Antimicrobial activity, cytotoxicity and DNA binding studies of carbon dots

https://doi.org/10.1016/j.saa.2018.02.030Get rights and content

Highlights

  • The stability of TCDs was investigated under different experimental conditions such as effect of pH, NaCl and irradiation time.

  • The biocompatible nature of TCDs was confirmed by an in vitro cytotoxicity test on L6 normal rat myoblast cells and MCF 7 human breast carcinoma cells using MTT assay.

  • Antimicrobial activity of TCDs is also demonstrated with bacterial and fungal cells.

  • Intercalation type of interaction between TCDs and calf thymus-DNA was indentified leads specific detection of DNA.

  • As a consequence of the work, we believed that TCDs can be used as thernastic agent for biological applications such as bioimaging, targeted drug delivery and antimicrobial agent.

Abstract

In recent years, quantum dots (QDs) are one of the most promising nanomaterials in life sciences community due to their unexploited potential in biomedical applications; particularly in bio-labeling and sensing. In the advanced nanomaterials, carbon dots (CDs) have shown promise in next generation bioimaging and drug delivery studies. Therefore the knowledge of the exact nature of interaction with biomolecules is of great interest to designing better biosensors. In this study, the interaction between CDs derived from tamarind and calf thymus DNA (ct-DNA) has been studied by vital spectroscopic techniques, which revealed that the CDs could interact with DNA via intercalation. The apparent association constant has been deduced from the absorption spectral changes of ct-DNA−CDs using the Benesi-Hildebrand equation. From the DNA induced emission quenching experiments the apparent DNA binding constant of the CDs (Kapp) have also been evaluated. Furthermore, we have analyzed the antibacterial and antifungal activity of CDs using disc diffusion assay method which exhibited excellent activity against E. coli and C. albicans with inhibition zone in the range of 7–12 mm. The biocompatible nature of CDs was confirmed by an in vitro cytotoxicity test on L6 normal rat myoblast cells by using MTT assay. The cell viability is not affected till the high dosage of CDs (200 μg/mL) for >48 h. As a consequence of the work, future development of CDs for microbial control and DNA sensing among the various biomolecules is possible in view of emerging biofields.

Introduction

Contemporary advances in carbon based nanomaterials have allowed for a significant number of applications in the state of the art fields such as medicinal and biological sciences [1]. They have been emerged as novel materials for diverse applications such as bioimaging, biosensing, drug delivery and antimicrobial fields owing to their unique properties such as facile and economic synthesis, high water solubility, biocompatibility, rich in fluorescence quantum yield, excitation dependent/independent emission nature, surface defects for interaction with others molecules and possible to surface passivation etc. [2,3]. Carbon based nanomaterials have been synthesized by both top-down (laser ablation, chemical vapour deposition etc.) and bottom-up (hydrothermal, electro deposition, solvothermal etc.) approaches from wide range of precursors of chemical as well as green resources [[4], [5], [6], [7], [8],10].

There are numerous reports available for the binding studies of DNA with various metal-ligand complexes using as a fluorescent probe [[11], [12], [13]]. The combination of carbon nanomaterials and biological molecules has attracted tremendous attention, where the former could present a versatile nanoscale interface for biomolecular recognition. We could find only a less details about the DNA interaction studies with carbon based materials those has also not been discussing the specific mechanism for the type of interaction. For instance, Nandi et al. explored the sensing of DNA from other biomolecules using sulphur functionalized graphene - EthBr template [14]. Some researchers have reported the specific interaction between nanoparticles and DNA or protein as well as other biomolecules. In 2009, Kathiravan and his co-worker reported the photoinduced interaction between colloidal TiO2 nanoparticles with ct-DNA by spectroscopic techniques and explored the binding constant as well as binding sites [15].

Development of new antimicrobial agents is necessary due to rise in the rate of infection by antibiotic-resistant microorganisms. The direct interaction between carbon based materials like graphene, reduced graphene oxide and graphene oxide with various cells such as bacterial and fungal has been frequently documented by eminent scientists [16]. In particular, extensive efforts have been devoted to exploring the broader applications of carbon based nanomaterials to microbial activities based on their electronics, optics with unique structure and biocompatible, non-toxic nature, economic and facile synthesis etc. [17,18]. Investigations on the antimicrobial activities of carbon dots are increasing in recent years which explore several mechanisms including oxidative stress and wrapping etc. [19,20]. There are only countable numbers of reports in the literature for the effective utilization of carbon dots for both antimicrobial activities and DNA binding studies. For instance, Meziani et al. reported [21] that bactericidal functions of carbon quantum dots derived from functionalization of commercial carbon nanopowder which inhibited the growth of E. coli. Thakur et al. [22] explored the synthesis of carbon dots from gum arabic and found that the bare C-dots did show potential antibacterial activity towards both gram positive and gram negative bacteria. Chun et al. [23] accounted the significant contribution of the nanocarbon present in turmeric smudge towards its antibacterial activity especially for E. coli.

Even though abundant investigations on carbon dots for various applications have been reported, systematic analysis on their antimicrobial property is still lacking. But there are extensive reports on the antimicrobial property of other carbonic nanomaterials such as graphene, graphene oxide, reduced graphene oxide, carbon nanotubes, fullerenes etc. have been made [16]. Moreover, evidences from the literature inferred that the antimicrobial activities of cabon based materials are not same which depends on the structure of the material [24].

Recently we have reported the synthesis of novel fluorescent carbon dots from tamarind (TCDs) for the study of photoinduced interactions towards the applications of energy conversion devices [25]. In that we have reported complete synthesis and characterization of the TCDs which exhibited the properties such as excellent water solubility, storage stability and 4% quantum yield with the particle size of 1–3 nm. The carbon dots are pH sensitive with negatively charged functional groups present on the surface. The reported TCDs demonstrated the unique nature of excitation dependent emission with the average lifetime of 3.62 ns.

Feng et al. reported the first example that photoluminescent carbon dots can induce right-handed B-DNA to left-handed Z-DNA under physiological salt conditions with sequence and conformation selectivity. Further studies indicate that carbon dots would bind to DNA major groove with GC preference. Inspired by carbon dots lighting up Z-DNA and DNA nanotechnology, several types of DNA logic gates have been designed and constructed based on fluorescence resonance energy transfer between photoluminescent carbon dots and DNA intercalators [26]. So with this introduction we aimed to study the antimicrobial activity of TCDs material and interested to analyze the DNA binding study of carbon dots (TCDs) with DNA- ethidium bromide (EthBr) system which will enable us to understand the type of interaction between the carbon dots with biomolecules. To the best of our knowledge, there are not many reports available in literature for the use of carbon dots derived from tamarind (TCDs) for both antimicrobial activity and DNA binding studies. We hope that this report will help the reader for effective usage of carbon nanomaterials as antibacterial and antifungal agents and furthermore for various biological studies as theranostic agent such as targeted delivery of drugs, cancer cell imaging and biosensors etc.

Section snippets

Materials and Methods

TCDs are synthesized by a reported method [25]. ct-DNA and ethidium bromide were purchased from Sigma–Aldrich and used as such without further purification. Milli-Q water was used to prepare the samples for spectral measurements.

DNA Binding Studies

DNA stock solution is prepared by dissolving 25 mg of ct-DNA in 10 mL of Milli-Q water with Tris (5 mM) and sodium chloride (50 mM) (pH is adjusted to 7.2 with hydrochloric acid), which is kept in the fridge for overnight. Further the solution was subjected to mild

Confocal Microscopy

Total volume 200 μL (1:1 v/v) of TCDs was mixed with C. albicans culture (fast growing stage) and kept in orbital shaker for 2 h at 22 °C. After that, fungal cells were centrifuged at 3000 rpm for 5 min and washed with PBS (pH 7.2) twice to remove the unessential TCDs. Approximately, 25 μL of solution was placed and spread onto the thin glass slide and mount in laser confocal microscope (Carl Zeiss, LSM 710, Germany). Morphology of the C. albicans cells was observed and laser images were collected

Stability of TCDs

Experimental procedure for the synthesis of TCDs and material characterizations were recently reported [25]. Before using TCDs in biological applications, it is necessary to check the stability by various parameters such as effect of pH, NaCl and irradiation time on the fluorescence intensity of TCDs. Fig. 1 shows that the effect of pH (3–11) on the fluorescence intensity of TCDs. The maximum emission intensity is observed at neutral pH, however, the intensity is not much varied at acidic as

Conclusion

For the first time we have reported the combined study of DNA binding and antimicrobial activity of carbon dots derived from tamarind as resource. Affinity of TCDs with DNA occurred via intercalation mode and there is true quenching of DNA-EthBr system in presence of TCDs. Competition between TCDs and EthBr to get bind with DNA is one of the significant reasons for quenching. Furthermore TCDs is used as effective antibacterial agent for E. coli bacteria. Moreover biocompatible and fluorescent

Acknowledgements

M. A. J thanks to DST-SERB, India for the project [Ref. No. SB/FT/CS-125/2013, Dt. 30/06/2014]. A. K thanks to Department of Science and Technology, India for DST-INSPIRE Faculty Award [IFA12-CH-78]. D. A. A and T. S thank DST-PURSE (DST Sanction Order No-SR/FT/LS-113/2009) for providing confocal facility. This work was also supported by grants from Science and Engineering Research Board, Dept. of Science and Technology (Ref No. DST-SERB/LS-248/2013) and Dept. of Biotechnology, Govt. of India

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