Facile bio-inspired synthesis of zinc sulfide nanoparticles using Chlamydomonas reinhardtii cell free extract: optimization, characterization and optical properties

1 Introduction

Zinc sulfide (ZnS) is a semiconductor with a wide band gap of 3.7eV [1]. Due to quantum confinement, nanocrystalline ZnS has novel properties compared to the bulk material. Its unique properties include a large surface to volume ratio, enhanced activity, and distinct optical and electronic properties. It finds extensive application in light emitting diodes [2, 3], as photocatalysts that may be used for environmental remediation [4] and have also been used as effective antimicrobial agents [5]. There is a constant need for new strategies that provide better control over the size and shape of semiconductor nanoparticles, especially since their optoelectronic properties critically depend on these factors [6].

Traditionally, physical and chemical methods such as pulsed laser deposition at 600°C, high energy milling and chemical precipitation have been used to synthesize semiconductor nanoparticles [7]. These methods have been popular because they have a number of advantages, for example, the size and shape control offered by the classical colloidal chemistry approach is hard to beat. However, they come with their own limitations, like the use of expensive equipment, hazardous materials and severe conditions. Moreover, the synthesis of nanostructures with a precise shape usually requires multiple steps and a copious amount of chemicals to create a suitable template. Following synthesis, it is necessary to remove the template and this can prove challenging as it involves a protracted, energy consuming process [8]. Green strategies are based on the use of benign, solvents, nontoxic chemicals and sustainable materials. Recently, there has been a growing interest in the use of biological materials for nanoparticle synthesis, as it is well known that they are capable of producing inorganic materials both intracellularly and extracellularly. Further, there have been reports on the synthesis of unique hierarchical nanostructures with outstanding shape and size control by employing biomolecules [9]. A biomimetic approach would be environmentally benign and more cost effective. In our study we have utilized the cell free extract of Chlamydomonas reinhardtii as the capping material to help synthesize the nanoparticles and the reaction was conducted in an aqueous solution at room temperature.

Diverse organisms or its components have been used in the synthesis of ZnS nanoparticles; for example, the immobilized phototrophic bacterium, Rhodobacter sphaeroides [10], latex of the plant Jatropha curcas and Fusarium oxysporum, a common fungus [11]. Ordered ZnS nanocrystal assemblies have been fabricated using a genetically modified P22 coat protein as a template [9]. To the best of our knowledge, there have been no reports until date regarding the application of algae in the synthesis of ZnS nanoparticles. Chlamydomonas reinhardtii is an alga that is abundant in fresh water. These harmless algae are easy to culture in large quantities as they do not require expensive media or exacting conditions and are safe for handling. The entire process can easily and economically be scaled up to produce up to 5 g/l of photoautotrophic biomass using low cost photobioreactors [12]. Their cost effectiveness and the fact that algal cells are rich in polysaccharides and glycoprotein matrices, composed of numerous functional groups (such as carbonyl, thiol, carboxyl, amidic and amino) which play a significant role in the reduction and stabilization of nanoparticles make them ideal for use in synthesis [13]. Zinc sulfate has been used to synthesize the nanoparticles in this study; it is commonly used as source of zinc in animal feeds and is generally considered safe [14].

One of the challenges in semiconductor nanoparticle synthesis has been the low yield; this is largely due to intracellular synthesis that required cell lysis and further downstream processing for nanoparticle purification [15]. In our study, we employ an extracellular approach that allows us to readily recover the nanoparticles without further downstream processing. The entire process is easily scalable, economical and makes use of environmentally safe materials.

By employing mild conditions in combination with nontoxic materials, we have developed a facile, green synthetic method to produce nanoscale ZnS. The optical properties of these nanoparticles were characterized using UV-visible spectroscopy, photoluminescence (PL) spectroscopy; the size and crystal structure were analyzed using high resolution transmission electron microscopy (HRTEM), high resolution scanning electron microscopy (HR-SEM), dynamic light scattering (DLS) and X-ray diffraction (XRD). Finally, the functional groups involved and the stability of the nanoparticles were determined by Fourier transform infrared (FTIR) spectroscopy and by measuring the zeta potential. We have discussed the plausible mechanism of nanoparticle formation involving proteins belonging to the oxidoreductive apparatus, their role in capping and subsequent stabilization.

2 Materials and methods

3 Results and discussion

ZnS nanoparticles were synthesized by a rapid, green method that employed the cell free extract of the ubiquitous fresh water algae C. reinhardtii. The nanoparticles were easily separated from the algal cell free extract by centrifugation and repeated washing (thrice). They did not require any additional processing and were used in subsequent experiments. Biosynthesis of ZnS nanoparticles was monitored using UV-visible spectroscopy under ambient conditions (room temperature). Over the course of 24 h, there was a change in color from a colorless solution to a pale solution with a whitish precipitate. Periodic sampling taken every 8 h showed the development of a shoulder at 300 nm. Over the course of the reaction, there was increase in absorbance and a mild red shift of approximately 10 nm. Following 24 h, there was no further increase in the absorbance or shift in the peak position indicating the completion of the reaction. The absorption spectra revealed a broad excitonic peak around 310 nm as can be observed in Figure 1; a similar broad excitonic peak has been observed during the synthesis of thioglycolate coated ZnS nanoparticles after 16 h [16]. This peak is significantly blue-shifted compared to the bulk material and could be explained by quantum confinement effects. As the particle size decreases, the wavelength of maximum exciton absorption (λmax) also decreases [17]. Other studies that have made use of biological sources for ZnS nanoparticle synthesis have reported similar results [18, 19]. When the absorption peak is located around 325 nm, particle size could be correlated to 4.5 nm. We observed larger particles of approximately 8 nm in size; this discrepancy could be explained by virtue of the broadness of the peak that indicates some amount of heterogeneity in particle size [20].

Figure 1:

UV-visible spectrum of zinc sulfide nanoparticles synthesized using Chlamydomonas reinhardtii cell free extract at room temperature over the course of 24 h.

There are a number of factors that play a key role in the synthesis of nanoparticles; these include temperature and pH whose effects have been studied in detail. The effect of temperature on the synthesis of ZnS nanoparticles was studied by varying the temperature up to 80°C as seen in Figure 2. At higher temperatures of 60°C and 80°C, no characteristic absorption peaks were observed, however broad peaks at 280 nm and 430 nm were observed. These likely correspond to algal proteins and chlorophyll a present in the algal cell extract [21]. At room temperature, a peak at approximately 307 nm was observed, corresponding to the conversion of bulk zinc sulfate to nanoscale ZnS. Approximately 1.5 g/l of ZnS nanoparticles from cell free extract was obtained; there was a lower yield at higher temperatures. It is likely that at higher temperatures, the algal proteins denature and are unable to interact and reduce the zinc sulfate solution, whereas at room temperature the proteins retain their functionality and are able to reduce and stabilize the as-formed nanoparticles.

Figure 2:

Effect of temperature on the synthesis of zinc sulfide nanoparticles using Chlamydomonas reinhardtii.

The role of surface charge on the formation of ZnS nanoparticles were studied by varying the pH and observing its effect on the absorption properties. At more alkaline pHs no peaks were seen and this could be attributed to greater solubility of the product preventing the formation of ZnS nanoparticles [22]. There have been a number of reports that have studied the activity of oxidoreductive proteins such as carbonic anhydrase and sedoheptulose 1,7 bisphosphatase under different pH conditions. They have observed an increase in the activity of these proteins under alkaline conditions compared to acidic pHs, thus indicating that these proteins are functionally active even at higher pHs [23, 24]. At a pH of 4.5, a broad peak around 325 nm was observed (Figure 3) indicating the formation of nanoparticles. It is highly probable that these nanoparticles would be larger in size, because at such low pH values, agglomeration is favored over nucleation. At a pH of 6.5, which is the physiological pH of the microalgae, a strong absorption peak around 310 nm was clearly observed. There was a high yield of nanoparticles at this pH; at more acidic pHs insubstantial amounts of nanoparticles were produced. The algal proteins play a critical role in the nucleation-growth processes and at this pH they act as a scaffold and help regulate the size of the nanoparticles.

Figure 3:

Effect of pH on the synthesis of zinc sulfide nanoparticles using Chlamydomonas reinhardtii.

The effect of the algal cell free extract in the synthesis of ZnS nanoparticles (Figure 4) was studied by varying the concentration of the extract, meanwhile the other conditions were maintained as follows – zinc sulfate solution (1 mm) with a pH of 6.5 at room temperature. When 0.5 ml of extract was used, no ZnS peak was observed, but a peak around 285 nm was seen and this corresponds to a protein component of the extract. As the concentration of the extract increased there was a slight red shift in the peak to approximately 290 nm; this would be indicative of the nascent formation of ZnS nanoparticles. Finally, when a concentration of 2.5 ml was used, a pronounced peak at 310 nm corresponding to the formation of nano-ZnS was observed; this peak was also significantly blue-shifted from bulk material and this could be ascribed to the quantum confinements.

Figure 4:

Effect of Chlamydomonas reinhardtii cell free extract concentration on the synthesis of zinc sulfide nanoparticles.

The fluorescent properties of the nanoparticles were determined using PL spectroscopy and the samples were excited at 325 nm. Two peaks were observed; a broad peak at 410 nm and a shoulder peak at 431 nm as seen in Figure 5. The peak at 410 nm may be attributed to the recombination between the sulfur-vacancy-related donor and the valence band [25], while the peak at 430 nm may be due to defect related emissions, specifically due to zinc related vacancies [17, 26]. This could tie in with the EDS results, which indicated reduced levels of zinc. A moderate blue shift in the emission (30 nm) band relative to that of bulk ZnS is further attestation to the size quantization effects. A previous study observed a similar emission profile with a peak at 420 nm and attributed this to the recombination of electron and holes in the trapped surface states located in the forbidden region of the bandgap [27].

Figure 5:

Fluorescence spectrum obtained from aqueous zinc sulfide nanoparticles at room temperature excited at 325 nm.

The HRTEM images as shown in Figure 6 indicate that the particles are spherical in shape. At lower magnification, there are a number of spherical particles clustered together. Analysis at higher magnifications indicated that they are actually a number of smaller particles that have been loosely assembled together. The particles were found to range from 8 nm to 12 nm and the average particle size was around 10 nm. The selected area electron diffraction (SAED) image (6D) showed bright concentric rings instead of sharp, well-defined spots indicating the polycrystalline nature of the nanoparticles that could be indexed with the zinc blende structure. A large number of spherical particles were observed during HRTEM analysis. Some amount of agglomeration of the sample was observed; a possible reason could be due to the association of particles during drying in the HRTEM sample preparation process [17]. ZnS nanoparticles that were synthesized using SDS as a capping agent were found to agglomerate; this was reasoned to be due to the large surface encompassed by the detergent that promoted agglomeration [28]. HR-SEM was used to study the surface morphology of the ZnS nanoparticles; at higher magnifications, large numbers of spherical nanoparticles measuring around 20 nm to 35 nm were observed (Figure 7). However, at lower magnifications, the particles appeared to possess an irregular morphology and were agglomerated. Energy dispersive X-ray spectroscopy (EDX) can be very informative about the structure or the elemental composition of the material that is being investigated. Elemental analysis of ZnS nanoparticles showed the presence of zinc and sulfur confirming that the synthesized nanoparticles were indeed ZnS (Figure 7D). Other peaks that were observed corresponding to carbon, oxygen and nitrogen are most likely due to proteinaceous matter capping the nanoparticles. The crystallographic nature and size of the nanoparticles were analyzed by powder XRD. Figure 8 shows the powder XRD profile of ZnS nanoparticles synthesized using the cell-free extract of C. reinhardtii. The product was found to exhibit the characteristic features attributed to the FCC structure of cubic ZnS. The profiles matched JCPDS powder diffraction file no. 651691. The characteristic peaks at 2θ values of 28.3, 46.9 and 56.1 correspond to (1 1 1), (2 2 0) and (3 1 1) planes for cubic ZnS. The particle size was calculated using Debye-Scherrers formula, which is:

Figure 6:

High-resolution transmission electron microscopy (HRTEM) images of zinc sulfide nanoparticles (A–C) from lower to higher magnifications; SAED pattern (D); particle size distribution graphs (E).

Figure 7:

High resolution scanning electron microscopy (HR-SEM) images of zinc sulfide nanoparticles at different magnifications (A–C); (D) EDX analysis of zinc sulfide nanoparticles; particle size distribution graphs (E).

Figure 8:

Powder X-ray diffraction (XRD) pattern of zinc sulfide nanoparticles synthesized using the cell free extract of Chlamydomonas reinhardtii.

where D represents mean crystallite size, λ is the wavelength of the copper target that was used, β represents the full width half maximum of the peak and θ is the diffraction angle. The particle size was found to be ~7 nm. The broadness of the peaks indicates the relatively small particle size.

DLS is a technique that depends on the interaction of light with particles [29]. The hydrodynamic size of the particles was determined using DLS and was found to be 68 nm for the aqueous preparation of ZnS nanoparticles (Figure 9A). Zeta potential is an indicator of the stability of the nanoparticle solution. For ZnS nanoparticles, it was measured to be -21.1 mV indicating its relative stability (Figure 9B). The large variation in sizes reported by DLS and HRTEM could be due to the capping agent that surrounds the nanoparticle. Further, HRTEM and XRD measure the inorganic core of the particle, since the organic shell is less electron dense in comparison to the inorganic core and thus transparent in HRTEM and XRD. This may account for the variation in measurements made using these techniques [29]. Zeta potential is indicative of both the surface charge and the local environment surrounding a nanoparticle. The proteins adsorbed on the surface of the nanoparticle may influence its zeta potential. There is a correlation between the amount of protein adsorbed and zeta potential [30]. FTIR analysis of the ZnS pellet revealed strong bands at 1630 cm^-1 and 1540 cm^-1 corresponding to amide I and amide II vibrational bands (Figure 10). The band at 1102 cm^-1 corresponded to the C-N stretch of aliphatic amines. The peak at 617 cm^-1 could be assigned to ZnS (i.e. sulfides) [31]. The broad band at 3220 cm^-1 could be assigned to O-H stretching. The peaks at 2850 cm^-1 and 2917 cm^-1 corresponded to C-H stretches in alkyl groups.

Figure 9:

Hydrodynamic size distributions of zinc sulfide nanoparticles with an average hydrodynamic radius of 68 nm as measured by dynamic light scattering (DLS) (A); zeta potential of zinc sulfide nanoparticles synthesized using Chlamydomonas reinhardtii cell free extract (B).

Figure 10:

Fourier transform infrared (FTIR) spectrum of zinc sulfide nanoparticles synthesized using algal cell free extract.

Earlier reports have observed that the carbonyl groups present in proteins have a strong affinity to metals and may thus mediate capping. Cyclic peptides present in plants have also been shown to be responsible for nanoparticle synthesis and stabilization [19]. Bacteria that synthesized ZnS nanoparticles were found to possess sulfate reduction systems, namely, enzymes like sulfate permease, ATP sulfurylase etc [18].

It is still unclear whether microalgae possess a similar sulfate reduction system, therefore to get an idea about the algal proteins involved in the formation of these nanoparticles, the sample was boiled with 1% SDS solution for 10 min in a water bath, following which it was run in a 12% SDS polyacrylamide gel. SDS is a detergent which helps in linearizing the polypeptides and facilitates their detachment from the surface of the nanoparticle. An intense band at approximately ~30 kDa was observed in Figure 11 and this could likely be oxygen enhancing evolver protein (OEE) 1. A few faint bands below 27 kDa were also observed and it is possible that these could be other oxygen evolving enhancer proteins (proteins 2 and 3) Another study that used the same algae in the synthesis of silver nanoparticles got similar results when they identified the proteins using MALDI MS-MS. These proteins are usually localized in the chloroplast and are frequently produced in response to stress especially from metals. Between 27–59 kDa, three faint bands were observed and these could be ATPases, carbonic anhydrase or sedoheptulose-1,7-bisphosphatase. ATPase or ATP synthase is a membrane bound ion transporter and like oxygen evolving enhancer proteins, is produced in response to metallic stress. Sedoheptulose-1,7-bisphosphatase is capable of capping the nanoparticles with its free -SH group that is produced by thioredoxin-mediated reduction [32]. All of these proteins form a major part of the oxidoreductive machinery in the algal cell and are likely to play a pivotal role in the synthesis and stabilization of the semiconductor nanoparticles. Further analysis would be required to specifically identify the proteins involved in chalcogenide nanosynthesis. However, the use of the same microalgae has been implicated in the synthesis of silver and gold nanoparticles and may play a similar role in this case with these proteins in fact acting as the capping agents.

Figure 11:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis of proteins bound to ZnS nanoparticles using 1% SDS solution. Lane 1 corresponds to the molecular weight markers. Lane 2 represents the protein profile of proteins that were bound to zinc sulfide nanoparticles after boiling with 1% SDS solution.

Based on these findings, we propose a mechanism as seen in Figure 12, by which C. reinhardtii is able to synthesize ZnS nanoparticles. Initially, the oxygen evolving enhancer proteins act as a reducing agent; these proteins exhibit thioredoxin activity and their thiol groups are involved in capping the nanoparticles [33]. These proteins possess two cysteine residues; the thiol group of one residue forms a transient heterodisulfide linkage with zinc sulfate, the second cysteine residue then attacks this linkage and releases the reduced ZnS nanoparticles [34]. Sedoheptulose-1,7- bisphosphatase may work in conjunction with these proteins to help in the capping process. The other proteins that are part of the algal redox machinery are likely to play a crucial role in the stabilization of these nanoparticles. The nanoparticle-protein corona is highly dependent on physicochemical features of the nanoparticle as well as the proteins that are bound to it. The binding of proteins on the surface of uncoated nanoparticles was found to considerably increase nanoparticle stability and this has been attributed to the increase in hydrophobic interactions as well as greater entropic gain [35]. Thus, based on these preliminary findings we can recognize the fundamental role of algal proteins in the biosynthesis of ZnS nanoparticles. Further studies into the specific role of these proteins and their interactions would help exercise greater control over the size and functionality of these nanoparticles.

Figure 12:

Mechanism describing the synthesis and stabilization of zinc sulfide nanoparticles by algal biomolecules.

4 Conclusion

This is the first report to describe a facile, low cost method to produce a large number of stable ZnS nanoparticles using the green algae C. reinhardtii. A large number of spherical ZnS nanoparticles measuring 8–12 nm were observed using HRTEM. Elemental analysis of the sample indicated that they contained zinc, sulfur and other elements that may be representative of the proteinaceous material capping the nanoparticles. The UV-visible, PL spectra emphasize the unique optical properties of these nanoparticles. Structural analysis by XRD showed the formation of pure ZnS nanoparticles with FCC structure. Analysis of proteins bound to the surface of the nanoparticles hinted at the pivotal role played by the oxidoreductive machinery of the microalgae.