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Sustainable-Resource-Based Carbon Dot−Silver Nanohybrid: A Strong Tool against Culex quinquefasciatus, a Common Disease Vector Shaswat Barua, Prasanta K. Raul,* Reji Gopalakrishnan, Bodhaditya Das, Vanlalhmuaka, and Vijay Veer

ACS Sustainable Chem. Eng. 2016.4:2345-2350. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/20/19. For personal use only.

Defence Research Laboratory, DRDO, Post Bag No. 2, Tezpur-784001, Assam, India ABSTRACT: With the evolution of material science, researchers are deeply concerned about the utility of sustainable resources for multifaceted advanced applications. Here we project an abundant, nonedible bioresource-based carbon dot−silver nanohybrid as a highly competent larvicidal agent against Culex quinquefasciatus. Mosquitoes have been the closest enemy of humankind for a very long time. Tropical areas around the globe suffer severe ailments due to mosquitovector-borne diseases. Japanese encephalitis, lymphatic filariasis, etc. are such fatal threats spread by Culex species. With the emergence of nanotechnology, the perspectives of conventional antivector materials have changed dramatically. The C-dot precursor used here was the roots of Cyperus rotundus, a very abundant grass species found in South Asian countries. The nanohybrid was synthesized by a simple thermal approach without the application of additional reducing agent. The nanohybrid was distributed within a narrow size window of 35% carbohydrate, with ipolamiide being predominant.21,25 Formation of C-dots from carbohydrate sources through hydrothermal methods has been reported in the literature.19It has been proposed that during the first phase of the process, hydrolysis and subsequent decomposition of the carbohydrates takes place, yielding different aldehydes, ketones, and carboxylic acids. Aromatization and carbonization of such products followed by nuclear bursts result in the C-dots.26 The C-dots thus generated contain a large number of surface hydroxyl groups. Such structures have a high affinity to form complexes with metal ions. This involves the release of a considerable number of protons and electrons, as reported in the literature.21,27,28 The released electrons can in turn help to reduce to Ag+ ions to Ag0. In this process, the simultaneous nucleation of silver and C-dots helps to generate the watersoluble CDS. Figure 1 schematically shows the overall process. Characterization. Simultaneous generation of C-dot and silver nanoparticles was evident from the UV−vis spectrum shown in Figure 2a. The C-dot UV absorption peak was

EXPERIMENTAL SECTION

Materials. C. rotundus roots were collected from the garden of the Defence Research Laboratory, Tezpur, Assam, India. They were sundried and ground to fine powder using a domestic blender. AgNO3 was procured from Merck, India and used as received. Preparation of the C-dot−Silver Nanhohybrid. C. rotundus root powder (2 g) was heated with 100 mL of water at 50 °C for 1 h. Then the extract was filtered using a muslin cloth. To the extract (50 mL) was added 0.169 g of AgNO3, and the mixture was stirred in a conical flask, firmly closed by a cotton plug, for 10 min at room temperature. The flask was then autoclaved for 2 h at 120 °C under a pressure of 15 psi. After the termination of the reaction time, the product was allowed to cool to room temperature. The dark-brown product thus obtained was the C-dot−silver nanhohybrid (CDS). This was then filtered and centrifuged at 960g (3000 rpm) for 10 min to discard the larger particles.19 Water was evaporated subsequently at room temperature to obtain the desired nanohybrid. The dried nanohybrid was then dissolved again in water to make a 1000 mg/L stock solution. Subsequent dilution of the stock solution yielded the required concentrations. Characterization. The formation of CDS was preliminarily evidenced with the help of a UV−vis spectrophotometer (U2001, Hitachi, Japan). Chemical functionalities associated with the nanohybrid were identified on the basis of FTIR spectra recorded on an FTIR spectrophotometer (Impact 410, Nicolet) after mixing with KBr pellets. The shape and size accord and the morphology of the hybrid were studied by a high-resolution transmission electron microscopy (HRTEM) on a JEOL microscope at an operating voltage of 200 kV. 2346

DOI: 10.1021/acssuschemeng.6b00015 ACS Sustainable Chem. Eng. 2016, 4, 2345−2350

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ACS Sustainable Chemistry & Engineering

Figure 2. (a) UV−vis spectra of CDS after 2 h of reaction and (b) FTIR spectrum of CDS.

eration. This implies that the C-dots imparted an excellent stabilizing effect to the particles. Previously a few biocompatible, bioderived materials such as chitin, chitosan, etc. were also used to stabilize silver nanoparticles, which enhanced their antimicrobial activity.29 However, because of the low contrast on the substrate, C-dots are poorly visible in the images. HRTEM and inverse fast Fourier transform (IFFT) images ascertained the layer spacing of a representative C-dot to be 0.32 nm (Figure 3d), which resembles most of the values reported in the literature.20 It was further noted that the diameters of the C-dots in close proximity to silver nanoparticles were larger than those of the unattached C-dots. This may be attributed to the high surface energy of the particles, which induces agglomeration within them. In Figure 4, red and blue borders clearly differentiate the

observed at 253 nm and is due to the n−π* transition of the CO bond. Oxygenaceous moieties present in the C-dot structure aided the growth of the peak. The typical surface plasmon resonance peak for silver nanoparticles was visible at 418 nm and is attributed to collective oscillation of the electron gas at the nanoparticle surface.19 Surface functionalities of C-dots are primarily responsible for their excellent water solubility. The FTIR spectrum (Figure 2b) showed the presence of different functional groups on the CDS surface. The bands at 3416, 2929, 1680, 1632, 1400, and 1060 cm−1 clearly indicate the existence of OH, C−H, CO, CC, C−O−C, and C−O groups in the CDS structure.19,25 TEM images (Figure 3a−c) clearly show that the C-dots were formed with very small diameters along with the comparatively larger silver nanoparticles. The diameters of the silver nanoparticles ranged from 5 to 18 nm, while those of the C-dots were 2−6 nm. Both types of particles were uniformly dispersed throughout without any sign of agglom-

Figure 4. Representative HRTEM images of C-dots (blue lines) and silver nanoparticles (red lines) held together by strong interactive forces.

metallic silver nanoparticles and C-dots, respectively. The overall TEM studies confirmed that the two types of particles were held together by strong interactive forces that mutually stabilize each other.16 Larvicidal Activity of the Nanohybrid. To investigate the effect of CDS, bioassays were carried out against C. quinquefasciatus mosquito larvae. The results indicated that the LC50 and LC90 values (95% fiducial limits) lie between 0.05 (0.045−0.055) and 0.211 (0.182−0.250) mg/L, respectively (Table 1). Silver nanoparticles synthesized using Tinospora cordifolia extract showed an LC50 of 6.34 mg/L against Anopheles subpictus, while Nelumbo nucifera extract-mediated ones showed an LC50 of 0.69 against C. quinquefasciatus.30,31

Figure 3. (a−c) TEM images of CDS. (d) HRTEM image of a C-dot. The inset is an IFFT image of the C-dot, showing the layer spacing. 2347

DOI: 10.1021/acssuschemeng.6b00015 ACS Sustainable Chem. Eng. 2016, 4, 2345−2350

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ACS Sustainable Chemistry & Engineering

Table 1. Dose-Dependent Larvicidal Activity of CDS As Indicated by Values of LC50 and LC90 against C. quinquefasciatus Mosquito Larvae dose (mg/L) 0.4 0.3 0.2 0.1 0.05 0.04 0.03 0.02 0.01

mortality (% ± SEmean) 100 92.95 83.08 69.92 56.28 42.19 32.38 21.49 7.80

± ± ± ± ± ± ± ±

LC50 (mg/L) (95% fiducial limits)

LC90 (mg/L) (95% fiducial limits)

0.05 (0.045−0.055)

0.211 (0.182−0.250)

4.90 2.18 5.42 4.10 3.44 4.48 3.29 3.73

However, these works used the crude plant extracts, which also exhibited larvicidal activity. Despite the number of literature reports based on larvicidal silver nanoparticles, it is rare to find such commercial products. The vital reasons may be the toxicity of the nanoparticles and their poor stability over the course of time. Thus, the present nanohybrid can be used to overcome both of these demerits. The highly biocompatible Cdots also act as a stabilizing agent for the silver nanoparticles by steric hindrance. During the experiment it was noted that the dead larvae turned faint black in color and settled at the bottom of the vessel (Figure 5a). Optical microscopy images taken at 36×

Figure 6. SEM images of (a) a representative control larva, (b) a CDStreated larva showing the disruption of the cuticular layer and intestine, (c) normal anal segments of a representative control larva, and (d) deposition of CDS onto the anal segments of a treated larva.

Anal segments were also found to be denatured in the CDStreated larvae. Deposition of the nanomaterials is clearly visible in the SEM image (Figure 6d). Thus, it is quite obvious from the optical microscopy and SEM studies that treatment with CDS completely destroyed the normal metabolic system in the Culex larvae. Silver nanoparticles can bind to compounds containing sulfur or phosphorus (e.g., DNA), which denatures the same or inhibits enzyme activities, consequently diminishing the membrane permeability. Thus, alteration of the proton motive force occurs, and the larval membrane gets damaged.32The most fascinating part of the study lies in the effective concentration of the nanohybrid applied for the larvicidal activity. A CDS concentration of only 0.05 ppm showed LC50, which may be regarded as a remarkable achievement in controlling C. quinquefasciatus, the notorious vector of many life-demanding diseases. Stability of the CDS. Environmental applications of nanomaterials demand the study of their stability. Accordingly, the stability of the present nanohybrid was monitored for 45 days under ambient conditions of temperature, pressure, and light by UV−vis spectroscopy. The spectra showed that the material was quite stable in dispersed form for more than 30 days from preparation (Figure 7a). It is clearly visible from the figure that on the 45th day of preparation, the characteristic UV absorption peak at around 418 nm broadens, indicating agglomeration of the nanoparticles.7 Thus, CDS may be endorsed as an effective larvicidal material in drains and

Figure 5. (a) Representative image of the experiment, showing where dead larvae settled. The inset shows a few dead larvae. (b) Optical microscopy image of a control larva and a CDS-treated larva, showing the accumulation of CDS within the larval body. (c, d) EDX spectra of representative (c) control and (d) CDS-treated larva.

zoom showed that the nanohybrid was accumulated by the treated larvae. The head, intestine, and gut were visibly black (Figure 5b). Contrarily, the control larva retained the normal stature. This affirmation was further supported by the EDX studies (Figure 5c,d), which showed the presence of up to 2.86 atom % silver in the treated larva. Similar observations were made in the SEM study. The SEM image of the control larva showed normal organization,8 whereas the treated larva faced severe damage of the cuticular membrane (Figure 6a,b). This allowed the intrusion of the nanohybrid into the larval body and subsequently ruptured the cellular organization of the gut and intestine. 2348

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Figure 7. (a) UV−vis spectra showing the stability of CDS on the 1st, 15th, 30th, and 45th days after preparation. (b) TGA thermogram of CDS.

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stagnant water bodies that would be stable against different environmental factors such as temperature, photoinduced oxidation, etc. Moreover, the thermal stability of CDS was another interesting study, which revealed that the onset degradation temperature of the nanohybrid is around 195 °C (Figure 7b; the TGA thermogram was analyzed by a Shimadzu TG 50 analyzer under a constant nitrogen flow (30 mL min−1) at a heating rate of 10 °C min−1). The same for silver nanoparticles and C-dots alone were reported to be 160 and 110 °C respectively.33,34 Thus, formation of the nanohybrid synergistically increased the thermal stability of the individual nanomaterials.

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CONCLUSION The utility of a sustainable-resource-based stable nanohybrid to control C. quinquefasciatus larvae was projected in the present investigation. The work demonstrated a facile method for the preparation of a C-dot−silver nanohybrid that may be adopted easily. The material was extremely effective at a concentration of only 0.05 ppm and stable against different environmental factors such as temperature, photoinduced oxidation, etc. The union of larivicidal silver nanoparticles and bioresource-based C-dots may permit the sustained utility of the nanohybrid for actual field applications after the toxicity accords are critically scrutinized.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

All of the authors significantly contributed to the reported work. The final version of the manuscript was approved by all of the authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to DBT, India for financial assistance through Grant BT/518/NE/TBP/2013 dated Dec 12, 2014. DRL, Tezpur and DRDO, New Delhi are duly acknowledged for providing research platform. SAIF, NEHU, Shillong is acknowledged for TEM imaging. 2349

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DOI: 10.1021/acssuschemeng.6b00015 ACS Sustainable Chem. Eng. 2016, 4, 2345−2350