Extracellular biosynthesis of biocompatible CdSe quantum dots

Abstract

An extracellular biosynthesis method has been developed to prepare cadmium selenide (CdSe) quantum dots (QDs) with strong fluorescence emission by incubating cheap Cd and Se inorganic salts with Escherichia coli (E.coli) bacteria. Ultraviolet–visible absorption spectra, photoluminescence (PL) spectra, and high‐resolution transmission electron microscopy analysis showed that the biosynthesised CdSe QDs have an average size of 3.1 nm, the excellent optical properties with fluorescence emission around 494 nm, and the good crystallinity. It was found that addition of 80 mg of mercaptosuccinic acid resulted in the formation of CdSe QDs with highest PL intensity. Furthermore, Fourier‐transform infrared spectra of as‐synthesised CdSe QDs confirmed the presence of a surface protein capping layer. The biosynthesised CdSe QDs were incorporated into the yeast cells as illustrated by laser confocal scanning microscopy images, showing a great potential in bio‐imaging and bio‐labelling application.

1 Introduction

Semiconductor nanocrystals known as quantum dots (QDs) with size of 1–100 nm have unique electric and optical properties owing to quantum confinement effect [1, 2, 3]. In recent decades, QDs with different sizes, compositions, and shapes have been extensively applied in many areas including light source in LED, solar cell, display materials, optic‐fibre amplifier, fluorescent DNA label, labels for cell imaging, optoelectronic transistor components, air and water purifiers, analytical tools for automatic chemical analysis [4, 5, 6].

To date, water‐based synthesis [7, 8] and organometallic method [9, 10] have been developed to synthesise highly fluorescent QDs. However, these two methods involve highly toxic chemicals and high temperatures [11]. To gain desirable biocompatibility, QDs are usually treated with a complicated surface modification process [5]. Recently, the biosynthesis strategy has attracted researchers’ attention because of environmental friendliness, biological compatibility, low temperature, and low cost [12]. Many biological organisms have been used for the preparation of QDs with precisely controlled sizes, shapes, and functionalities [12]. CdTe QDs have been prepared by using Saccharomyces cerevisiae and Escherichia coli (E.coli) [13, 14]. The synthesis of PbS QDs by Torulopsis spp. has been reported [15]. Ag nanocrystals have been synthesised by using Bacillus licheniformis and Aspergillus fumigatus [16, 17]. CdS nanoparticles have been obtained by using Fusarium oxysporum [18]. These approaches are carried out at ambient temperature and considered as environmental‐friendly method. It is noted that this synthesis strategy is divided into intracellular synthesis and extracellular synthesis based on the location of nanocrystals inside or outside the cells. Intracellular biosynthesis of cadmium selenide (CdSe) QDs with different fluorescence emission wavelengths has been realised, however, a complicated procedure including cell washing, cell disruption, and removal of cell fragments is required to extract CdSe nanocrystals [19]. By contrast, the complicated treatment is avoided by the extracellular biosynthesis route, thus facilitating the harvesting and isolation of bio‐grown QDs. In addition, various resources are available for biosynthesis, such as plants, bacteria, fungi, yeast, and viruses [20]. Bacteria are commonly selected for preparing QDs since they are beneficial for handling and extracting QDs [21]. E.coli is a gram‐negative bacterium that is widely used as a model organism in biological studies and can be easily cultured under aerobic or anaerobic conditions at 37°C. Therefore, it is desirable to develop a biosynthesis approach that produces fluorescent QDs extracellularly using E.coli without complicated after treatment procedure.

As one of the most important metal ion semiconductors, CdSe nanocrystals have been intensively studied for energy, electronics, and biological applications due to their tunable size‐dependent emission within the whole visible range [22, 23]. Compared to conventional fluorescent dyes, CdSe QDs possess the advantages of high photostability, controllable and narrow emission bands, and a high quantum yield, which allow for the application of CdSe QDs as fluorescent marker in bio‐imaging of living cells [24]. In this work, based on the extracellular biosynthesis route, CdSe QDs have been produced by incubating cheap Cd and Se salts with a widely used bacterium‐E.coli. The CdSe QDs are grown in vitro, which simplifies the collection step only by centrifugation. The obtained samples show good water solubility, stability, and biocompatibility. This work not only offers an economically and environmental‐friendly method for preparing QDs with good biocompatibility, but also extend bio‐imaging and bio‐labelling applications of biosynthesised QDs.

2 Materials and methods

2.1 Materials

Peptone, yeast extract, sucrose, sodium chloride (NaCl), sodium hydroxide (NaOH), sodium nitrate (NaNO₃), sodium phosphate dibasic dodecahydrate (Na₂ HPO₄ ˑ12H₂ O), ethanol (anhydrous, ≥99.95%), potassium dihydrogen phosphate (KH₂ PO₄), dipotassium hydrogen phosphate (K₂ HPO₄), ammonium chloride (NH₄ Cl), magnesium sulphate heptahydrate (MgSO₄ ·7H₂ O), glucose (C₆ H₁₂ O₆), cadmium chloride hemipentahydrate (CdCl₂ ·2.5H₂ O), mercaptosuccinic acid (MSA), sodium citrate tribasic dihydrate (C₆ H₅ O₇ Na₃ ·2H₂ O), potassium chloride (KCl), hydrochloric acid (HCl), Rhodamine 6G, and sodium selenite (Na₂ SeO₃) were purchased from commercial suppliers and used without further purification. Double distilled water was used for all experiments.

2.2 Culture of E.coli and yeast bacteria

E.coli bacteria were grown with aeration at 37°C on a rotary shaker (200 r/min) in Luria‐Bertani medium containing 5 g of peptone, 2.5 g of yeast extract, 5 g of NaCl, pH = 7.0. After growth of bacteria overnight, the cultures were centrifuged at 4000 r/min for 15 min to extract E.coli bacteria. Then, E.coli was transferred into a modified Czapek's (M9) medium, which was incubated at 37°C on a rotary shaker (200 r/min). In a typical procedure for preparing M9 medium, 15 g of Na₂ HPO₄ ˑ12H₂ O, 7.5 g of KH₂ PO₄, 2.5 g of NH₄ Cl, and 1.25 g of NaCl were added into 500 ml of H₂ O, followed by diluting for five times to form a solution, in which 1 ml of MgSO₄ ·7H₂ O (1 mol/l) and 10 ml of 20 wt% glucose were introduced per litre of the diluted solution. When the absorbance of bacteria cultures reached 0.6 at 600 nm (A ₆₀₀  = 0.6), the cultures were centrifuged at 4000 r/min for 15 min to harvest E.coli bacteria for the biosynthesis of CdSe QDs, and the obtained E.coli bacteria are marked as second‐generation E.coli bacteria. Before use of M9 medium, all the solutions were sterilised in high‐pressure steam sterilisation pot at 121°C for 15 min.

Yeast bacteria were grown with aeration at 37°C on a rotary shaker (200 r/min) in yeast extract peptone dextrose medium containing 5 g of yeast extract, 10 g of peptone, 10 g of glucose, pH = 7.0. After growth of bacteria overnight, the cultures were centrifuged at 4000 r/min for 15 min to extract yeast bacteria. Then, yeast bacteria were added into the Czapek's medium consisting of 15 g of sucrose, 1 g of NaNO₃, 0.5 g of K₂ HPO₄, 0.25 g of KCl, 0.25 g of MgSO₄ ·7H₂ O, followed by incubation at 37°C on a rotary shaker (200 r/min). After incubation for 1 day, the cultures were centrifuged at 4000 r/min for 15 min to harvest yeast bacteria for cell imaging of CdSe QDs. Before use, the Czapek's medium was sterilised in high‐pressure steam sterilisation pot at 121°C for 20 min.

2.3 Biosynthesis of CdSe QDs

Second‐generation E.coli bacteria were added to 100 ml of M9 medium in a conical flask. Afterwards, 8 ml of CdCl₂ solution (0.04 mol/l), 800 mg of C₆ H₅ O₇ Na₃ ·2H₂ O, 1.5 ml of Na₂ SeO₃ solution (0.02 mol/l), and certain amount of MSA were added in the system under stirring, followed by incubation at 37°C on a rotary shaker (200 r/min). After certain reaction time, the reaction solution was withdrawn and centrifuged at 4000 r/min for 15 min to remove E.coli bacteria, and the resulting supernatant was further centrifuged at 10,000 r/min for 30 min to extract CdSe QDs. The CdSe QDs were washed with 50 wt% ethanol by centrifugation (10,000 r/min, 30 min) for three times to remove residual chemicals.

2.4 Characterisation of biosynthesised CdSe QDs

The tested QDs samples biosynthesised by using 80 mg of MSA were taken from the reaction vessel after different incubation times and were centrifuged to remove bacteria without any further narrowing of their size distribution. After incubation for different times, the supernatant of centrifuged E.coli bacteria culture was collected for ultraviolet–visible (UV–vis) absorption measurement, which was performed on a Perkin Elmer Lambda 750 spectrophotometer to monitor the growth of CdSe QDs (Fig. 1). For CdSe QDs grown at different times, the supernatant of centrifuged E.coli bacteria culture was collected for photoluminescence (PL) measurement (Fig. 2) that was carried out by using Shanghai Leng Guang Technic F96PRO spectrofluorimeter with an exciting wavelength of 400 nm. Transmission electron microscopy (TEM) samples were fabricated by dropping on copper grids and drying overnight at room temperature. The high‐resolution TEM (HRTEM) images were taken with a JEM 2100 (JEOL, Japan) electron microscope operating at 200 keV. The dried QDs were mixed with KBr to produce pellets. Fourier‐transform infrared (FTIR) spectra were obtained with a HP Laser Jet P2035.

Fig. 1.

UV–vis absorption spectra of extracellularly biosynthesised CdSe QDs after incubation with E. coli at 37°C in the open air for different times

Fig. 2.

PL spectra of extracellularly biosynthesised CdSe QDs after incubation with E. coli at 37°C in the open air for different times

2.5 Characterisation of E.coli

After incubation at 37 °C for 5 days, the reaction solution for the biosynthesis of CdSe QDs was centrifuged at 10,000 r/min for 30 min to precipitate the E.coli cells, which were washed by M9 medium for three times. Then, the morphology and structure of E.coli were observed by using an Olympus BX 43 fluorescence microscope.

2.6 Calculation of PL quantum yields

PL quantum yields of biosynthesised CdSe QDs were calculated by comparison with Rhodamine 6G in anhydrous ethanol according to the procedure reported in [9]. The calculation was implemented by following the formula (e.g. (1)) as displayed below:

$${\phi }_x={\phi }_{\mathrm{st}}{\left(\frac{M_x}{M_{\mathrm{st}}}\right)}^2{\left(\frac{{\eta }_x}{{\eta }_{\mathrm{st}}}\right)}^2$$ (1)

where ${\phi }_x$ and ${\phi }_{\mathrm{st}}$ correspond to quantum yields of CdSe QDs and Rhodamine 6G, respectively (${\phi }_{\mathrm{st}}$  = 95%); $M_x$ and $M_{\mathrm{st}}$ denote the integral area of fluorescence spectra divided by the absorbance at 350 nm for CdSe QDs and Rhodamine 6G; ${\eta }_x$ and ${\eta }_{\mathrm{st}}$ represent the refractive index of solvents for CdSe QDs and Rhodamine 6G.

2.7 Cell imaging test

The washed CdSe QDs were transferred into the yeast culture, which was prepared by adding the yeast bacteria into 50 ml of Czapek's medium until the absorbance of yeast culture reached 0.6 at 600 nm (A ₆₀₀  = 0.6). The resulting system was exposed to incubation for 2 h. Then, the yeast cells were harvested by centrifugation (4000 r/min, 15 min) and removal of supernatant, and the fluorescence micrographs were captured via laser scanning confocal microscopy measurement by an Olympus FV1000 instrument equipped with an Ar/Kr laser.

3 Results and discussion

3.1 UV–vis absorption and PL spectra analysis

In this study, the E.coli bacteria without exogenous protein were selected for study on biosynthesis of CdSe QDs, which were obtained via an extracellular growth approach by incubating cheap inorganic salts CdCl₂ and Na₂ SeO₃ with E.coli cells in M9 medium at mild temperature (37°C). The production cost of CdSe QDs was calculated as ∼2.43 USD/mg. The UV–vis absorption and PL spectra of the extracellularly biosynthesised CdSe QDs were recorded to monitor the growth of the QDs during the incubation process. The QDs samples tested were taken from the reaction vessel after different incubation times and were centrifuged to remove bacteria without any further narrowing of their size distribution. All samples showed well‐resolved absorption maxima of the first electronic transition (Fig. 1). After incubation for 1 day at 37°C, CdSe QDs show an intense narrow absorption at 407 nm from the first electronic transition (Fig. 1) and a strong emission centred at 493 nm in the PL spectra (Fig. 2). The absorption edge moves to a shorter wavelength at 2nd day, and then is slightly shifted to longer wavelength at 3rd day. A little bit larger red shift of absorption edge occurs at 4th day, indicating the slight growth of CdSe QDs during incubation, which may be ascribed to the effect of Ostwald ripening [25]. As the incubation time is increased to 5 days, absorption edge does not move towards longer wavelength, but exhibits a slight blue shift, meaning that the growth of CdSe QDs has been completed. The intensity of PL band is increased at 2nd day, and then is gradually decreased until 5th day, while emission maximum peak moves to slightly shorter wavelength (491 nm) at 2nd day, and then continuously shifts to a little longer wavelength (494 nm) at 5th day. The enhanced PL intensity at 2nd day is attributed to the better crystallisation of QDs during growth process. In the following nanocrystal growth, a few dislocations and defects may be formed to lower the PL intensity at 5th day. The luminescence of CdSe QDs is dominated by the near band edge luminescence and the quantum yield of biosynthesised CdSe QDs with an emission peak at 494 nm is about 35% (by comparison with Rhodamine 6G in anhydrous ethanol according to the procedure reported in the previous reference [9]), which is much higher than that observed for CdSe nanocrystals obtained by refluxing method [26]. UV–vis absorption and PL spectra demonstrate that CdSe nanocrystals with strong blue emission (∼ 494 nm) can be produced using E.coli. However, QDs have a slight shift of absorption edge and no obvious change in emission wavelength as the incubation time is tuned. In addition, the biosynthesised CdSe QDs exhibit an excellent stability, which is possibly due to the surface capping layer of proteins secreted by E.coli.

It should be noted that the stabiliser agent MSA plays a key role in passivation to prevent nanoparticles aggregation and precipitation through forming Cd‐thiol surface layers at the surface of nanocrystals. The amount of MSA used for biosynthesis of CdSe QDs may affect the surface defects and crystallisation of QDs, which can be reflected by PL spectra. As illustrated in Fig. 3, CdSe QDs had a relatively low PL intensity centred at 503 nm when 40 mg of MSA was utilised in the synthesis process, suggesting that the crystallisation of CdSe QDs is undesirable, and some defects exist in the nanocrystals. Once the amount of MSA was increased to 60 mg, CdSe QDs showed an enhanced PL intensity with the maximum emission at 495 nm. As the amount of MSA was continuously increased to 80 mg, PL intensity of CdSe QDs became largest while the maximum emission was shifted to 492 nm, indicating the desirable crystallisation and decrease in surface defects. However, a further increase of the amount of MSA to 100 mg resulted in lower PL intensity while the maximum emission slightly moved to 491 nm. This is possibly attributed to the fact that more dithio‐Cd complex is formed in the precursor, thereby causing a decrease in the amount of monothiol‐Cd complex, which has an important effect on PL intensity [27].

Fig. 3.

PL spectra of extracellularly biosynthesised CdSe QDs after incubation with E. coli at 37°C in the open air by using different amounts of MSA

(a) 40 mg, (b) 60 mg, (c) 80 mg, (d) 100 mg

3.2 TEM analysis

The morphology and crystallinity of the biosynthesised CdSe QDs were analysed by TEM. Fig. 4 displays the TEM and HRTEM images of the biosynthesised CdSe QDs incubated for 5 days at 37°C, revealing that the CdSe QDs are well‐dispersed single‐crystalline nanoparticles with an average size of 3.1 nm (Fig. 4 a), which is illustrated by the size distribution of CdSe QDs in Fig. 4 c. HRTEM image (Fig. 4 b) shows that the QDs have clear lattice planes with spacing of 0.21 nm, corresponding to the d (111) spacing of wurtzite structure of bulk CdSe. This also suggests that the individual nanoparticle is a single crystal. It is acknowledged that the size and dispersity of nanoparticles are related to the rates of nucleation and growth as well as the extent of agglomeration. The good dispersity of CdSe QDs may be also ascribed to surface capping proteins secreted by E.coli.

Fig. 4.

CdSe QDs extracellularly biogrown with E.coli cells at 37°C for 5 days

(a) TEM image at 5 nm scale, (b) HRTEM image at 2 nm scale, (c) Size distribution of 51 CdSe QDs

3.3 FTIR spectral analysis

The FTIR spectrum was used to identify the chemical composition on the surface of the biosynthesised CdSe QDs. The biosynthesised CdSe QDs show two infrared absorption bands centred at 1657 and 1578 cm⁻¹ (Fig. 5), which are referred as the amide I and amide II absorptions of protein molecules, respectively. It confirms that biosynthesised CdSe QDs are capped with proteins that improve the biocompatibility of the QDs. It should be noted that no protein was added to the culture medium, so the proteins capped on QDs come from the E.coli bacteria during the incubation process. E.coli bacteria used in the biosynthesis not only provides an environmental‐friendly reaction system for the growth of uniform CdSe nanoparticles, but also secretes proteins that strongly bind metal to form a coating layer on CdSe QDs, which can prevent the agglomeration of the nanoparticles, so the QDs keep stable in the dispersion medium. In addition, the proteins can capture the unreacted Cd²⁺ in Cd containing waste stream from bacteria, and are finally precipitated out to avoid the pollution caused by Cd²⁺ through adding saturated NaCl solution, thus guaranteeing the environmental friendliness.

Fig. 5.

FTIR spectrum of extracellularly biosynthesised CdSe QDs incubated with E. coli cells at 37°C for 5 days

3.4 Fluorescence microscopy analysis

The toxic effect of CdSe QDs on E.coli was investigated by using fluorescence microscope. The fluorescence microscopy image (Fig. 6) shows that E.coli cells incubated for 5 days are more slender and filamentous in comparison with normal E.coli cells. Elongated E.coli cells can usually be found to adapt to the environments with strong heavy metal stress or limited nutrients. It is suggested that CdSe QDs have toxicity for the growth metabolism of E.coli. Moreover, yellow‐green emission is observed from the E.coli cells, indicating that the biosynthesised CdSe QDs are well dispersed in the E.coli cells. This demonstrates that the entry of QDs into E.coli bacteria for labelling may be achieved by permeabilising their outer layer following an endocytosis pathway as proposed in the previous study [13].

Fig. 6.

Fluorescence microscopy image of the E.coli cells incorporated with CdSe QDs after incubation at 37°C for 5 days

3.5 In‐vitro cell imaging

Laser confocal scanning microscopy under excitation by an Ar/Kr laser with bright field imaging was used to capture the in‐situ bio‐images of the yeast cells incubated at 37°C for 2 days with CdSe QDs (Fig. 7). Green emission from bio‐grown CdSe QDs is observed in the yeast cells (Fig. 7 a), showing that the QDs have been incorporated into the yeast cells. Interestingly, the overlaid image (Fig. 7 c) shows that the CdSe QDs are well distributed in the cytoplasm and nucleus of the yeast cells and no QDs are found in the cell membrane. This result is different from the intracellular biosynthesis of CdSe QDs [19], which are mainly dispersed in the membrane and cytoplasm of the cells. The confocal images indicate that the biosynthesised CdSe QDs have good biocompatibility for a potential application of in‐vitro cell imaging, which may act as an efficient alternative or complementary tool to conventional organic dyes. Furthermore, it is speculated that the good biocompatibility of CdSe QDs may facilitate the uptake of QDs into cells when the CdSe QDs are extracellularly biosynthesised by using E.coli bacteria, which are similar to the extracellular synthesis mechanism as revealed in the previous report [13].

Fig. 7.

Confocal images of the yeast cells incorporated with the CdSe QDs at 37°C for 2 days

(a) Recorded under excitation by an Ar/Kr laser giving green emission, (b) Bright field image, (c) Overlaid image

4 Conclusion

In summary, a simple and efficient extracellular biosynthesis method has been developed to prepare CdSe QDs by incubating E.coli cells with cheap Cd and Se precursors. The extracellularly bio‐grown CdSe QDs possess an average size of 3.1 nm and good crystallinity. The biosynthesised CdSe QDs have intense fluorescence and the emission is centred at ∼494 nm. It has been found that CdSe QDs with highest PL intensity were produced in presence of 80 mg of MSA. E.coli ‐secreted proteins are confirmed to cap on surface of CdSe QDs, which exhibit an excellent biocompatibility and are well dispersed in the cells as illustrated by in‐situ bio‐images of the yeast cells. In comparison with other approaches, the method allows for one‐step preparation of biocompatible CdSe QDs with high fluorescence emission in aqueous solution at relatively low temperature (37°C). Moreover, the proteins capped on the QDs provide high biocompatibility and functional groups for further chemical modification. As a result, these protein‐capped CdSe QDs are expected to be very useful for bio‐imaging and bio‐labelling applications.