Abstract
Nowadays, bioactive nanomaterials have been attracted the researcher’s enthusiasm in various fields. Herein, Diplocyclos palmatus leaf extract-derived green-fluorescence carbon dots (DP-CDs) were prepared using the hydrothermal method. Due to the strong fluorescence stability, the prepared DP-CDs were coated on filter-paper to make a fluorometric sensor-strip for Fe3+ detection. After, a bandgap-narrowed DP-CDs/TiO2 nanocomposite (DCTN) was prepared using the methanolic extract of D. palmatus. The prepared DCTN exhibited improved photocatalytic bacterial deactivation under sunlight irradiation. The DCTN-photocatalysis slaughtered V. harveyi cells by the production of reactive oxygen species, which prompting oxidative stress, damaging the cell membrane and cellular constituents. These results suggest the plausible mode of bactericidal action of DCTN-photocatalysis under sunlight. Further, the DCTN has shown potent anti-biofilm activity against V. harveyi, and thereby, DCTN extended the survival of V. harveyi-infected shrimps during the in vivo trial with Litopenaeus vannamei. Notably, this is the first report for the disinfection of V. harveyi-mediated acute-hepatopancreatic necrosis disease (AHPND) using nanocomposite. The reduced internal-colonization of V. harveyi on the hepatopancreas as well as the rescue action of the pathognomonic effect in the experimental animals demonstrated the anti-infection potential of DCTN against V. harveyi-mediated AHPND in aquaculture.
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Introduction
The tainting of water bodies by heavy metals has become a severe ecological threat globally1. The development of exceedingly selective sensors has been attracting vast welfare for scrutinizing heavy metals due to their substantial environmental impacts because of severe toxicity and mobility2. Among various heavy metals, Iron (Fe3+) is the third most abundant element on the earth and considered as a crucial transition metal in cellular systems, which plays an important role in biological systems3. Furthermore, both deficiency and overabundance of Fe3+ are associated with serious diseases in humans4. Normally, this metal can be detected through various analytical techniques, including atomic absorption spectroscopy (AAS), mass spectrometry (MS), emission spectroscopy (ES), gas chromatography (GC), and voltammetry. However, these techniques are expensive, time-consuming, and inaccessible worldwide, particularly in developing countries5. Therefore, some fluorescent sensors, such as organic dyes, quantum dots (QDs), metal nanoclusters, and metal–organic framework probes have been reported for metal sensing6. Among these sensors, QDs have great attention due to their high aqueous solubility, robust chemical inertness, easy functionalization, high resistance to photo-bleaching, low toxicity, and good biocompatibility7. In particular, carbon quantum dots (CQDs) are innovative semiconductor carbon nanoparticles with a size of less than 10 nm that are mostly self-processed with sp2 carbon, oxygen, and other doped heteroatoms8. CQDs have pulled in expanding consideration in various fields because of their exceptionally stable fluorescence, photo-stability, quantum yield, low cost, water solubility, and positive biocompatibility9. Further, CQDs are highly established fluorescent-materials as promising candidates for a wide scope of potential applications, including metal sensors, bio-imaging, photocatalysts, and optoelectronic devices10. Due to the environmental compatibility, the CQDs emerge to be the best alternative to Cd2+/Pb2+-based QDs in electroluminescent LEDs and also considered as next-generation luminophore for future LEDs11. In addition, CQDs are exclusively utilized for glucose sensing, DNA testing, and dopamine sensing through electrochemical approach12. In recent, CQDs are used as efficient fluorophores for high-performance tandem-luminescent solar concentrators13,14,15.
A variety of methods has been utilized to synthesize CQDs, including hydrothermal, electrochemical, microwave, discharge-emission, plasma-treatment, and laser-ablation mediated synthesis16. Among these, hydrothermal method offers several advantages such as relatively one-step synthetic procedure, inexpensive, environmental friendliness, and high dispersion in solution4. In the present study, a biocompatible CQDs were synthesized from the aqueous leaf extract of Diplocyclos palmatus through hydrothermal approach. D. palmatus is a creeper-type tropical medicinal plant, mainly distributed in Malaysia, Australia, Africa, South China, and India. In previous, our group has been reported their protective efficacy on Serratia marcescens infection as well as anti-photoaging property using Caenorhabditis elegans model17.
On the other hand, the bacterial contamination in water bodies is one of the critical human health issues around the world18. Similarly, the bacterial contamination in aquaculture has practiced relatively many bacterial diseases owing to the lack of suitable treatment, resulting in cause serious economic loss and affecting sustainability of aquaculture industries19. Importantly, Vibrio species such as Vibrio harveyi, V. vulnificus, V. parahaemolyticus, and V. alginolyticus are frequently present in the seawater, and thereby associated with a number of infectious diseases, which are affecting a wide range of aquatic creatures including fishes, crustaceans, and molluscs20. Notably, V. harveyi is a gram-negative serious pathogen can able to cause several diseases to fish and shrimp, particularly Penaeus monodon and Litopenaeus vannamei21. This bacterium has well renowned to cause diseases such as acute hepatopancreatic necrosis disease (AHPND), vibriosis, septic hepatopancreatic necrosis (SHPN), skin ulcer, gastroenteritis, vasculitis, Bolitas negricans, eye lesions, and deep dermal lesions22. The biofilm formation and virulence factor production by bacterial pathogens attributed to their pathogenesis, which regulated by cell-density dependent gene expression, known as quorum sensing23. Therefore, antibiotics have been used to control bacterial infections in aquaculture so far. However, as the development of drug-resistance in bacterial pathogens due to the frequent use of antibiotics, a promising alternative approach is needed to control bacterial diseases in aquaculture24.
TiO2-based photocatalysis has offered an innovative platform to annihilate bacterial contamination in the water bodies using light irradiation25. However, the practical applications of TiO2 in solar-light is confined because of the wide bandgap (~ 3.2 eV), which requires UV-light (λ ≤ 387 nm) for its activation26. Therefore, modulating the bandgap absorption to the visible region is needed for practical functioning under direct sunlight26. Since UV-light comprises only 4–5% of the solar spectrum, whereas 40% of solar-light is in the visible region27. Hence, in the present study, a bandgap-narrowed carbon dots/TiO2 nanocomposite (DCTN) was prepared using the D. palmatus methanolic extract for photocatalytic bactericidal applications. Due to the antibiofilm behaviour against V. harveyi, the prepared nanocomposite also subjected to evaluating for its anti-infection potential against acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei for valuable application in aquaculture.
Results and discussion
Structural and optical properties of synthesized DP-CDs
In Fig. 1a, the HR-TEM analysis revealed the structural morphology of the prepared DP-CDs, wherein DP-CDs were spherical with a uniform size. Then, DLS analysis showed that the DP-CDs were mostly eight nanometers size (Fig. 1b). In Fig. 1c, the SAED pattern of an individual particle indicated the crystallinity of the DP-CDs with a lattice parameter of 0.347 nm, corresponding to the (002) diffraction plane of graphitic (sp2) carbon28. XRD analysis revealed a broad (002) peak at 26.13° 2θ with a d-spacing of 3.49 Å, which confirmed the graphitic nature of the DP-CDs (Fig. 1d). In addition, the Raman spectrum characterized the defects or disorders in DP-CDs (Fig. 1e). The relative intensity of the D-band (amorphous) and G-band (crystalline) was ID/IG = 0.842, which showed that the synthesized DP-CDs were partially amorphous because of the presence of hydroxyl functional groups. The FT-IR spectrum showed the functional groups present on the surface of DP-CDs in Fig. 1f. The characteristic absorbance peaks at 3389 cm–1, 2925 cm–1, 1718 cm–1, 1619 cm–1, 1405 cm–1, 1181 cm–1, and 618 cm–1 were detected on the surface of the DP-CDs, indicating the presence of –OH stretching, C–H stretching, C=O stretching, C=C stretching, C–N stretching, C–O stretching, and C–OH vibrations, respectively29. In addition, XPS was used to examine the constituents of the surface functional groups of DP-CDs. In Fig. 1g, the high-resolution C1s XPS spectrum of DP-CDs was deconvoluted into four peaks at 284.7 eV, 285.1 eV, 285.6 eV, and 287.1 eV, which were assigned to C=C/C–C, C–N, C–O–C, and C=O/C–O bonds, respectively. The N1s spectrum displayed a peak at 400.7 eV, which denotes the pyrrolic N atoms (Fig. 1h). The O1s spectrum revealed two peaks at 532.4 eV and 535.1 eV, which indicates the presence of C=O and C–O (Fig. 1i). Moreover, the obtained FT-IR and XPS data reveal the surface wettability of the prepared DP-CDs. The presence of hydroxyl and carboxyl (hydrophilic) groups in the surface of DP-CDs, which makes surfaces super-hydrophilic nature30,31,32. Hence, the hydrophilic surface-wettability nature of DP-CDs is the reason for superior water solubility in aqueous solution33. Further, it is known that when plant extracts are hydrolyzed at high temperature, intermolecular and/or intramolecular dehydration will occur between the –OH, –COOH, and –H groups present in leaf extract and further polymerization, carbonization, and aromatization processes will lead to the formation of aromatic sp2 carbon34.
In Fig. 2a, the UV–Vis absorption spectrum revealed a peak centered at approximately 270 nm, which was assigned to the π–π* transitions of aromatic C=C bonds in sp2 hybridization, and is a fingerprint of CQDs structures28. The fluorescence (FL) intensity of the DP-CDs under different excitation wavelengths, ranging from 360 to 450 nm, was measured. The obtained spectra demonstrated the maximum emission of 520 nm at 420 nm, which reveals the excitation-dependent emission of DP-CDs (Fig. 2b, c). Furthermore, the quantum yield of the DP-CDs was 31.4%. In addition, the stability of the DP-CDs under various pH conditions, high ionic strengths, different temperatures and various solutions was examined. The obtained result shows that the DP-CDs exhibited invariable FL intensity under the pH range of 2–12 (Fig. 2d). Similarly, DP-CDs maintained their intensity while increasing the ionic strength (Fig. 2e). In addition, the FL stability of DP-CDs under different temperatures as well as various solvents was analyzed. As shown in Fig. S1, the DP-CDs exhibits reduced-FL intensity while increasing temperature, which indicated the temperature-dependent FL emission of the DP-CDs. Moreover, the DP-CDs have stable FL emission under various solutions such as water, methanol, ethanol, acetone, chloroform, and petroleum ether (Fig. S2). It shows that the high FL emission was observed in water as well as methanol solution compared to other solutions. On the other hand, the DP-CDs showed a constant intensity even up to 1 h of UV-irradiation, whereas the well-known FL probe, fluorescein isothiocyanate (FITC) showed a decrease in intensity (Fig. 2f), which confirmed the photo-stability of the DP-CDs. In addition, the DP-CDs exhibited stable FL emission on filter paper and maintained their stability for a long time, as shown in Fig. 2g.
On–off based Fe3+ sensing of DP-CDs coated fluorometric sensor-strip
Stable FL intensity of carbon dots could be useful to detect heavy metals in the form of fluorescence probe28. In order to predict the selectivity of the prepared DP-CDs in solution, the variation in FL intensity (I–I0) of DP-CDs was calculated in the presence of various metal ions (50 µM), including Cr2+, As2+, Hg2+, Zn2+, Pd2+, Fe3+, Ba2+, Cu2+, Ag+, Fe2+, and Al3+ (Fig. 3a, b). The result showed that Fe3+ abruptly quenched the FL intensity of DP-CDs and insignificant changes were observed with other ions. Then, the sensitivity of DP-CDs towards the concentration of Fe3+ was assessed. The sensing efficiency (changes in I/I0) of DP-CDs showed a good linear relationship with the Fe3+ concentrations (Fig. 3c), and the limit of detection for Fe3+ ions in solution was 10 μM. The FL emission of DP-CDs arises from radiative recombination under excitation35. Further, when binding with metal ions, the valence electrons in the metal ions that give sensitivity by forming strong interactions among the CQDs28. Herein, the high selectivity of Fe3+ ions probably due to the high affinity with the surface carboxyl groups (C–OH, C–O, and C=O) of the DP-CDs and thereby, forming a complex between Fe3+ ions and DP-CDs36. The complex formation by Fe3+ ions might affect the surface energy traps of the DP-CDs, which accelerates the non-radiative electron/hole recombination via an effective electron transfer process, and resulting cause FL quenching ultimately28, as shown in Fig. 8 Scheme-1.
Then, filter paper-based DP-CDs coated fluorometric sensor-strip was designed and made by drawing circular reaction pots using a wax liner37, as shown in Fig. 8 Scheme-2. Because, the paper-based analytical device (PAD) is a potential diagnostic tool due to its low-cost, user-friendliness, biodegradability, ease of fabrication, biocompatibility, and requirement of a smaller workforce without an external pump or power supply38,39,40. The selectivity of the prepared sensor-strip towards different metal ions was examined. Figure 3e shows digital photographs of the FL intensity of a reaction zone of a sensor-strip toward different ions. The FL intensity was captured using a Canon EOS 90D DSLR camera and quantified using Image-J software. As shown in Fig. 3d, e, similar FL quenching was observed with Fe3+ in sensor-strip, which was in agreement with our previous result. During the sensitivity trial, the intensities of the reaction zones in the sensor-strip decreased gradually in the presence of increasing concentrations of Fe3+ ions (Fig. 3f, g). The minimum detection limit was reported to be 10 µM based on visual differentiation and Image-J quantification. In conclusion, this part of the work suggests that the prepared fluorometric sensor-strip could act as an easy-handling and promising microfluidic-device for the onsite detection of Fe3+ ions.
Synthesis and characterization of DP-CDs loaded TiO2 nanocomposite (DCTN)
On the other hand, a bandgap-narrowed DP-CDs/TiO2 nanocomposite (DCTN) was successfully prepared using D. palmatus methanolic leaf-extract as a capping and stabilizing agent under hydrothermal condition. Our previous finding has reported the phytochemical profiling of D. palmatus methanolic leaf-extract using gas chromatography-mass spectrometry (GC–MS) analysis17. Based on the report, the major compounds such as palmitic acid, tocopherols, and phytol are antioxidant compounds having –OH functional groups, which acted as reducing and capping agents during the nanocomposite synthesis. In characterization studies, the FE-SEM analysis revealed the spherical shape of the prepared nanocomposite in Fig. 4a. Further, the HR-TEM analysis clearly showed that the coupled structure of the DCTN was spherical (Fig. 4b). Then, the lattice spacing of the 0.347 nm of (002) plane and 0.356 nm of (101) plane established the presence of DP-CDs and TiO2 in the nano-spheres. In addition, AFM analysis also confirmed the spherical-structure of the prepared DCTN in the 3D arrangement and revealed the size, which was ~ 78.3 nm on average (Fig. S3). The XRD patterns revealed reflections at 27.4°, 36.1°, 39.1°, 41.2°, 44.1°, 54.4°, 56.6°, 63.1°, 64.1°, and 69.8° 2θ, which were indexed to the (110), (100), (101), (200), (111), (210), (211), (220), (002), (310), and (112) crystallographic planes of rutile TiO2 (Fig. S4). This data clearly showed that doping of DP-CDs does not alter the lattice structure of TiO2 during composite preparation. In the FT-IR spectrum, the characteristic peaks at 3389 cm–1, 2925 cm–1, 1618 cm–1, 1405 cm–1, and 1181 cm–1, indicate the presence of –OH, C–H, C–O, C–N, and C–OH groups, respectively (Fig. 4c). The broad absorption below 1000 cm−1 represents the combination of Ti–O–Ti and Ti–O–C vibrations41. This suggests that coupling between the TiO2 and DP-CDs developed through these bonds. The high-resolution C1s XPS spectrum of DCTN was deconvoluted into four peaks at 280.9 eV, 283.1 eV, 284.6 eV, and 285.8 eV, which were assigned to the Ti–O–C, C–C/C–H, C=C, and C–OH/C–O–C bonds, respectively (Fig. 4d). The N1s spectrum has a peak at 399.1 eV, which represents the C–N bonding (Fig. 4e). In the Ti 2p high-resolution spectrum, two peaks at 458.7 and 464.9 eV indicated the binding energies of Ti2p 3/2 and Ti2p 1/2, respectively (Fig. 4f). The O1s spectrum showed two peaks at 530.1 eV and 531.6 eV, which were assigned to Ti–O–Ti/Ti–O–C and C=O bonds, respectively (Fig. 4g). Overall, the XPS and FT-IR data suggest the strong interaction between DP-CDs and TiO2 via the formation of Ti–O–C bonds. The UV-DRS spectra show that the DP-TiO2 nanoparticles did not absorb in the visible region between 400 and 800 nm, whereas DCTN exhibited an extended absorbance in the visible light region (Fig. 4h), which indicates that DCTN has better photocatalytic activity under direct sunlight42. In addition, the bandgap of DCTN was estimated to be 2.87 eV using the Tauc equation, which means that DCTN has a smaller bandgap than DP-TiO2 (3.25 eV) and P25 TiO2 (3.15 eV) (Fig. S5).
Photocatalytic bacterial-deactivation of DCTN under sunlight
The photocatalytic bacterial deactivation of DCTN under sunlight was evaluated using Vibrio harveyi (MTCC 7771) as a model bacterium. It was found that the time-dependent bacterial killing was observed in the presence of DCTN photocatalyst under sunlight irradiation (Fig. S6). In Fig. 5a, the result depicted that the untreated V. harveyi barely shows self-degradation (~ 3%), whereas the complete killing of V. harveyi was noted within 240 min in the presence of DCTN photocatalyst. The bacterial degradation efficacy of DCTN under sunlight was higher than that of commercial P25-TiO2 and prepared DP-TiO2. Moreover, there was no bacterial killing in the presence of DCTN, P25-TiO2, and DP-TiO2 under dark conditions. This result clearly revealed that the bacterial killing of V. harveyi accomplished by the photocatalytic activity and not by their antibacterial activity of DCTN. In addition, the photocatalytic bactericidal activity of environmental water samples also performed. The obtained result shows that DCTN-photocatalysis significantly inactivated the bacterial load in the natural seawater (Fig. S7) as well as tap-water (Fig. S8), within 240 min under sunlight irradiation. complete.
Similarly, Bonnefond et al.43 have achieved bacterial deactivation of Escherichia coli within 240 min using acrylic/TiO2 nanocomposite under sunlight irradiation. Sharma et al.44 reported that TiO2–Fe2O3 nanocomposite has exhibited complete bacterial killing within 120 min under sunlight. In another hand, Lin et al.45 have deactivated E. coli cells within 360 min using iodine-modified TiO2 under visible light irradiation. Yadav et al.46 have successfully inactivated E. coli and Salmonella abony cells under 300 and 360 min of visible light irradiation, respectively. Ouyang et al.47 have effectively disinfected E. coli cells within 120 min using C70–TiO2 hybrid thinfilm under visible light irradiation. Similar to our finding, Zeng et al.48 have reported that carbon dots-TiO2-rGO nanocomposite rapidly destroyed E. coli cells within 60 min under simulated solar-light irradiation. Taking everything into account, the present study is the first study was reported the photocatalytic deactivation of aquatic pathogen-V. harveyi with its mechanism insights, which was achieved within 240 min under sunlight irradiation. Hence, due to the successful and efficient bacterial killing efficacy, the present study profoundly suggests the prepared DCTN as an proficient photocatalyst for water disinfection process using sunlight irradiation.
Mode of ROS induced bactericidal action during DCTN photocatalysis
The active mechanisms of photocatalytic mediated bacterial deactivation using TiO2-based photocatalysis are well known. Briefly, the photogenerated active free radicals induce oxidative stress that damage the cell membranes as well as the cellular components, including proteins, lipids, enzymes, DNA, and other biomolecules49, and ultimately cause bacterial death, as shown in Fig. 8 Scheme-3. Therefore, the generation of ROS stress inside V. harveyi cells plays a pivotal role in the photocatalysis-mediated bacterial deactivation process50. Therefore, the intracellular ROS level in V. harveyi during DCTN-photocatalysis under sunlight irradiation was measured using a DCFDA-probe method. The result shows that the intracellular ROS level in the photocatalyzed cells increased extensively compared to the normal cells at the initial time point (Fig. 5b). Subsequently, the intensity of ROS decreased gradually with increasing experimental time, which indirectly confirming the bacterial death during DCTN-photocatalysis51. The predominant stimulation of intracellular ROS stress in V. harveyi cells indicates the ROS-mediated bacterial killing upon DCTN-photocatalysis under sunlight irradiation.
At the same time, the cell morphology was observed by SEM to better understanding the deleterious effect of ROS-induced bactericidal action on V. harveyi52. The SEM results revealed the uninterrupted, damage-free, and usual morphology of gamma-like bacteria in the control samples (Fig. 5c), whereas, broken cells along with remarkable changes, such as membrane damage caused by forming pits and holes in their cell membrane in the photocatalyzed samples, which are evidently visualized the cell-membrane dameges during the DCTN-photocatalysis. Further, the disruption of the cellular components was confirmed by FT-IR spectroscopy (Fig. 5d), which showed a significant decrease in the region of 600–800 cm−1, indicating the breakdown of glycoside linkages of polysaccharide molecules in the cell membrane53. The reduction at 1087 and 1238 cm−1 related to the breakdown of bacterial membrane phospholipids in photocatalyzed V. harveyi. The reduced intensity at 1550 and 1645 cm−1 denotes the damage to the amide-I and amide-II bonding of proteins and peptides54. The perceptible decrease in the intensity around 2750–3050 cm−1 indicates the damage to the fatty acid contents in the cell membrane in the photocatalyzed cells17. Overall, these results hypothesized that the inactivation of V. harveyi was accomplished mainly by the apparent damage of the cell membrane as well as the subsequent removal of cellular components by oxidative stress during the DCTN-photocatalysis.
Identification of ROS generation during DCTN photocatalysis under sunlight
The prepared DCTN showed improved visible light absorption due to the narrowed bandgap compared to the commercial P25-TiO2 and prepared DP-TiO2, as shown in Fig. S5. The results of FT-IR and XPS analysis also revealed that the significant decrease in bandgap could be associated with the chemical bonding between TiO2 and DP-CDs by Ti–O–C bond formation in the DCTN. Several studies also reported that the presence of Ti–O–C bonding offers charge transfer and can extend light absorption to longer wavelengths55,56. At the same time, DP-CDs can act as an electron acceptor to trap the electrons from the conduction band of TiO2 and hinder electron–hole recombination, which leads to more charge carriers to produce reactive oxygen species (e.g., OH· and O2·−)57. These reactive species are very strong oxidants that promote bacterial deactivation under sunlight irradiation.
The formation of photocatalytically generated ROS by the DCTN photocatalyst under sunlight irradiation was confirmed using the 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and terephthalic acid (TA) method. In the XTT assay, O2·− reduces XTT to XTT-formazan and shows a specific absorption peak at 470 nm. The XTT-formazan concentration indicates the formation of reactive species (O2·−). The results showed that the formation of O2·− radicals in DCTN photocatalysis is much higher than that in DP-TiO2 and P25 TiO2 under sunlight irradiation (Fig. S9). Similarly, TA reacts with OH· radicals to form 2-hydroxyterephthalic acid (TA), which emits an FL signal at 420 nm58. The results showed that DCTN exhibited an enhanced FL intensity of TA compared to DP-TiO2 and P25 TiO2, which validates the enhanced production of OH· radicals under sunlight irradiation (Fig. S10).
Anti-infection activity of DCTN against V. harveyi-caused acute hepatopancreatic necrosis disease (AHPND)
It is well-known that aquaculture is an important food-producing sector around the globe59. About 75% of the global production of aquatic-products comes from the Asian countries, including China, Thailand, Vietnam, Indonesia, and India60. However, the bacterial disease called acute hepatopancreatic necrosis disease (AHPND) mainly caused by V. harveyi intensely affects shrimp production, which leading to global economic losses61. During the AHPND pathogenesis, V. harveyi colonizes on the shrimp hepatopancreas (HP), in which it causing tissue destruction and dysfunction of the HP and digestive organs62. Hence, there is an increasing attention in the development of effective anti-infection agents to control the Vibrio mediated bacterial infections in aquaculture through anti-biofilm or anti-quorum sensing (anti-QS) approach in recent years23,24.
In vitro anti-biofilm and anti-virulence activity of DCTN
Biofilms are an aggregated growth pattern of bacterial pathogens, which acts vital role in their pathogenesis to cause infectious diseases to the host63,64. Hence, hindering biofilm formation by anti-biofilm agents has considered a promising mode to trim-down the infection rate in aquaculture23. Recently, Soowannayan et al.65 have successfully protected AHPND infected shrimps (P. vannamei) using Vibrio biofilm inhibitors. Therefore, the antibiofilm activity of the DCTN was assessed using in vitro assay17. The obtained result reveals that the DCTN effectively inhibited the biofilm formation of V. harveyi to the level of 85% at 50 μg/ml concentration (Fig. 6a). Further to confirm the anti-biofilm activity against V. harveyi, in-situ visualization of biofilms was performed using light microscopic and CLSM analysis54. In light microscopic results, the untreated V. harveyi has a thick coating of biofilms on glass slides; whereas, a perceptive diminution of biofilm cells was obtained in DCTN treated slides (Fig. 6b). Then, the dynamic complexity of biofilm architecture was observed using CLSM analysis66. The result depicts that the highly complex architecture was obtained in the untreated control sample; whereas, a reduced biofilm architecture was obtained in the DCTN treated sample (Fig. 6c), which strongly authenticated the anti-biofilm potential of DCTN against V. harveyi.
In addition, the bacterial pathogenicity also related to the secretion of QS-regulated extracellular virulence enzymes such as hemolysin and protease, which are facilitate the bacteria to defend against the host immune response24. In the current study, DCTN inhibited the hemolysin and protease production of V. harveyi to the level of 75 and 72%, respectively (Fig. S11). This section of the present study concluded that DCTN has the potential to inhibit QS-regulated biofilm formation as well as virulence factor production in V. harveyi.
In vivo toxicity level of DCTN in P. vannamei
Before going to assess the in vivo anti-infection activity, the toxicity level of DCTN was studied using a survival assay with whiteleg shrimp (Penaeus vannamei). It was found that DCTN has not shown any reduction in the survival of P. vannamei at 12.5 µg/ml concentration, whereas 80% of survival was observed at 50 µg/ml treatment (Fig. 6d). Hence, we have selected 12.5 µg/ml dosage for subsequent in vivo experiments. Further, the toxic level of commercial P25-TiO2 as well as DP-TiO2 at 12.5 µg/ml concentration was tested. The result shows that both DP-TiO2 and P25-TiO2 has significant toxicity to P. vannamei (Fig. S12). So, this part of the work strongly suggests that the prepared DCTN is more biocompatible than commercial P25-TiO2 and DP-TiO2.
In vivo anti-infection activity against V. harveyi-caused AHPND
To appraise the in vivo anti-infection efficacy of DCTN on V. harveyi-caused AHPND in P. vannamei, the survival assay was performed with infected animals. Figure 6e shows that the survival of infected shrimp without any treatment was 20%; whereas, the survival of shrimp treated with 12.5 µg/ml of DCTN was increased up to 83.33% in 96 h experiment. In Fig. 7a, the pathognomonic symptoms of AHPND such as pale and shrunken HP and empty gut were observed in the V. harveyi infected group65; wherein, these infectious lesions were significantly abridged in the DCTN treated group. This result evidently authorizes the in vivo disease protection efficacy of DCTN against V. harveyi caused AHPND.
The survival extension of infected shrimps consummated by hindering the V. harveyi accumulation inside the shrimp HP. Therefore, the V. harveyi accumulation inside the HP was analyzed using the CFU assay. The result displays a huge amount of V. harveyi colonization (8.63 × 104 CFU) observed in the infected shrimp; whereas, significantly reduced bacterial colonization (1.89 × 104 CFU) observed in the DCTN treated group (Fig. 7b). This result concluded that the DCTN-treatment effectively reduced the V. harveyi colonization, and thereby, rescued the infected shrimps from the infectious effect of AHPND.
In histopathological analysis, hematoxylin and eosin (H&E) stained HP tissues of shrimp reveal that the massive sloughed and elongated HP, collapsed tubule epithelia and severe hemocyte infiltration in the V. harveyi infected shrimp (Fig. 7c), which characterizes the factual pathognomonic symptoms of AHPND in shrimp culture67. In contrast, the HP tissue of treated with DCTN showed normal HP histology, which similar to the uninfected control group. Overall, the obtained results suggest that the bioactive DCTN can be exploited to care for acute hepatopancreatic necrosis disease in shrimp aquaculture.
Conclusions
A simple, low-cost, and facile approach was developed to prepare fluorescent carbon dots using a plant extract with a quantum yield of 31.4%. The prepared DP-CDs showed strong FL stability and photo-stability. Consequently, the FL intensity decreased upon binding with Fe3+ions, which prompted the development of an uncomplicated on–off fluorometric sensor-strip for Fe3+ detection. Subsequently, DP-CDs/TiO2 hybrid-spheres (DCTN) were prepared for photocatalytic application. As expected, DCTN showed superior photocatalytic activity in bacterial deactivation under sunlight because of the narrowed bandgap. The DCTN effectively deactivated V. harveyi by the photogenerated ROS stress, which damaged the cell membrane and cellular components, resulting in bacterial death under sunlight irradiation. On the other hand, the DCTN exhibited admirable anti-biofilm activity against V. harveyi. Further, the DCTN not showed any significant toxicity in P. vannamei, which indicates that the prepared DCTN is more biocompatible than commercial P25-TiO2 and DP-TiO2. Consequently, the DCTN showed excellent disinfection activity against V. harveyi-induced AHPND in P. vannamei. The lifespan extension of V. harveyi-infected shrimps was achieved by reducing the internal accumulation of V. harveyi inside the HP upon the DCTN treatment. The histopathology study also validated the rescue action from the pathognomonic effects of V. harveyi-caused AHPND in the treated shrimps. Overall, the present study recommends that the prepared bioactive plant-derived carbon dots-TiO2 nanocomposite (DCTN) present a great prospect in photocatalytic water disinfection, along with an innovative biological-application in the treatment of V. harveyi-mediated acute hepatopancreatic necrosis disease for shrimp aquaculture.
Methods
Materials
The leaves of D. palmatus (L.) were shade dried and ground into a fine powder. Subsequently, 10 g of leaf powder was extracted sequentially using 100 ml of distilled water and methanol. The aqueous extract was used for carbon dot synthesis, and the methanolic extract was used as a capping and stabilizing agent for the nanocomposite synthesis. Titanium isopropoxide (C12H28O4Ti, > 97% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification as a precursor for the carbon dot/TiO2 composite synthesis. All the chemicals and reagents used in the current study were purchased from Sigma-Aldrich. Whatman No.1 (#3) filter papers were used for the preparation of the sensor strip.
Preparation and characterization of carbon dots
In the experiment, 100 ml of an aqueous extract of D. palmatus was transferred to a Teflon-lined autoclave and sealed tightly to maintain the intrinsic pressure. The autoclave was heated to 180 °C for 30 min. After a dark brown solution formed, it was allowed to cool to room temperature. Finally, the solution was centrifuged, and the supernatant was filtered through a 0.2 μm filter membrane to eliminate the micron-sized particles. The final solution was lyophilized to obtain solid carbon dots and stored at 4 °C for subsequent studies.
The structural morphology was investigated by high-resolution transmission electron microscopy (HRTEM, JEM 2011, JeolCo.). The particle size was observed using a Zeta-sizer (Nano ZS, Malvern Instruments, United Kingdom). The crystallinity was investigated by X-ray powder diffraction (XRD, PAN analytical X-Pert PRO). Raman spectroscopy was carried out using an imaging spectrograph STR 500 mm focal length laser Raman spectrometer at room temperature. The elemental composition was investigated by energy-dispersive X-ray (EDX, SU-70, Hitachi) attached to the FE-SEM (SU-70, Hitachi) instrument. The surface chemical composition was observed by X-ray photoelectron spectroscopy (XPS, PHI 5600ci) using AlKα radiation (hv = 1486.6 eV, Thermo Fisher Scientific). XPS curve fitting and elemental analyses were performed using a Casa XPS 2318 PR1-0 instrument. The surface functional groups were investigated using Fourier Transform Infrared (FT-IR, Thermo scientific iS4) spectroscopy using the KBr compressed pellet method. The fluorescence intensity of the DP-CDs was observed using a Shimadzu RF-5310 PC spectrofluorophotometer. The quantum yield (QY) of the DP-CDs was measured using quinine sulphate (QY: 54%; dissolved in 0.1 M H2SO4 aqueous solution) as a standard, and the yield was calculated using the following formula:
where φ, A, I, and η is the QY, optical density, integrated emission intensity, and solvent refractive index, respectively. The subscript ‘c’ refers to the DP-CDs solution, and subscript ‘s’ refers to the quinine sulphate solution.
Metal sensing of Fe3+ using DP-CDs in solution
To determine the sensing selectivity of the as-prepared DP-CDs towards metal ions, the FL intensity was measured in the presence of various aqueous metal chloride solutions with a concentration of 50 µM (Cr2+, As2+, Hg2+, Zn2+, Pd2+, Pd2+, Fe3+, Ba2+, Cu2+, Ag+, Mg2+, and Al3+) at room temperature28. The DP-CDs were diluted with distilled water to a concentration of 1 mg/ml. In the experiment, the DP-CDs solution was mixed with one of the above metal solutions in equal volumes, and the intensity was measured using a multilabel reader (Molecular Device Spectramax M3, Softmax Pro V5 5.4.1 software) at Ex420 nm and Em520 nm. For the sensitivity assessment, the FL intensities of DP-CDs were calculated with different concentrations of Fe3+ to predict the limit detection limit68.
Preparation of DP-CDs coated sensor-strip
For paper-based sensor strip preparation, the design of the microfluidic device was drawn on filter paper with a pencil (length diameter of reaction zone = 5 mm). A wax coating was made over the pencil marks with a wax marker. Subsequently, the wax-coated papers were placed on a hot plate at 120 °C for 2 min to fix the wax figure and pull in the hydrophobic boundaries. Subsequently, 20 µl of an aqueous DP-CDs dispersion was dropped within the boundaries on the filter paper and allowed to dry at room temperature69. The prepared sensor strips were now ready for the sensing application. Subsequently, 10 µl of a metal ion solution was placed on the sampling part and allowed to dry at room temperature. The FL intensity of the sampling area was observed using an ultraviolet inspection cabinet and captured with a Canon EOS 90D DSLR camera. Finally, the FL intensity of the captured images was measured using ImageJ software.
Preparation and characterization of carbon dots-TiO2 nanocomposite (DCTN)
For DCTN preparation, 50 ml of a methanolic extract of D. palmatus and the DP-CDs solution (100 mg) were added to 50 ml of titanium isopropoxide (10 mM) and stirred for 30 min. The mixture was autoclaved at 120 °C for 30 min. A precipitate of the resultant-suspension was dried overnight at 180 °C. Finally, the resulting precipitate powder was calcined at 400 °C for 3 h. For material characterization, the structure of the prepared DCTN was examined by HR-TEM and FE-SEM. The 3D morphology and the size of the DCTN were observed by atomic force microscopy (AFM, Agilent Technology) in contact mode. Compositional characterization studies were performed with FT-IR spectroscopy and XPS. The absorbance characteristics of DCTN were analyzed by UV–Vis diffuse reflectance spectroscopy (UV-DRS) using a UV–Vis–NIR spectrophotometer (Shimadzu UV-3600).
Photocatalytic bacterial deactivation of DCTN under sunlight
Assessing the bacterial growth of V. harveyi during DCTN photocatalysis
The photocatalytic disinfection efficacy of DCTN was evaluated using Vibrio harveyi (MTCC 7771) as the model bacterium. The photocatalytic experiments were performed under direct sunlight irradiation between 11.00 am and 2.00 pm (light intensity was 983 ± 46 W m−2, measured using a photometer) from April to July. For this experiment, V. harveyi was cultured in 100 ml Luria Bertani (LB) medium overnight at 37 °C. The bacterial cells grown in the media were harvested using centrifugation and washed twice with PBS. The cell pellets were re-suspended in 100 ml of saline water (1% NaCl). Subsequently, 50 mg of DCTN was added to the V. harveyi suspension and placed in the dark for 10 min. The bacterial suspension with the DCTN photocatalyst was then exposed to direct sunlight. Every 60 min, 100 µl of the reaction samples were taken and spread on an LB agar plate. Each plate was incubated at 30 °C for 24 h, and the bacterial colonies were counted after the incubation period (Fig. 8).
FE-SEM analysis
For FE-SEM analysis, the bacterial samples were harvested using a centrifuge after 240 min sunlight irradiation. The bacteria were then fixed with a 2% glutaraldehyde solution for 4 h and washed twice with distilled water. The fixed samples were dehydrated with increasing concentrations of an ethanol solution (20, 40, 60, 80, and 100%) and dried overnight. Then, the prepared samples were observed in field emission scanning electron microscopy (FE-SEM) instrument (Zeiss ultra FE-SEM instrument).
Measurement of intracellular ROS in bacteria
The intracellular ROS generation in the bacterial cells was determined using the DCFDA method70. Briefly, 1 ml of treated and untreated culture was centrifuged at 5000 rpm for 15 min, and the supernatant was removed. Subsequently, 20 μg/ml of DCFDA was added and kept for 1 h at 37 °C. Finally, the fluorescence intensity was recorded at an excitation and emission wavelength of 485 nm and 535 nm, respectively, using a multilabel reader (Molecular Device Spectramax M3, Softmax Pro V5 5.4.1 software).
FT-IR analysis
In FT-IR analysis, the cell pellets of V. harveyi before and after photocatalysis were harvested and washed with 1% PBS. The KBr pellets were then prepared separately with these cell pellets. The FT-IR (NicoletTM iS5, Thermo Scientific, U.S.A) spectra were recorded in the range, 400–4000 cm−1, with a resolution of 4 cm−1 using OMNIC Software71.
ROS analysis in water under sunlight
An XTT assay and TA method were performed to confirm the generation of ROS in the photocatalytic reaction under sunlight irradiation72. In the 2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) probe-based confirmation of O2·− generation, 100 µM XTT was used as an indicator. During the photoreaction, 1 ml of the experimental water was collected, and the intensity of the orange-colored XTT-formazan was measured at 470 nm using a multilabel reader (Molecular Device Spectramax M3, Softmax Pro V5 5.4.1 software). Terephthalic acid (10 μM, Sigma-Aldrich) was used as an indicator of OH· radical detection. In general, TA binds with OH· radicals to form the fluorescent product, 2-hydroxyterephthalic acid (hTA). Therefore, the fluorescence intensity of hTA was measured at Ex 310 nm and Em420 nm using a multilabel reader.
Assessing the anti-infection efficacy of DCTN against V. harveyi-caused AHPND
Biofilm assay
In biofilm assay, 10 µl of an overnight culture of V. harveyi was added to 1 ml of sterile LB broth in 24-well MTP containing different concentrations of DCTN (10–50 µg/ml). After 24 h incubation, the planktonic cells were discarded, and adhering biofilm cells were mildly rinsed with distilled water. Then, the biofilm cells were stained with 0.2% crystal violet (Himedia, India) and suspended in 1 ml of 20% glacial acetic acid. Finally, the intensity was measured at OD570 nm using UV–Vis spectroscopy and calculated the inhibition percentage73.
In-situ visualization of biofilms
For light microscopic analysis, the biofilm assay was carried out with glass slides (1 × 1 cm). Then, the glass slides were rinsed twice with distilled water and stained with 0.2% crystal violet. After that, the glass slides were observed under a light microscope with an attached digital camera (Nikon Eclipse Ti 100) at 400 × magnification74. For confocal laser scanning microscopy (CLSM) image analysis, the biofilms were stained with 0.1% acridine orange and observed under CLSM (Zeiss LSM 710, Carl Zeiss, Germany) at the magnification of 200 ×.
Hemolysin assay
In hemolysin assay, 10 µL of V. harveyi culture was inoculated into 1 ml of LB broth containing the various concentrations of DCTN (10–50 µg/ml) and incubated at 28 °C for 24 h. Then, the cell-free culture supernatants (CFCS) were collected using centrifugation. After, 100 µl of CFCS was added to 900 µl of 2% sheep erythrocytes in phosphate buffer saline (PBS). The mixture was placed in ice for 20 min and centrifuged to collect supernatants. Finally, the absorbance of hemoglobin was measured at 530 nm using a UV–Vis spectroscopy23,24.
Protease assay
In protease assay, 10 µl of V. harveyi culture was inoculated into 1 ml of LB broth containing the different concentrations of DCTN (10–50 µg/ml) and incubated at 28 °C for 24 h. After incubation time, the 75 µl of CFCS was added to 125 µl of 2% azocasein in 0.25 M Tris (pH 8.0) and incubated for 30 min at 37 °C. Subsequently, the reaction was stopped using 10% trichloroacetic acid and the supernatant was collected through centrifugation. Finally, the absorbance of the supernatant was measured at 440 nm using a UV–Vis spectroscopy24.
Toxicity assessment using P. vannamei survival assay
Healthy Whiteleg shrimps (P. vannamei) (body length: 4.5 ± 0.26 cm) were purchased from Allwin hatchery, Pondicherry, India and acclimatized for a week. Then, ten numbers of shrimp were separately transferred to 20 l plastic bucket containing different concentrations of DCTN, P25-TiO2, and DP-TiO2 in 5 l of sterile seawater (30 ppt) at 27 °C. Without any material supplement considered as normal control. The mortality was counted and the survival percentage was calculated after 96 h of the experiment for assessing their toxicity level. Experiments were performed triplicates and no feeding was provided during the experiments61.
V. harveyi challenge study
For in vivo challenge test, shrimps were infected with 1 × 108 CFU/ml for 4 h. Then, the infected shrimps were transferred into a new 20L plastic bucket containing 12.5 µg/ml of DCTN in 5 l sterile seawater, named as treated group. Without DCTN treatment is considered an infected group. The mortality of experimental animals was counted and the survival percentage was calculated after 96 h. The experiments were performed triplicates and no feeding was provided during the experiments61. To assess the reduction of V. harveyi colonization, the shrimp hepatopancreas HP was collected and homogenized with sterile PBS. Then, the homogenized samples were allowed to spread plating and the colonies were counted after incubation23.
Histopathology analysis
The experimented shrimps were collected and immediately fixed in 10% (v/v) phosphate-buffered formalin containing 4 g/l of NaH2PO4, 6.5 g/l Na2HPO4 in 100 ml of 40% formalin in 900 ml of distilled water. Then, the shrimps were preserved in 70% ethanol till processing. Then, the formalin-fixed animals were allowed to sectioning using a microtome and tissue embedding. Finally, the cut sections were subjected to hematoxylin and eosin (H&E) staining and they were observed under a light microscope with an attached digital camera (Nikon Eclipse Ti 100)65.
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Acknowledgements
The author Rajaiah Alexpandi sincerely acknowledges Rashtriya Uchchatar Shiksha Abhiyan (RUSA) - Phase 2.0, Government of India [F. 24-51/2014-U, Policy (TN Multi-Gen), Dept of Edn, GOI] for financial support through RUSA 2.0 Ph.D. Fellowship. The author Rajaiah Alexpandi also wish to thank Dr. Balaji Murugesan, Department of Industrial Chemistry, School of Chemical Sciences, Alagapppa University for his guidance during material synthesis. The authors acknowledge BIF (funded by DBT, India, GOI; File No. BT/BI/25/012/2012, BIF), DST-FIST (Grant No. SR/FST/LSI-639/2015(C)), UGC-SAP (Grant No. F.5-1/2018/DRS-II (SAP-II)), DST-PURSE (Grant No. SR/PURSE Phase 2/38 (G)) and RUSA-Phase 2.0 [F. 24-51/2014-U, Policy (TN Multi-Gen), Dept. of Edn, GOI] for providing the infrastructure and instrumentation facilities. The authors also acknowledge University Science Instrumentation Centre (USIC), Alagappa University for providing the instrumentation facilities for characterization studies.
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R.A. and A.V.R. conceptualized and designed the experiments. R.A. performed the experiments. R.A., A.V.R., S.K.P., C.V.V.M.G., and H.-J.K. analyzed the results. R.D. helped more during in vitro experiments. R.A. wrote the manuscript. A.V.R., S.K.P., C.V.V.M.G., and H.-J.K. revised the manuscript. A.V.R. approved the final version of the manuscript. All authors participated in the discussion and commented on the paper.
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Alexpandi, R., Gopi, C.V.V.M., Durgadevi, R. et al. Metal sensing-carbon dots loaded TiO2-nanocomposite for photocatalytic bacterial deactivation and application in aquaculture. Sci Rep 10, 12883 (2020). https://doi.org/10.1038/s41598-020-69888-x
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DOI: https://doi.org/10.1038/s41598-020-69888-x
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