Advances in Materials Science and Engineering

Advances in Materials Science and Engineering / 2020 / Article

Research Article | Open Access

Volume 2020 |Article ID 9718706 |

Thi-Diem Bui, Quang-Liem Nguyen, Thi-Bich Luong, Van Thuan Le, Van-Dat Doan, "Utilization of Mn-Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus", Advances in Materials Science and Engineering, vol. 2020, Article ID 9718706, 10 pages, 2020.

Utilization of Mn-Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus

Academic Editor: Zhiping Luo
Received17 Mar 2020
Accepted10 Apr 2020
Published30 Apr 2020


In this study, Mn-doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti-E. coli antibody and anti-MRSA antibody via protein A supported by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria-Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

1. Introduction

Development of semiconductor nanocrystals or quantum dots (QDs) has been attracted great attention in the past decades due to their potential applications in molecular and cell imaging, biological probes, solar conversion components, optoelectronic components, and light-emitting devices [13]. Hitherto, Cd-based QDs (e.g., CdS, CdSe, and CdTe) have been widely investigated due to their outstanding optical and electrical characteristics such as high photoluminescence quantum yield, broad absorption range, narrow and size-tunable emission, and photostability [47]. However, the inherent toxicity of cadmium has restricted the wide applicability of Cd-based QDs in biological applications. Therefore, many research groups have paid attention to develop Cd-free QDs for optoelectronics and biological applications [813]. Among Cd-free type II–VI semiconductors, ZnSe with a bandgap at room temperature of 2.71 eV [14] are considered as promising fluorescent materials due to their good optical characteristics, remarkable biocompatibility, and stability [9, 15, 16]. Recently, transition-metal-doped ZnSe semiconductors have been intensively developed to increase fluorescence efficiency of bare ZnSe [1720]. The selection of transition-metal ions can be relied on their ion radius and bandgap energy. Among them, Mn2+ is commonly used as a doping agent for various types of semiconductor type II–VI thanks to the following: (i) the replaced positions by Mn2+ inside the lattice of the doped material can be examined by electron paramagnetic resonance; (ii) pure and strong dopant emission is observed due to Mn2+4T1⟶6A1 transition [21]. Besides, the bonds with Mn2+ on the surface of the nanocrystals have formed trapping states, affecting fluorescence and its quantum efficiency [22]. Therefore, as the passive surface states become stable, the QD’s emission ability also becomes better [23, 24].

One of the most well-known methods to stabilize the QDs surface is to wrap one or two layers of semiconductors with a larger bandgap [24, 25]. Semiconductors selected to be sheathed should meet the following conditions: (i) the bandgap width of the shell must be greater than that of the core, so the confined carriers can be remained in the core of the nanocrystals; (ii) the lattice constant should be similar to that of the core so that the shell does not change too much at the junction between the two substances. Furthermore, a thick shell surrounding the nanocrystals will limit the trapped carriers on their surface, and adding a shell of semiconductors with a larger bandgap can increase the efficiency and improve their durability [23, 26, 27]. Compared to another popular II–VI material, ZnS with a large bandgap energy ranging from 3.56 to 3.76 eV has outstanding photoelectric and luminescent properties with extensive applications [16]. Thus, the coating of a ZnS layer around Mn-doped ZnSe QDs could improve its optical properties.

Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA), two common pathogens, especially in food poisoning, are considered as a cause of infection of hospitals and communities because of the high possibility of causing death to patients and long-time treatment [2831]. To detect these bacteria, the traditional culture methods are still used as a gold standard; however, it takes 3–5 days to get the desired results. Many methods of molecular biology such as polymerase chain reaction (PCR), including real-time PCR and pentaplex PCR, have been applied to quickly detect E. coli O157:H7 and MRSA [3235]. However, false-negative results were still recorded in many cases due to several components contained in samples that can inhibit the PCR reaction when using this method for direct detection of bacteria. Fast and accurate detection methods of pathogens are an indispensable need in the current context, especially in developing countries, as the number of hospital infections and food poisoning is increasing day by day. The quick diagnosis will give a significant contribution to the right treatment, fast treatment map for improving patient’s health. To solve the abovementioned problems, currently, the application of nanotechnology in biomedical research, namely, the rapid diagnosis of pathogenic microorganisms by nanotechnology is being intensively invested. In this context, the fluorescent labeling based on colloidal quantum dots has been increasingly developed due to many advantages over other methods, especially in the real-time study of biological processes [3638]. Kloepfer et al. in 2003 initiated the use of QDs for bacterial labeling [39]. The nature of fluorescent labeling is of the possibility of conjugating the QDs to various types of biomolecules such as proteins and antibodies that are specifically coupled with respective antigens of bacteria. QDs have since been used for labeling and detection of numerous bacteria including Mycobacterium bovis, Bacillus Calmette-Guérin, E. coli O157:H7, Salmonella typhimurium, Bacillus anthracis, Staphylococcus aureus, Mycobacterium tuberculosis, and Listeria monocytogenes [40].

In our previous study, QDs of ZnSe/ZnS:Mn/ZnS were successfully synthesized and applied in the detection of E. coli O157:H7 and MRSA using the fluorescent labeling method [41]. However, the synthesis process was quite complicated and the obtained suspensions of QDs were unstable over time, causing difficulties in practical applications. In this study, a fluorescent labeling for rapid detection of E. coli O157:H7 and MRSA by using more stable Mn-doped ZnSe/ZnS core/shell QDs (CSQDs) conjugated with antibody was provided. The water-dispersed Mn-doped ZnSe/ZnS CSQDs were prepared in the aqueous phase using polyethylene glycol (PEG) as a suitable surface stabilizer. The as-synthesized CSQDs were carefully characterized by means of X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Fourier transform-infrared spectroscopy (FT-IR), UV-visible (UV-vis) spectrophotometry, and photoluminescence (PL) spectroscopy before being labeled with antibodies for the detection of E. coli O157:H7 and MRSA. Protein A and 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were used as crosslinkers to covalently conjugate antibodies onto the CSQDs surface. The compatibility of CSQDs with antibodies and the ability of the CSQD-labeled antibodies toward E. coli O157:H7 and MRSA bacteria were also evaluated. The structure of synthesized CSQDs and its application are illustrated in Scheme 1.

2. Materials and Methods

2.1. Materials

Zinc chloride (ZnCl2, 99.9%), manganese(II) chloride (MnCl2, 99.9%), sodium borohydride (NaBH4, 99.9%), selenium powder (Se, 99.9%), zinc acetate dihydrate (Zn(OAc)2 2H2O, 99.9%), sodium sulfide nonahydrate (Na2S.9H2O, 99.9%), sodium hydroxide (NaOH, 99.9%), polyethylene glycol-1500 (PEG-1500, 99.9%), and 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, C8H17N3, 99.9%) were purchased from Merck Co. Protein A was supplied by Sigma Co. Antimethicillin-resistant S. aureus antibody [332/423] (ab62742) and anti-E. coli O157:H7 antibody (FITC) (ab31550) were purchased from ABcam Company. Two strains of E. coli O157:H7 and MRSA were provided by the Institute of Applied Materials Science, Vietnam Academy of Science and Technology, Ho Chi Minh City. The strains were stored at −20°C in 20% glycerol until their use. Before use, the strains were cultured on Luria-Bertani (LB) medium at 37°C overnight, and the initial concentration of bacteria was determined by using the plate counting method. The bacterial strain cultures were then washed and diluted to a final concentration of 106 CFU/mL by phosphate buffered saline (PBS), which was prepared by dissolving 8 g NaCl, 0.2 g KCl, and 1.44 g KH2PO4 in 1L distilled water and adjusted its pH to 7.4.

2.2. Synthesis of Mn-Dopped ZnSe/ZnS CSQDs

The synthesis process can be divided into two stages as follows:Stage 1. Synthesis of ZnSe:Mn as the core: zinc chloride (0.1000 g) and a calculated amount of manganese chloride were dissolved with 10 mL of H2O (solution 1). PEG (0.05 g) was added to 80 mL of H2O and stirred for 15 min at 50°C (solution 2). Then, solutions 1 and 2 were transfered to the three-neck flask and adjusted the pH to 7 by dilute NH4OH 0.01 M solution. The reaction mixture was thoroughly stirred and degassed by inert gas of N2 during the reaction time and kept the reaction temperature at 80°C (solution 3). NaHSe solution was prepared from Se powder (0.4 g), NaBH4 (0.3 g), and water in a vacuum jacketed reaction vessel. The as-prepared NaHSe solution was then injected quickly into the abovementioned reaction solution 3 and kept continuously stirring at 80°C for 90 min.Stage 2. Shell coating process by ZnS for the core: 5 mL solution of Zn(OAc)2.2H2O (0.05 g) and 5 mL solution of Na2S.9H2O (0.03 g) were dropped down slowly one after another into the reaction vessel at stage 1. The reaction mixture was then kept stirring for 90 min at 80°C. Next, the mixture was cooled down and then aged at room temperature for 24 hours and stored for next experiments. The ZnSe:Mn/ZnS samples with a doped amount of Mn ranged from 1% to 11% were named as ZnSe:1% Mn/ZnS to ZnSe:11% Mn/ZnS, respectively.

2.3. Characterization Methods

The crystal structure of Mn-doped ZnSe/ZnS CSQDs was analyzed by X-ray powder diffraction (XRD) using a D8 Advance-Bruker diffractometer with CuKα emission sources (λ = 1.5418 Å). Photoluminescence (PL) intensity and the fluorescence performance were examined on a WGY-10 fluorospectrophotometer (Tianjin Gangdong Scientific and Technical Development CO., Ltd, PRC) using a Xe lamp as the excitation source. The PL measurements were recorded between 400 and 800 nm with an excitation wavelength of 350 nm. The best excitation wavelength for fluorescence was determined via UV-Vis maximum absorbance on an Optizen 2120 UV spectrophotometer. Morphology and size of CSQDs were determined by transmission electron microscopy (TEM) on a JEM 2100F with an acceleration voltage of 200 kV. The presence of functional groups in the product were determined by Fourier transform-infrared spectroscopy (FT-IR) using a FT-IR spectrometer (Brucker Equinox 55). Finally, the dynamic diameter of QDs in colloidal solution was examined by a Horiba SZ-100 nanoparticle analyzer (Japan) using dynamic light scattering (DLS) technology.

2.4. Preparation of CSQDs-Antibody Conjugates and Labeling

Conjugation of the anti-E. coli O157:H7 antibody and anti-MRSA antibody to CSQDs were achieved by using protein A and an EDC crosslinker. The method for conjugating proteins and antibody to QDs is described in detail in previous studies [42,43]. Typically, 30 μL of the synthesized CSQDs suspension was mixed with 50 μL of protein A (2 mg/mL) and 20 μL of EDC (20 mg/mL) at a temperature of 4°C for 5 hours. Then, a calculated volume of anti-E. coli O157:H7 antibody (5.5 mg/mL) or anti-MRSA antibody (1 mg/mL) was added in the abovementioned mixture to obtain a suspension with antibody concentration of 5 μg/mL. The obtained complex was shaken constantly at 4°C for 24 hours. For the bacterial detection test, 20 μL of the resulting CSQDs-antibody complex was added to 100 μL of the bacteria suspensions with different concentrations in the range of 101–106 CFU/mL and reacted at room temperature for the time interval from 5 to 30 min. After the selected time period, the suspension containing CSQDs-antibody-bacteria was centrifuged at 5000 rpm for 5 min, washed twice with PBS buffer, and then dissolved again in 100 μL of PBS. Finally, the studied samples were spread on glass slides and observed under a fluorescence microscope (Leica M205 FCA).

3. Results and Discussion

3.1. Characterization of Materials
3.1.1. Characterization of ZnSe:Mn Cores

The Mn2+ content is an important factor affecting the optical properties of the prepared ZnSe:Mn QDs core. A series of ZnSe:Mn samples with the different concentrations of Mn were synthesized under the other experimental conditions fixed and then characterized to find the optimum Mn content in the QDs core. The light absorption of ZnSe:Mn solutions with different Mn concentrations was investigated by UV-Vis spectrophotometry. As shown in Figure 1(a), a small absorption peak at around 320 nm was assigned to the formation of an exciton of the intrinsic ZnSe nanocrystals. The negligible increase in intensity and the slight shift in the absorption spectra of ZnSe:Mn QDs as increasing the amount of Mn element revealed the formation of a bigger QDs particle size [44, 45].

The PL emission spectra of ZnSe:Mn samples at different Mn concentrations are presented in Figure 1(b). It can be seen that the PL emission spectra show the emission peaks at 595 nm corresponding to Mn2+4T1-6A1 transition [21, 22]. At 1% doped Mn concentration, the characteristic orange luminescence intensity assigned to the Mn2+ center is weak. As the concentration of doped Mn increases, the luminescence intensity of the QDs nanoparticles increases and reaches optimum at Mn concentration of 5%. However, the further increase in doped Mn concentration leads to a decrease in luminescence intensity. It could be explained by the fact that the higher the concentration of Mn, the more the interaction between Mn-Mn, resulting in reducing fluorescent efficiency. At 11% Mn concentration, the obtained fluorescent light has a faint orange color. Thus, doped Mn concentration dramatically affects the luminescence intensity and luminescent color of quantum dots. The XRD patterns of ZnSe:Mn samples presented in Figure 1(c) show three broad diffraction peaks at 27.21°, 45.57°, and 53.27° corresponding to the planes (111), (220), and (311) of the cubic zinc blende of ZnSe nanocrystals [38, 42]. In addition, the small shift of the diffraction peaks of the ZnSe:Mn to larger angles compared to the bare ZnSe (ICDD PDF no. 01-071-5977) caused by the incorporation of Mn2+ ions into the ZnSe nanocrystal lattice was observed. The doping Mn2+ ions into the host ZnSe did not totally cause a crystal phase transformation. However, there were several small sharp peaks appeared in the XRD patterns of ZnSe samples with low concentrations of doped Mn. It could be explained by the formation of two crystalline phases of the zinc blende and hetaerolite, therein hetaerolite played a role of secondary phase [27]. It is noted that the sample with a 5% Mn possesses the highest PL intensity; therefore, the concentration of 5% doped Mn QDs was chosen as a premise for next steps of the synthesis. The EDX spectrum of ZnSe:5% Mn (Figure 1(d)) indicates that the main components of the synthesized ZnSe:Mn sample are Zn and Se elements with 44.8 and 20.5%, respectively. The existence of Mn element in the product has also been found to be about 4.8%, indicating that Mn successfully integrated into ZnSe structure. In addition, the EDX peaks of C and O elements also appeared, confirming that the PEG stabilizer was attached to the surface of luminescent QDs.

3.1.2. Characterization of ZnSe:Mn/ZnS CSQDs

Figure 2(a) shows the XRD patterns of ZnSe, ZnSe:5% Mn cores, and ZnSe:5% Mn/ZnS CSQDs. It can be seen that the XRD patterns of the three samples are similar and have the diffraction peaks at around 27.31°, 45.57°, and 53.27°, which are indexed as the cubic zinc blende structure as mentioned above. The XRD peaks of ZnSe:5% Mn/ZnS CSQDs tend to be broadened and shift toward a larger angle in comparison to that of the ZnSe:5% Mn cores. This may be due of the size effect caused by the ZnS shell surrounding the ZnSe:Mn core QDs. The similar results have been published in many previous studies [46, 47].

The FTIR spectra of the ZnSe, ZnSe:5% Mn, and ZnSe:5% Mn/ZnS QDs stabilized by PEG are given in Figure 2(b). The bands at 3422 (broad), 1600, 1452, 890, and 504 cm−1 are assignable to stretching frequencies for O-H, C=O, C-H, and C-C, respectively. These functional groups demonstrated the existence of PEG on the surface of QDs, which is responsible for the good dispersion of QDs in water and makes them more compatible with bacterial cells. The band at 673 cm−1 appeared only on the ZnSe:5% Mn/ZnS FTIR spectrum is attributed to the Mn-S-specific vibrations, confirming a partial replacement of Zn2+ by Mn2+ ions [48].

Figure 2(c) shows comparison of PL spectra of the bare ZnSe, ZnSe:5% Mn, and ZnSe:5% Mn/ZnS samples. From Figure 2(c), it can be seen that the ZnSe sample exhibited the very weak PL intensity with maximum at around 485 nm, while the ZnSe:5% Mn/ZnS CSQDs demonstrated increased PL intensity as compared to the ZnSe:5% Mn cores and bare ZnSe, indicating the positive role of ZnS in increasing PL intensity for the CSQDs. The enhancement in PL intensity of the CSQDs, as explained in the previous reports [46, 49], is due to the significant reduction in the number of defects in the ZnSe:5% Mn core surface by the growth of an additional ZnS shell on the cores. Besides, the ZnS shell coating also led to a shift of the PL peak at 595 nm for the ZnSe:5% Mn cores to the wavelength of about 605 nm.

The TEM images of the ZnSe:5% Mn core and ZnSe:5% Mn/ZnS core/shell with its dynamic particle size distribution are presented in Figure 3. The synthesized ZnSe:5% Mn cores appear as spherical particles and their average diameter is 7 nm. The average diameter of ZnSe:5% Mn/ZnS CSQDs is about 8.5 nm, which is larger than that of the cores, indicating the formation of ZnS shell on the ZnSe:Mn cores. As shown in the insets of Figure 3, the QDs have uniform dynamic particle size distribution, and this confirmed that the PEG surfactant played a perfect role in helping the particles disperse better in the suspension. The uniformity of ZnSe:5% Mn/ZnS CSQDs is also consistent with the particle size distribution diagram through its narrow bottom width. It has been also found that the ZnSe:Mn cores and ZnSe:5% Mn/ZnS CSQDs have an average dynamic particle size of 125.5 nm and 164.2 nm, respectively. The significant difference in dynamic particle size and the size recorded by TEM images is related to the PEG layer acting as an effective stabilizer for QDs.

4. Application of CSQDs for Bacteria Detection

The ability of CSQDs labeled with E. coli O157:H7 and MRSA was examined with different bacteria concentrations (101, 102, 104, and 106 CFU/mL) at the reaction time ranging from 5 to 30 min. It was observed that the clear fluorescence signals began to be recorded for all studied concentrations after 15 min. The highest fluorescence signals in the detection of E. coli O157:H7 (Figure 4(a)) and MRSA (Figure 4(b)) with the lowest concentration of 101 CFU/mL were obtained after 30 minutes of exposure. Besides, the achieved labeling efficiency of E. coli O157:H7 and MRSA with CSQDs was found to be high, approximately 100%.

Figure 5 shows TEM images of E. coli O157:H7 and MRSA with and without CSQDs. It can be seen that the morphology of E. coli O157:H7 (Figure 5(a)) and MRSA (Figure 5(b)) is clearly visible due to the presence of CSQDs. It is well known that biological samples as well as bacteria belong to a class of nonconductive materials; therefore, it is difficult to show its images on an electron microscope. Here, the synthesized CSQDs can act as a conductive sputter coating, which improved the quality of TEM images of bacterial cells. Without CSQDs, E. coli O157:H7 and MRSA were almost impossible to detect by TEM (Figures 5(c) and 5(d)). Interestingly, no evidence for rupture and collapse of bacterial cells was observed in the TEM images of E. coli O157:H7 and MRSA conjugated with CSQDs, demonstrating that the CSQDs do not exhibit cytotoxicity against the aforementioned strains at used concentration. Therefore, the synthesized CSQDs are eligible to be applied as an effective fluorescent labeling material.

5. Conclusions

In summary, Mn-doped ZnSe/ZnS core/shell quantum dots were successfully synthesized and used for selective fluorescent labeling and detection of E. coli O157:H7 and MRSA. The synthesized CSQDs had an average particle diameter of 8.5 nm and exhibited high luminescence. The CSQDs conjugated well with the anti-E. coli O157:H7 antibody and anti-MRSA antibody via protein A and EDC crosslinkers. The CSQDs-antibody complex allowed intensive detection of the bacteria at levels down to 101 CFU/mL within 30 min. The labeling efficiency of E. coli O157:H7 and MRSA with CSQDs-antibody complex was approximately 100%. Thus, the provided CSQDs-antibody complex can be used as an effective and useful tool for detection of pathogenic bacteria by the fluorescence method in the clinical microbiology and biomedicine field as well as in dairy products to ensure the food safety.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interests.


The authors acknowledge the Industrial University of Ho Chi Minh City, Vietnam, for supporting this work (funding no. 184.HH01). The authors also would like to thank the Department of Science and Technology of Ho Chi Minh City for their support to complete this work.


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