A novel biocompatible titanium–gadolinium quantum dot as a bacterial detecting agent with high antibacterial activity

nanomaterials Article A Novel Biocompatible Titanium–Gadolinium Quantum Dot as a Bacterial Detecting Agent with High Antibacterial Activity Vishma Pratap Sur 1,2,*,†, Aninda Mazumdar 1,2,†, Amirmansoor Ashrafi 1,2 , Atripan Mukherjee 1,2, Vedran Milosavljevic 1,2 , Hana Michalkova 1, Pavel Kopel 3, Lukáš Richtera 1,2 and Amitava Moulick 1,2,* 1Department of Chemistry and Biochemistry, Faculty of AgriSciences, Mendel University in Brno, Zemedelska 1/1665, CZ-61300 Brno, Czech Republic; [email protected] (A.M.); amirmansoor[email protected] (A.A.); [email protected] (A.M.); [email protected] (V.M.); [email protected] (H.M.); [email protected] (L.R.) 2Central European Institute of Technology (CEITEC), Brno University of Technology, Purkynova 123, CZ-62100 Brno-Královo Pole, Czech Republic 3Department of Inorganic Chemistry, Faculty of Science, Palacky University, 17. listopadu 12, CZ-771 46 Olomouc, Czech Republic; [email protected] *Correspondence: [email protected] or [email protected] (V.P.S.); [email protected] or [email protected] (A.M.); Tel.: +420-545-133-350 (V.P.S.); +420-545-133-350 (A.M.) †These authors contributed equally to this work. Received: 18 March 2020; Accepted: 16 April 2020; Published: 17 April 2020   Abstract: In this study, the titanium–gadolinium quantum dots (TGQDs) were novel, first of its type to be synthesized, and fully characterized to date. Multiple physical characterization includes scanning electron microscopy (SEM), scanning electrochemical microscope (SCEM), x-ray fluorescence, spectrophotometry, and dynamic light scattering were carried out. The obtained results confirmed appropriate size and shape distributions in addition to processing optical features with high quantum yield. The synthesized TGQD was used as a fluorescent dye for bacterial detection and imaging by fluorescent microscopy and spectrophotometry, where TGQD stained only bacterial cells, but not human cells. The significant antibacterial activities of the TGQDs were found against a highly pathogenic bacterium (Staphylococcus aureus) and its antibiotic resistant strains (vancomycin and methicillin resistant Staphylococcus aureus) using growth curve analysis and determination of minimum inhibitory concentration (MIC) analysis. Live/dead cell imaging assay using phase-contrast microscope was performed for further confirmation of the antibacterial activity. Cell wall disruption and release of cell content was observed to be the prime mode of action with the reduction of cellular oxygen demand (OD). Keywords: bacterial resistance; titanium–gadolinium quantum dots; bacterial detection; antibacterial activity; SECM 1. Introduction Antibiotic resistance in bacteria is a major challenge to medical sciences and the priority list by World Health Organization (WHO) categorizes it into critical, high, and medium [ 1 ]. Research for the past two decades have produced only two new types of antibiotics: lipopeptides and oxazolidinones, although majorly overcoming antibiotic resistance has not been achieved yet. The present situation of bacterial infections is considered to be a threat to human society due to the evolving strains of Nanomaterials 2020,10, 778; doi:10.3390/nano10040778 www.mdpi.com/journal/nanomaterials Nanomaterials 2020,10, 778 2 of 18 Staphylococcus aureus with resistance to broad-range antibiotics such as methicillin and vancomycin [ 2 ]. The infections caused by the resistant bacteria are usually treated with existing antibiotics, which usually exerts side effects with their incomplete dosage and improper use. The overuse and misuse of antibiotics are the key factors contributing to antibiotic resistance [ 3 ]. The overuse of antibiotics, especially taking antibiotics even when they are not the appropriate treatment, promotes antibiotic resistance [ 4 ]. On the other hand side, the effects can be varied, for example, vomiting, nausea (feeling like one may vomit), diarrhea, bloating and indigestion, abdominal pain, loss of appetite, a raised, itchy skin rash (urticaria, or hives), coughing, wheezing, tightness of the throat, feeling lightheaded or faint, breathing difficulties such as fast, shallow breathing, wheezing, a fast heartbeat, clammy skin, confusion and anxiety, collapsing or losing consciousness [ 5 ]. Furthermore, some antibacterial agents can elicit adverse effects against the hepatic system, where the types of liver damage induced by antibacterial agents cover cytotoxic, injury, cholestatic injury, mixed cytotoxic and cholestatic injury, steatosis, chronic, active hepatitis, and cirrhosis [ 6 ]. This further increases the complexity in the development of the resistance against these molecules, assisting it in being a global health problem [ 7 , 8 ]. Thus, the importance of developing new treatment strategies or alternatives for antibiotics against these multidrug resistant bacterial strains are the major demands in 21st century medical science [ 9 – 11 ]. To overcome the antibiotic resistance and absolute necessity of finding an alternative to existing antibiotics with negligible toxicity, proper biocompatibility, and fulfil the needs of clinical research, novel antibacterial agents like nanoparticles, peptides, metabolites, oligonucleotides, and other biogenic or chemical compounds have been introduced [ 8 , 12 – 17 ]. Nanoparticles, nowadays, are the focus of medical sciences with various applications like drug delivery, tracking, and alternatives to antibiotics such as nanomedicine [ 18 – 20 ]. However, nanoparticles also have adverse effects toward human health. Nanoparticles exert various kinds of toxicity, they can be cancerous, affect our immune system, liver, spleen, can generate cellular ROS, damage our DNA, or they can also affect our metabolism through blocking or disrupting various enzymatic pathways [21,22]. The detection and imaging of bacteria by staining has been a reliable method for more than a century. Commercial unavailability of any bacteria specific fluorescent stain has limited the process of fluorescent labelling. Most of the existing stains follow a general mode of action, where it stains almost all types of cells including mammalian cells, or it is artificially designed for specific target staining. For example, BacLight green is a bacterial specific stain that contains SYTO9, frequently used in live/dead cell imaging of bacteria, but SYTO9 is an intercalating membrane permeable green stain that stains all cells containing nucleic acid [23]. Moreover the vital stain, fluorescein diacetate (FDA) is based on intracellular hydrolysis of FDA and in the case of living cells, the non-fluorescent FDA is converted into fluorescein, a green fluorescent compound, indicating the cell viability [ 24 , 25 ]. The detection of bacteria in human tissue during infection development, or the host pathogen interaction study by using a fluorescent dye is quite challenging due to the generalized mode of action of commercialized organic dyes. Thus, the necessity of developing a bacteria specific stain to improve the bacterial detection and imaging is required and long awaited. Nanoparticles (NPs) can be derived from various sources in a desirable nanometer (nm) size. Due to their enhanced retention and permeability, NPs have a broad range of clinical applications along with their ability to accumulate the sites of infection by enhancing retention and the permeability effect [ 26 ]. NPs with few modifications help in site specific drug delivery by reducing target toxicity [ 27 ]. Quantum dots (QDs) are a type of nanoparticles with the ability to fluoresce, which is helping in the development of new detection and imaging techniques [ 27 – 29 ]. A few novel QDs have been reported to have antibacterial [ 30 – 32 ] and anticancer properties [ 33 , 34 ], which elevates their value even more in the field of biomedicine with some drawbacks like toxicity, inflammation, and stability [ 27 ]. Titanium dioxide (TiO 2 ) based nanocomposites have been reported to have antimicrobial activity against Escherichia coli,Staphylococcus aureus,Streptococcus sobrinus,Pseudomonas putida,Pseudomonas aeruginosa, and Listeria innoculim due to the production of reactive oxygen species (ROS), which can inactivate organic and inorganic pollutants and consequently inactivate the microorganisms, even Nanomaterials 2020,10, 778 3 of 18 where environmental and economic factors have raised their volume [ 35 – 38 ]. The gadolinium based nanoparticles also have antimicrobial activities against Bacillus subtilis,E. coli, and Staphylococcus aureus [ 39 , 40 ]. Though both gadolinium and TiO 2 nanoparticles are biologically active, no study has tested their real implications and effects against vancomycin resistant Staphylococcus aureus (VRSA) and methicillin resistant Staphylococcus aureus (MRSA) yet. In this study, we used titanium and gadolinium together to synthesize a titanium–gadolinium quantum dot (TGQG), the first of its kind, and its characterization was undertaken using dynamic light scattering, spectrophotometry, X-ray fluorescence, scanning electrochemical microscope (SECM), and scanning electron microscope (SEM). Thereafter, TGQD was used as a stain in the detection of VRSA using a fluorescence microscope. Furthermore, the antibacterial activity was tested against the pathogenic strains of vancomycin resistant S. aureus (VRSA), methicillin resistant S. aureus (MRSA), and the mechanism of action was also investigated. Finally, the cytotoxicity test was performed and conclusions were drawn. 2. Materials and Methods 2.1. Chemicals 2-Pyridinecarboxaldehyde, diethylene triamine, gadolinium (III) nitrate, titanium (IV) butoxide, isopropanol, and methanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Muller Hinton broth (MH) was obtained from HiMedia Laboratories Pvt. Ltd., Thane, India. 2.2. Chemicals and Synthesis of Titanium–Gadolinium Quantum Dots (TGQDs) The preparation of the gadolinium Schiffbase was according to our previous studies with necessary modification [ 28 ]. Initially, 1.08 mL of diethylene triamine and 1.9 mL of 2-pyridinecarboxaldehyde were mixed and heated under reflux in 35 mL of methanol (MeOH) for 6 h. To prepare the desired solution of Schiffbase [(2-[(E)-2-pyridylmethyleneamino]-N-[2-[(E)-2-pyridylmethylene-amino]ethyl] -ethanamine)], the solution was cooled and the volume was made up to 50 mL using MeOH. In a separate beaker, 5 mL of gadolinium nitrate aqueous solution (90 mg/mL) was mixed with 10 mL of MeOH, which was subsequently mixed with 5 mL solution of the Schiffbase. Finally, the solutions were mixed on hot plate magnetic stirrer for 2 h at 40 ◦ C and the volume was made up to 100 mL with deionized water. The Gadolinium (Gd)–Schiffbase solution was stored at 25 ◦ C. To prepare the titanium dioxide (TiO 2 ) solution, 50 mL of isopropanol and 50 µ L of titanium butoxide was put into a glass beaker and placed on a magnetic stirrer for 48 h, and the clear solution turned into a milky white solution without precipitation. Then, the aqueous phase synthesis of quantum dots was carried out by mixing the Gd–Schiffbase solution with TiO 2 in a beaker. The Gd–Schiffbase and TiO 2 solutions were mixed in the ratio of 1:1. Later, 2 mL of the solution was taken in a glass tube and heated for 10 min at 80 ◦ C, 300 W, (ramping time, 10 min) under microwave irradiation (Multiwave 3000, Anton Paar GmbH, Graz, Austria) to prepare the TG quantum dots. The samples were filtered using 3 kDa (Amicon Ultra 0.5 mL centrifugal filters) filters and further filtered through a 0.22 µ m membrane. To remove the unreacted initiators, the solution was dialyzed against deionized water several times, then the TGQD were dried under a vacuum dry system. 2.3. Characterization of TGQD The prepared TGQDs were visualized under a UV transilluminator at excitation wavelengths ( λex ) of 312 nm and 270 nm (Transilluminator Multiband TFX-35.MC, Torcy, France). The fluorescence and absorbance spectra of the TG QDs were obtained using a microplate (UV plate, 96 well; Corning Incorporate, Corning, NY, USA) in Tecan Infinite m200 PRO (Männedorf, Switzerland). A total of 100 µ L of the samples were used for the measurements. The fluorescence spectrum was measured using an excitation wavelength ( λex ) of 230 nm, the emission wavelength ( λem ) range was 280–850 Nanomaterials 2020,10, 778 4 of 18 nm, and the absorbance spectrum of the TGQDs was measured from 230 to 1000 nm. The QDs were observed under a fluorescence microscope with a UV filter for visualization. The photoluminescence quantum yield of the TGQDs was determined using the reference as rhodamine 6G according to a reported protocol [41]. The scanning electron microscopy (SEM) instrument (TESCAN Company, Brno, Czech Republic, EU) was used to observe the TGQDs under the following conditions: high vacuum mode (10–3 Pa), voltage of 15 kV, and work distance of 3 mm [ 28 ].The scanning electrochemical microscopy (SECM) measurements were performed using a CHI 900 setup (CH Instrument Inc., Austin, TX, USA). A Pt ultramicroelectrode (d=10 mm) was the SECM tip (RG factor =10), a bare glassy carbon (GC) covered with 10 µ L Nafion 1%, and a GC immobilized with TGQDs and covered with 10 µ L Nafion 1% served as the SECM substrates. The tip was positioned near the substrate using the probe-approach curve (PAC) technique and a potential of 500 mV was applied to the tip with a scan rate of 0.5 µ m/s. A platinum wire was the counter electrode and the reference electrode was Ag/AgCl (3 M KCl). A solution of 1 mM of ferrocenemethanol (FcOH) in 0.1 M KCl was used as the redox mediator. All potentials were referred to the reference electrode. TGQDs were also characterized using an elemental analyzer SPECTRO XEPOS energy dispersive x-ray fluorescence (ED-XRF) spectrometer (SPECTRO Analytical Instruments GmbH, Kleve, Germany) equipped with a 10 mm 2 Si-Drift Detector with Peltier cooling and a 75 µ m Be side window was employed. The instrument uses a Pd-target end window tube at a maximum power of 50 W and a maximum voltage of 50 kV. Spectral resolution of the instrument (FWHM) was <170 eV for Mo K α (measured under input count rate 10,000 pulses). SPECTRO XEPOS was operated and data were evaluated by means of the software Spectro X-Lab Pro, Version 2.5, Kleve, Germany. For the excitation of light elements (Mg–V, 25 kV), a HOPG (highly oriented pyrolithic graphite) crystal target was used. For the determination of heavier elements, a Mo secondary target (Cr–Y, Hf–U, 45 kV) and Al 2 O 3 polarization target (Zr–Ce, 49.5 kV) were used. The sample (1.0 mL) was dried directly at 70 ◦ C in a sample cup (32 mm in diameter) on polypropylene thin-film (Specac Ltd., Kent, UK) and measured in vacuum using the so-called Turboquant method (fundamental parameters method) [ 42 ]. Fourier transform infrared (FTIR) spectra were measured using a Thermo Scientific Nicolet iS5 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an iD5 Diamond ATR accessory over a wave number range of 4000–550 cm –1 . The average size of the TG QDs, the size distribution, and zeta potential were determined by quasielastic laser dynamic light scattering (DLS) with a Malvern Zetasizer (NANO-ZS, Malvern Instruments Ltd., Worcestershire, UK). Initially, 1.5 mL of an aqueous solution of TGQDs (1 mg/mL) was poured into a polystyrene latex cell and measured at a temperature of 25 ◦ C with a detector angle of 173 ◦ , a wavelength of 633 nm, a refractive index of 0.30, and a real refractive index of 1.59 [28]. 2.4. Application of TGQD on Bacteria for Detection and Killing The bacterial strains Staphylococcus aureus (S. aureus) (NCTC 8511) [ 43 , 44 ], vancomycin resistant Staphylococcus aureus (CCM 1767) [ 19 , 45 ], and methicillin resistant Staphylococcus aureus (MRSA) ST239:SCCmec IIIA) [ 46 ] were obtained from the Czech Collection of Microorganisms, Faculty of Science, Masaryk University, Brno, Czech Republic and from England, with the cooperation of the University of Cambridge [ 19 , 43 – 46 ]. The bacterial cells were cultivated in Muller Hinton (MH) broth medium, pH 7.4. The bacterial cultures were cultivated overnight at 37 ◦ C in a shaking incubator. Bacterial optical density was adjusted to 0.1 absorbance (0.5 MacFarland standards) at 600 nm for the subsequent experiments [45–50]. The absorbance spectral scan was recorded of the bacterial sample for their detection by using Tecan Infinite m200 PRO immediately after preparing the samples. Bacterial culture was adjusted up to 0.012 absorbance (OD600) and incubated with TGQD for 5 min, then the bacterial sample incubated with TGQD was placed on a spectrophotometric plate and the plate was scanned under the Tecan Infinite m200 PRO. Nanomaterials 2020,10, 778 5 of 18 Furthermore, the bacterial detection via TGQD was carried out through microscopic analysis. The bacterial sample (VRSA) was stained with TGQD (15 µ g/mL) and Images were obtained using an optical Olympus BX51 fluorescence microscope equipped with 40×phase contrast lens. Thereafter, further confirmation was obtained by treating bacteria, bacteria–human cell co-culture with TGQD, and microscopic images were captured. For the co-culture and the bacterial sample, VRSA was added in a 24-well cell culture plate in the presence of PNT1A (human cells) cells with 60% confluency, incubated for 1 h at 37 ◦ C. The co-culture system containing bacteria and human cells together was stained by TGQD (15 µ g/mL), then the plate was used for microscopy. The microscopy was performed by using an microscopic inverted Olympus IX 71S8F3 fluorescence microscope (Olympus Corporation, Tokyo Japan), which was equipped with an Olympus UIS2 series objective LUCplanFLN 40X (N.A. 0.6, WD 2.7–4 mm, F.N.22), and a mercury arc lamp X-cite 12 (120W; Lumen Dynamics, Mississauga, ON, Canada) was used. As the control, bacterial cells and the human cell line PNT1A were also stained with TGQD and observed under a microscope. The bactericidal effect of TGQD was analyzed by scanning electrochemical microscopy (SECM). where a Petri dish containing the VRSA treated with TGQD was subjected to the SECM study. The Petri dish was coated with Poly-L-Lysine and then the VRSA cells were added to the Petri dish for their attachment with the Poly-L-Lysine coated Petri dish surface. The Petri dish was filled with MH Broth, which was composed of beef infusion form 300.00 g/L, casein acid hydrolysate 17.50 g/L, starch 1.50 g/L, and KCl 7.45 g/L. The probe electrode (Pt 10 µ m) was inserted in the Petri dish and the cyclic voltammetry (CV) was recorded. Standard isolates of S. aureus, VRSA, and MRSA were cultivated and used for this experiment. All bacterial isolates were grown in Mueller Hinton broth in a shaking incubator at 37 ◦ C. The susceptibility of bacterial cultures against TGQD detection was performed by the unaided eye [ 45 , 46 , 51 , 52 ]. The minimum inhibitory concentration (MIC) is the lowest concentration that inhibits bacterial growth. The MIC of TGQD was obtained by adding TGQD from lower to higher concentrations in microplate wells, mixed with bacterial cultures (0.1 O.D. equivalent to 0.5 MacFarland), and incubated at 37 ◦ C for 24 h. The final working concentration gradient of TGQD in the microplate wells were 45 µ g/mL, 55 µ g/mL, 62.5 µ g/mL, 85 µ g/mL, and 125 µ g/mL. The lowest concentration of TGQD added in the microplate well and the microplate well showed almost no bacterial growth (transparent medium with no turbidity), which was counted as the MIC value of TGQD against that specific bacterium. The control was the bacteria without TGQD treatment. The growth curve analysis for S. aureus, VRSA, and MRSA were performed for further confirmation of antibacterial activity of TGQD. Standard isolates of S. aureus, VRSA, and MRSA were cultivated and used for this experiment. All bacterial isolates were grown in MH broth in a shaking incubator at 37 ◦ C. The control was the bacterial culture with no TGQD treatment. The antibacterial activity of the TGQDs were measured by Bioscreen C MBR (Oy Growth Curves Ab Ltd., Helsinki, Finland) for 24 h at 37 ◦ C using a multichannel pipette system in the 10 × 10 Honeycomb optical microplate well systems (Oy Growth Curves Ab Ltd., Helsinki, Finland) [ 51 , 53 – 56 ]. Different concentrations of TGQDs (45 µ g/mL, 55 µ g/mL, 62.5 µ g/mL, 85 µ g/mL, 125 µ g/mL) were added to bacterial cultures (0.1 absorbance equivalent to 0.5 McFarland) in the microplate wells. The microplate with the samples was then incubated in the growth curve analyzer (Bioscreen C MBR) for 24 h and the results were evaluated accordingly [46]. Furthermore, the cell leakage assay was carried out. The bacterial sample was treated with TGQD and incubated at 37 ◦ C for 4 h, which showed the cells treated with the TGQD were precipitated after 4 h. The treated samples as well as the untreated samples were centrifuged for cell debris removal and the supernatant was separately kept in a micro-centrifuge tube for quantification of DNA and RNA by a spectrophotometer (Tecan Infinite m200 PRO). The wavelength of 260 nm was used for the DNA and RNA quantification. For the DNA quantification, the samples were treated with RNase, and for the RNA quantification samples, we treated them with DNase, and purified them in a column purification system. After column purification, the absorbance spectra were recorded at Nanomaterials 2020,10, 778 6 of 18 260 nm. The presence of DNA and RNA were calculated according to a 260/280 ratio. According to the 260/280 ratio, ~1.8 is generally accepted as pure for DNA and ~2.0 is generally accepted as pure for RNA [ 57 – 61 ]. The DNA in the supernatant was used as a template with 16S rRNA primers (16S Forward-ACTGGGATAACTTCGGGAAAC, and 16S reverse-CAGCGCGGATCCATCTATAA) to perform PCR amplification, and the confirmation was done using agarose gel electrophoresis with 1.3% agarose gel [ 45 ]. The positive control was genomic DNA and the negative control was the supernatant from the untreated bacterial sample. To confirm the presence of all types of live cells (bacterial and human cells), SYTO9 was used. Thereafter, an optical Olympus BX51 fluorescence microscope equipped with a 40 × phase contrast lens was used to study the antibacterial activity of the TGQD against VRSA. Initially, the samples were incubated with bacteria and TGQDs (respective MIC) for 4 h at 37 ◦ C in a shaking incubator. Two different kinds of fluorescent dyes were used: SYTO9 and propidium iodide (PI) for live and dead cell staining in equal proportion. Fluorescence dyes were added to the sample and observed under microscope. 2.5. In Vitro Cytotoxicity Testing Assay The MDA-MB-231 (mammary gland adenocarcinoma cells) and HBL-100 (mammary gland epithelial cells) human cell line were used to study the cytotoxicity of TGQDs. The MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study the cell viability. The cells were maintained in RPMI-1640 medium with 10% fetal bovine serum, supplemented with penicillin (100 u/mL) and streptomycin (0.1 mg/mL). In a microtiter plate, each well was filled with 5000 cells in 50 µ L medium, followed by 24 h incubation at 37 ◦ C with 5% CO 2 . After 24 h incubation with TGQDs, 10 µ L of MTT (5 mg/mL in PBS) was added, and incubated for 4 h at 37 ◦ C with 5% CO 2 . When this incubation period was over, MTT containing medium was replaced and 100 µ L of 99.9% dimethyl sulfoxide (v/v) was added for 5 min. The absorbance was taken at 570 nm by an Infinite m200 PRO reader [46,62] 3. Results and Discussion 3.1. Synthesis and Characterization of TGQDs The characterization was initiated by measuring the fluorescence and absorbance spectra of the TGQDs, as shown in Figure 1a. The Gd–Schiffbase was mixed with TiO 2 in a 1:1 ratio and cooked at 80 ◦ C to form TGQDs, which when observed under microscopy showed a blue field due to its high fluorescence as observed under a UV filter (Figure 1b). The TGQD under UV transilluminator ( λ=312 nm ) showed high fluorescence intensity with a bluish white color (Figure 1c). The fluorescence emission maximum and the absorbance maximum of the TGQDs were observed at λ =375 nm and λ=262 nm , respectively. Furthermore, it was characterized by x-ray fluorescence spectrophotometry using the secondary target as Mo. The results indicate specific peaks that showed the presence of Gd and Ti in the sample, as shown in Figure 1d. Moreover, the particle size distribution, average particle size, and the zeta potential of the prepared TGQDs were analyzed using SEM and DLS (Figure 2). The results revealed that the size of the TGQDs were found to be in the range of 45 ± 2 nm to 95 ±2 nm (Figure 2a) and the value of the zeta potential showed that the value was highest at 58.7 ± 0.13 mV (Figure 2c). The size determined by DLS was in good agreement with the SEM image and data, as shown in Figure 2b. Finally, the FTIR spectrum of TGQD (Figure S3) indicates the presence of the different vibrations of the functional group, showing the presence of all the bond and functional groups present in the gadolinium nitrate and titanium butoxide, initial component of TGQD (Figures S1 and S2). In titanium, the functional group NO 3 has asymmetric and symmetric stretching from 1369 to 1381 cm −1 and 1340 to 1267 cm −1 [ 63 ]. In the case of titanium butoxide absorptions at 1125 cm −1 (Ti–O–C vibration), the intensity of the bands was from 1490 to 1340 cm −1 [ 64 ]. The SECM images obtained are shown in Nanomaterials 2020,10, 778 7 of 18 Figure 3. The scale of the current measured on the bare glassy carbon (GC) was from 0.09 to 0.2 nA. The GC immobilized with TGQD showed a current range mainly from 0.6 to 0.7 nA. Thus, it can be concluded that the current interval in both cases was the same (almost 1 nA). Hence, the inhomogeneity of both electrodes is comparable. It can be concluded that the homogeneity of the both electrodes is mainly affected by the Nafion layer. However, the current increased significantly in the presence of the TGQD. This can be attributed to the good electrochemical conductivity of the synthesized TGQD, which is due to low electrochemical resistance of their metallic components. Moreover, the charge of the QDs may influence their electrochemical conductivity. However, we found that the TGQDs were water soluble and showed high stability (fluorescence measurement) in solution, even after four months from its prepared time. Nanomaterials 2020, 10, x FOR PEER REVIEW 7 of 19 found that the TGQDs were water soluble and showed high stability (fluorescence measurement) in solution, even after four months from its prepared time. Figure 1. (a) The fluorescence spectrum is given in the blue line and the absorbance spectrum is given in the green line. (b) Titanium-Gadolinium Quantum dot (TGQD) observed under the UV filter of the fluorescence microscope. (c) TGQD shows a bluish white color under UV transilluminator; (d) X–ray fluorescence (XRF) spectrum confirms that the synthesized TGQDs contained both gadolinium and titanium metal. Figure 1. ( a ) The fluorescence spectrum is given in the blue line and the absorbance spectrum is given in the green line. ( b ) Titanium-Gadolinium Quantum dot (TGQD) observed under the UV filter of the fluorescence microscope. ( c ) TGQD shows a bluish white color under UV transilluminator; ( d ) X–ray fluorescence (XRF) spectrum confirms that the synthesized TGQDs contained both gadolinium and titanium metal. Nanomaterials 2020,10, 778 8 of 18 Nanomaterials 2020, 10, x FOR PEER REVIEW 8 of 19 Figure 2. (a) Zeta size of the TGQD. (b) SEM analysis of TGQD with 20 µm scale bar;.(c) Zeta potential of TGQDs. Data represent the mean ± SD, n = 5. Figure 2. ( a ) Zeta size of the TGQD. ( b ) SEM analysis of TGQD with 20 µ m scale bar;.( c ) Zeta potential of TGQDs. Data represent the mean ±SD, n=5. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 19 Figure 3. (a) Scanning electrochemical microscopy (SECM) image of glassy carbon electrode covered with Nafion. (b) SECM image of glassy carbon immobilized with QDs and covered with Nafion. The applied parameters: the tip electrode scan rate: 20 µm/s, the applied potential on tip: 0.5 V, the tip— substrate distance: 10 µm, quiet time: 30 s, the scanned surface area 100 µm × 100 µm. The measurements were carried out in a solution of 1 mm FcOH and 0.1 M KCl. 3.2. Detection of Bacteria Furthermore, according to the spectrophotometry it is clear that the bacterial cells were detected by applying TGQDs, where bacterial cells presented a 0.012 O.D. value, whereas absorbance from only bacteria and only QDs were lower than the QD mixed sample. Lower concentrations (0.012 O.D. concentration) of the bacterial sample were detected through the spectrophotometric method (Figure S4). The bacterial sample was stained with TGQD, which gave a high bluish white fluorescence under the UV filter of the fluorescence microscope (Figure S5). Figure 3. ( a ) Scanning electrochemical microscopy (SECM) image of glassy carbon electrode covered with Nafion. ( b ) SECM image of glassy carbon immobilized with QDs and covered with Nafion. The applied parameters: the tip electrode scan rate: 20 µ m/s, the applied potential on tip: 0.5 V, the tip—substrate distance: 10 µ m, quiet time: 30 s, the scanned surface area 100 µ m × 100 µ m. The measurements were carried out in a solution of 1 mm FcOH and 0.1 M KCl. Nanomaterials 2020,10, 778 9 of 18 3.2. Detection of Bacteria Furthermore, according to the spectrophotometry it is clear that the bacterial cells were detected by applying TGQDs, where bacterial cells presented a 0.012 O.D. value, whereas absorbance from only bacteria and only QDs were lower than the QD mixed sample. Lower concentrations (0.012 O.D. concentration) of the bacterial sample were detected through the spectrophotometric method (Figure S4). The bacterial sample was stained with TGQD, which gave a high bluish white fluorescence under the UV filter of the fluorescence microscope (Figure S5). Detection (through imaging) of the VRSA in the presence of PNT1A (coculture imaging) was carried out under fluorescence microscopy. The samples were stained with a very small amount (10 to 15 µ g/mL) of TGQD. The VRSA cells in the presence of PNT-1A were stained with TGQD and SYTO9 to understand the dye specificity (whether TGQD is specific toward bacteria or not) toward bacteria and human cells. The bright field image (Figure 4a) showed the presence of bacteria and human cells (indicated by arrow) in the field of observation. Figure 4b shows that TGQD positively stained the bacterial cells, but PNT1A cells were unstained and not visible in the field. In Figure 4c,e (merged image of Figure 4a,c), it can be seen that the SYTO9 stained both the VRSA and the PNT1A cells, which confirmed that SYTO9 does not have a cell specific staining nature. In contrast, TGQD specifically stained the bacterial cells, which was clearly visible in Figure 4b,d (merged image of Figure 4a,b) and f (merged image of Figure 4b,c). Finally, from Figure 4f (the merged image of Figure 4b,c), it can be clearly concluded that STYO9 is neither specific toward bacteria nor human cells (because both bacterial and human cells were stained), whereas the TGQD is a bacteria specific stain (only bacterial cells were stained). According to the best of our knowledge, there is no such TGQD that can stain only bacteria and not the human cells. This experimental data was further validated by control cell imaging (Figure S6) where TGQD stained VRSA cells showed high fluorescence under a fluorescence microscope, but the PNT1A cells were unstained and no fluorescence was observed. Nanomaterials 2020, 10, 778 9 of 18 only bacteria and only QDs were lower than the QD mixed sample. Lower concentrations (0.012 O.D. concentration) of the bacterial sample were detected through the spectrophotometric method (Figure S4). The bacterial sample was stained with TGQD, which gave a high bluish white fluorescence under the UV filter of the fluorescence microscope (Figure S5). Detection (through imaging) of the VRSA in the presence of PNT1A (coculture imaging) was carried out under fluorescence microscopy. The samples were stained with a very small amount (10 to 15 µg/mL) of TGQD. The VRSA cells in the presence of PNT-1A were stained with TGQD and SYTO9 to understand the dye specificity (whether TGQD is specific toward bacteria or not) toward bacteria and human cells. The bright field image (Figure 4a) showed the presence of bacteria and human cells (indicated by arrow) in the field of observation. Figure 4b shows that TGQD positively stained the bacterial cells, but PNT1A cells were unstained and not visible in the field. In Figures 4c,e (merged image of Figures 4a,c), it can be seen that the SYTO9 stained both the VRSA and the PNT1A cells, which confirmed that SYTO9 does not have a cell specific staining nature. In contrast, TGQD specifically stained the bacterial cells, which was clearly visible in Figures 4b, d (merged image of Figures 4a,b) and f (merged image of Figures 4b,c). Finally, from Figure 4f (the merged image of Figures 4b,c), it can be clearly concluded that STYO9 is neither specific toward bacteria nor human cells (because both bacterial and human cells were stained), whereas the TGQD is a bacteria specific stain (only bacterial cells were stained). According to the best of our knowledge, there is no such TGQD that can stain only bacteria and not the human cells. This experimental data was further validated by control cell imaging (Figure S6) where TGQD stained VRSA cells showed high fluorescence under a fluorescence microscope, but the PNT1A cells were unstained and no fluorescence was observed. Figure 4. The microscopic images. (a) VRSA in presence of PNT1A cells; (b) VRSA in presence of PNT1A cells stained with TGQDs and imaged under UV filter; (c) VRSA mixed with PNT1A cells stained with SYTO9 and imaged under fluorescence microscope through 485/498 filter; (d) Merged image of (a) and (b); (e) Merged image of (a) and (c); (f) Merged image of (b) and (c). 3.3. Antibacterial Activities The antibacterial activity of TGQDs was initiated by the determination of the MIC of TGQD, which was performed by the broth micro-dilution method against VRSA, MRSA, and S. aureus. The Figure 4. The microscopic images. 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