence of silver or copper in the QDs. For the silver doped QDs, the average doping percentage was 4.1 ± 0.5 for the Ag5%CdTe and 12.3 ± 1.6 for the Ag10%CdTe QDs. In the case of the copper doped materials, 2.4 ± 0.3 for the Cu5%CdTe and 11.4 ± 0.9 for the Cu10%CdTe QDs.

MIC determination
The antifungal studies were carried out using the CLSI protocol M27-A4. This is the first research in which the MIC was determined using a broth based CLSI protocol. Figure 5 shows the results applying the procedure for C. albicans ATCC 10231 and C. tropicalis ATCC 750. After 24 h of incubation at 30°C small dots and turbidity  indicated yeast growth in the positive control and some of the QDs concentrations also show turbidity indicating growth and negative antifungal properties. Figure 5 shows the 96 well plate in which some wells show turbidity and others do not in which the obtained QDs have antifungal effect at a significant QDs concentration. In case C. albicans pure QDs and those doped with silver, 5% and 10% show no growth at 20 mg l −1 , on the other hand, those doped with copper no growth was observed since 10 mg l −1 of each 5% or 10% doped QDs. In case of C. tropicalis, 50 mg l −1 of pure CdTe were required for fungal inhibition. Nevertheless, for both 5% and 10% Silver doped QDs and also 5% Copper doped QDs 20 mg l −1 show no fungal growth, but in case of 10% Copper doped QDs only 10 mg l −1 was required. It seems that Copper doped QDs show more inhibition effects than silver doped materials.

MFC determination
Minimum fungicidal concentration was determined in fresh SGA, MFC was calculated after incubating each sample observing which petri dish had fungal growth. The pure CdTe QDs were analyzed from 20 mg l −1 to 500 mg l −1 for C. albicans and for C. tropicalis from 50 mg l −1 to 500 mg l −1 in which no turbidity was observed in the MIC studies. In this case, the petri dishes of the 500 mg l −1 samples for both yeast did not show any growth, indicating that this concentration has a fungicidal effect for both Candida.
Copper doped materials show great fungicidal effects. In case of the 5% Copper doped QDs, 20 mg l −1 was required for both yeast. On the other hand, fungicidal effects were observed at 10 mg l −1 when 10% Copper doped QDs were studied.
For silver doped material, the same method was carried out and from 20 mg l −1 for both yeast and doping percentages. In this case, it was observed that for 5% Silver doped QDs, 50 mg l −1 was needed to promote fungicidal effect in C. albicans and 20 mg l −1 for C. tropicalis. The samples at 10% Silver doping show fungicidal effect at 20 mg l −1 for both yeast. These results are compared in table 1.
The fungicidal effect is considered to be caused by the generation of reactive oxygen species (ROS) and free radicals by the CdTe. Nevertheless copper and silver can generate ROS species. On the other hand, copper has shown that it can link to DNA molecules leading to cross-linking within to disrupt biochemical processes and cause bacterial and fungal death [22,23].

Conclusions
It was demonstrated that copper and silver doped QDs can be used for antifungal applications against C. albicans ATCC 10231 and C. tropicalis ATCC 750. This is the first paper in which antifungal effect was studied using a CLSI protocol based in broth medium. The QDs show fungistatic and fungicidal effects in a determined concentration. All QDs were characterized by XRD and TEM to observe the structure properties, the best antifungal effect was observed at 10 mg l −1 using the 10% Cu doped QDs for both yeast which, in comparison to Ag doped QDs despite they show great antifungal effects, are cheaper.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).