Metal sensing-carbon dots loaded TiO2-nanocomposite for photocatalytic bacterial deactivation and application in aquaculture

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.

Figure 1

(a) HR-TEM images of the structural characterization of the as-prepared DP-CDs (Inset: Lattice d-spacing of 0.347 nm represents the (002) diffraction plane of sp² carbon. (b) DLS measurement of DP-CDs in aqueous solution. (c) SAED pattern of individual particles of the DP-CDs. (d) XRD pattern of the DP-CDs revealed a (002) peak at 26.13° 2θ with a d-spacing of 3.49 Å, confirming the graphitic nature of the prepared DP-CDs. (e) Raman spectroscopic investigation of the defects or disorder in DP-CDs. (f) FT-IR analysis of functional groups on the surface of the DP-CDs. (g) C1s, (h) N1s and (i) O1s XPS high-resolution spectrum of DP-CDs.

Figure 2

(a) UV–Vis absorbance spectrum of DP-CDs. (b) Fluorescence (FL) spectrum of DP-CDs (Inset shows a visual photograph of FL emission under daylight and UV-light irradiation). (c) FL emission spectra of DP-CDs at different excitation wavelengths with a 10 nm increments, from 360 to 460 nm. (d) FL intensity of DP-CDs under different pH conditions. (e) FL intensity of DP-CDs under various salt concentrations from 0 to 1 M in solution. (f) FL stability of DP-CDs under photoleaching with UV-irradiation. (g) Photographs showing FL emission of DP-CD-painted drawing of the authors (SKP, AVR, and RA) on filter paper.

Figure 3

(a) Selectivity experiment on the changes in FL intensity (I/I₀) of DP-CDs solution in the presence of different ion solutions. (b) Optical images of the DP-CD solution in the presence of these metal ions (50 µM). (c) FL response of DP-CDs solution in the presence of different concentrations of Cd²⁺ and Fe³⁺ (I/I₀ corresponds to the changes in FL intensity in the absence and presence of Cd²⁺ and Fe³⁺ ions in solution). (d) Selectivity experiment of the DP-CDs in filter paper-based sensor strip. The graph shows the normalized intensity of DP-CDs after dropping of various ions solutions (50 µM) on the reaction zone. (e) Optical image of the FL intensity of DP-CDs sensor strip in the presence of different metal ions under UV-light captured by a Canon DSLR camera. (f) The graph represents the FL intensity changes (I/I₀) of DP-CDs in the presence of different concentrations of Cd²⁺ and Fe³⁺ on the prepared DP-CD-coated sensor strips. (g) Optical images showing the sensitivity experiment for the identification of a low detection limit of DP-CDs sensor strip toward Cd²⁺ and Fe³⁺ ions.

Figure 4

(a) FE-SEM and (b) HR-TEM analysis of the DCTN. (c) FT-IR analysis of functional groups on the surface of the DCTN. (d) C1s, (e) N1s, (f) Ti 2p and (g) O1s XPS high-resolution spectrum of DCTN. (h) UV-DRS absorbance spectra of DCTN and DP-TiO₂.

Figure 5

(a) Photocatalytic deactivation of V. harveyi over DCTN at different time points under sunlight exposure. (b) The graph represents the intracellular ROS level in V. harveyi during DCTN photocatalysis under sunlight (detected by DCFDA method). (c) FE-SEM analysis of DCTN photocatalysis-induced cell damage in V. harveyi upon sunlight irradiation (240 min). The yellow color arrow indicates the membrane damage and the red color arrows indicate the leakage of cellular components. (d) FT-IR analysis of the disruption of cellular components upon DCTN photocatalysis in V. harveyi upon sunlight irradiation. The result of the FT-IR spectra illustrates the reduction in the regions, such as (a) glycoside linkages of polysaccharide molecules in the cell membrane (600–800 cm⁻¹), (b) bacterial membrane phospholipids (1087 and 1238 cm⁻¹), (c) amide linkage from proteins and peptides (1550–1645 cm⁻¹), and (d) fatty acids in the cell membrane (2700–3100 cm⁻¹) in the photocatalyzed V. harveyi.

Figure 6

(a) Par graph represents the percentage of biofilm inhibition by DCTN on V. harveyi. (b) The image indicates the light microscopic observation of biofilm inhibition upon DCTN treatment. (c) CLSM analysis further endorses the anti-biofilm potential of DCTN against V. harveyi at 50 µg/ml concentration. (d) The graph reveals the survival percentage of shrimp (P. vannamei) in the presence of DCTN with various concentrations. (e) The graph shows the survival percentage of V. harveyi infected animals upon DCTN treatment at the selected dosage (12.5 µg/ml).

Figure 7

(a) Photographs reveal the pathognomonic symptoms of V. harveyi-caused AHPND in shrimp and rescue action of DCTN treatment. (b) The representative image for the reduction of V. harveyi colonization inside the HP by DCTN treatment. (c) The histopathology images of hematoxylin and eosin (H&E) stained hepatopancreatic (HP) tissues of the experimented shrimps.

Figure 8

(Scheme-1) The possible mechanism of FL quenching of the DP-CDs upon binding with Fe³⁺ ions. (Scheme-2) The schematic representation of DP-CDs coated fluorometric sensor-strip preparation. (Scheme-3) Schematic diagram of the photocatalytic deactivation of V. harveyi using DCTN photocatalyst under sunlight irradiation.