CeO₂-CePO₄ and Ag@CeO₂-CePO₄ nanocomposites from Penaeus semisulcatus for heavy metals sensing, UV shielding and cytotoxic applications

2.1 Materials

(NH₄)₂Ce(NO₃)₆ and (Ce(NO₃)₃·6H₂O) (99.9 %, AR Grade) were purchased from Sisco Research Laboratories. AgNO₃ (≥99.5 %, AR Grade) was procured from Sigma-Aldrich. EDTA disodium salt, C₁₀H₁₄N₂Na₂O₈·2H₂O (AR grade, 99.0 %), CoCl₂·6H₂O, CuSO₄·6H₂O, NiCl₂·6H₂O (SRL, 99.0 %), CrCl₃ anhydrous (99.9 %), FeCl₃ anhydrous (99.9 %), HgCl₂ (99.5 %), Cd(NO₃)₂·4H₂O (98.0 %), Pb(NO₃)₂ (99.0 %) and (NH₄)₂Fe(SO₄)₂·6H₂O (99.9 %) were acquired from Sigma Aldrich. The chemicals were utilized in their original form without any modifications.

2.2 Collection of P. semisulcatus and preparation of extract

Fresh and live P. semisulcatus were collected by trawl fishing from the Gulf of Mannar in Thoothukudi, Tamil Nadu, India. Shrimp shells and heads were removed and cleaned with copious amounts of water. Finally, the flesh part of the P. semisulcatus was dissected into tiny pieces and a 30 g portion of the flesh was weighed and then gently warmed to 40 °C in a water bath with 100 mL of bidistilled water for a duration of 10 min, followed by gradual cooling to ambient temperature. The resulting extract, obtained by filtering the supernatant through Whatman No. 41 filter paper, was utilized in the production of NCs.

2.3 Biosynthesis of CeO₂-CePO₄ NCs

To a solution of ceric ammonium nitrate (2.7412 g/100 mL, 0.05 M), an equal volume of the P. semisulcatus extract was slowly added dropwise (1:1 v/v) under magnetic stirring, followed by heating at 60 °C for 1 h. The yellow gel formed was further stirred in the magnetic stirrer for another 2 h at 1500 rpm. The protein-rich CeO₂-CePO₄ NCs was then centrifuged, extensively washed with water and air dried (labelled as CeNC@T1). Powdered CeNC@T1 was annealed at 550 °C for 3 h (labelled as CeNC@T2) and again annealed at 800 °C for 2 h (labelled as CeNC@T3) to obtain CeO₂-CePO₄ NCs.

2.4 Biosynthesis of Ag@CeO₂-CePO₄ NCs

Ag@CeO₂-CePO₄ NCs were synthesized adapting two methods. In the first method, AgNO₃ (0.08494 g) was dissolved in the prepared yellow gel (section 2.3) and exposed to sunlight while stirring magnetically for another 2 h. In an alternate method, an equal volume of P. semisulcatus extract was added to a 0.005 M solution of AgNO₃ and left in sunlight for 1 h. AgNPs thus prepared were added to the above yellow-coloured gel and magnetically agitated for another 2 h. The prepared gel was then centrifuged and washed with plenty of water and air dried. Finally, it was ground and further annealed at 800 °C for 2 h and labelled as Ag-CeNC@T3. The second method was followed owing to the more intense colour of AgNPs.

2.5 Biosynthesis of CePO₄ NC

CePO₄ NC was prepared by dissolving 2.1661 g of cerium nitrate hexahydrate (Ce(NO₃)₃·6H₂O) in 100 mL of water (0.05 M) and 100 mL of the P. semisulcatus extract was added dropwise slowly (1:1 v/v) while stirring magnetically. The reaction mixture was further subjected to heating at 60 °C for 1 h. This resulted in the formation of a white gel which was further stirred for 2 h using a magnetic stirrer at 1500 rpm. The resultant CePO₄ NC was further centrifuged, washed several times with water and subjected to air drying (CePO₄NC@T1).

A schematic representation for the synthesis of different NCs mediated by P. semisulcatus extract is presented in Scheme 1.

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Scheme 1

Schematic representation of P. semisulcatus extract mediated synthesis of NCs.

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2.6 Characterization

UV–Visible Diffuse Reflectance Spectra (UV–Vis DRS) of synthesized NCs were captured using a UV–Visible spectrophotometer (JASCO, V-650 model) between 200 and 900 nm, and emission studies were carried out using a JASCO FP-8300 spectrofluorometer. A Shimadzu 8400S model instrument was used to record the Fourier Transform Infrared (FT-IR) spectra of dried shrimp extract and NCs in the range of 4000–400 cm⁻¹ in KBr pellets. The crystallinity of the NCs was investigated using X-ray Diffraction (XRD) spectra obtained from an X'Pert Pro-PAnalytic Diffractometer with CuKα radiations (30 mA, λ = 1.5406 Å and 40 kV). Raman analysis was performed using a Horiba XploRA PLUS Raman microscope with laser wavelength 638 nm, and Energy Dispersive X-ray (EDX) analysis of NCs was done using Bruker Nano, GmbH, D-12489 instrument with an accelerating voltage of 30 kV. X-ray Photoelectron Spectroscopy (XPS) analysis was carried out to identify the chemical state of each element using PHI VersaProbe III instrument, which used a monochromatic Al-Kα X-ray source (hν = 1486.6 eV) with constant analyzer energy (26 eV) and binding energy was calibrated to 284.6 eV, which corresponds to the C-1s atom. High Resolution Transmission Electron Microscopy (HR-TEM) and Selected Area Electron Diffraction (SAED) measurements of the NCs were performed on JEOL 2100F and JEOL JEM 2100 PLUS instruments with an accelerating potential of 200 kV. HR-TEM images were acquired by dispersing the sample in an alcoholic medium that was cast onto copper grids and dried at ambient temperature.

2.7 Sensing of metal ions

To assess the potential of NCs as fluorescent sensors, different metal ions were examined at neutral pH under ambient conditions. For this, 10 mg of the NCs were dispersed in 30 mL bidistilled water (0.33 mg/mL) by continuously stirring with a magnetic stirrer throughout the experiment. 1 mL was taken from the dispersion setup and mixed with equal volume of target metal ions solution, to a total volume of 2 mL (0.05 M). The mixture was shaken for one minute, and the fluorescence spectra were then obtained with excitation at 220 nm. The fluorescence intensity of bio-derived NCs was enhanced by Pb²⁺, Hg²⁺ and Cd²⁺ and hence their potential to detect these metal ions was studied in detail in the concentration range of 5.0x10^-9 M to 4.0x10^-5 M. The Limit of Detection (LOD) was calculated using the formula: LOD = 3σ/ slope, where σ denotes the standard deviation of the blank (n = 10) (Gracia et al., 2022; Daphne et al., 2023; Gracia et al., 2023).

2.8 Evaluation of Sun protection factor (SPF) and cytotoxicity

SPF was detected using the method of Khan et al. (Khan, 2018) and the procedure is reproduced in S1 for quick reference. The in vitro screening of cytotoxicity of CeNC@T1 and CeNC@T2 was done by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Gerlier and Thomasset, 1986) (S2). The cell viability was expressed using the formula: $$Cellviability\left(\%\right)=\frac{\mathit{Average}\mathit{abs}or\mathit{bance}\mathit{of}\mathit{treated}\mathit{cells}}{\mathit{Average}\mathit{absorbance}\mathit{of}\mathit{control}}\times 100$$

3.1 X-ray diffraction analysis

XRD spectra of NCs delineated in Fig. 2. show typical peaks of CeO₂ and CePO₄ phases. The 2θ values of well-defined diffraction peaks along with crystallographic (hkl) planes are given in Table S1. The peaks reflect cubic fluorite structure of ceria (ICDD card no. 00–034-394) with fm̅3m space group (Vinothkumar et al., 2018). CePO₄ is expected to be in monoclinic phase at 550 °C. However, all the distinct peaks associated with monoclinic phase of CeO₂ are not observed in the diffraction pattern at this temperature owing to the convolution. Formation of CePO₄ on the surface of CeO₂ influences the particle size of nanoceria (Noor et al., 2021).

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Fig. 2

XRD patterns of (a) CeNC@T1, (b) CeNC@T2, (c) CeNC@T3 and (d) Ag-CeNC@T3.

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In CeNC@T1, the prophenoloxidase peaks associated with the shrimp extract are located at 13.71 and 32.40 (Ishwarya et al., 2016), the peaks at 30.44 and 64.53 are due to lectin and the peak at 32.40 is attributed to peptide crustin (Sivakamavalli et al., 2020). Diffraction pattern of CeNC@T3 shows characteristic peaks corresponding to cubic CeO₂ NPs and monoclinic CePO₄ NPs and exhibits 66.56 % crystallinity. The peaks at 28.53, 33.06, 47.46, 56.33, 59.08, 69.42, 76.61 and 79.09 correspond to CeO₂ phases (ICDD card no. 00–034-394) (Vinothkumar et al., 2018). Monoclinic phase of CePO₄ is confirmed by diffraction peaks at 12.31, 21.44, 26.99, 42.02, 45.96 and 52.65 (JCPDS card no.32–0199) (Noor et al., 2021).

Ag-CeNC@T3 shows characteristic diffraction peaks corresponding to CeO₂ at 28.55, 33.09, 47.50, 56.39, 59.19, 69.43, 76.33 and 79.17 which account for (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), (4 0 0), (3 3 1) and (4 2 0) planes respectively (ICDD card no. 00–034-394) (Vinothkumar et al., 2018). In Ag-CeNC@T3, peaks due to Ag⁺ ion are detected at 34.42 (Yang et al., 2016) and 41.09 (Shi et al., 2016). The peaks located at 12.03, 21.21, 26.95, 31.15, 42.22, 46.06, 48.56, 51.81 correspond to the (1 0 0), -(1 1 1), (1 2 0), (1 0 2), -(1 0 3), (2 1 2), (1 0 3) and -(2 3 2) planes of CePO₄ NPs (JCPDS card no.32–0199) (Verma and Bamzai, 2014).

A study of literature reveals that phosphorus in the extract of P. semisulcatus can serve as the precursor for the CePO₄ phase (Noor et al., 2021). The presence of CePO₄ phase has been confirmed by the XRD patterns. Additionally, EDX analysis further supports this finding by indicating that CeNC@T3 contains 4.52 % phosphorus (Fig. S1). The crystallite size of the NCs is ascertained using Debye-Scherrer’s formula represented as D = kλ/β cos θ, where D, k, λ, β and θ represent the crystallite size, Scherrer constant (0.9), wavelength of X-ray beam, FWHM and the diffraction angle respectively. The average particle size of CeNC@T1, CeNC@T2, CeNC@T3 and Ag-CeNC@T3 has been found to be 4.78 nm, 6.51 nm, 44.29 nm and 32.73 nm respectively. With a marked increase in temperature from 550 °C to 800 °C, hexagonal phase of CePO₄ transforms to monoclinic phase, which is accompanied by an increase in size of the nanoparticles.

3.2 UV–Vis DRS analysis

UV–Vis DRS spectral analysis of P. semisulcatus derived NCs was studied between 200 and 900 nm, and the results are compiled in Fig. 3. CeNC@T1, CeNC@T2 and CeNC@T3 show broad peaks centered at 352, 356 and 352 nm, humps around 256, 250 and 256 nm respectively, and a broad tail extending up to 800 nm. A closer examination of the CeNC@T3 spectrum reveals the presence of additional humps at 212 and 304 nm. The absorption spectra of CeNC@T2 and CeNC@T3 show an almost identical absorption edge in the visible region exhibiting a small shift in λ_max. The difference observed in the slope of the absorption curve is attributed to the heightened degree of crystallinity in both the oxide and phosphate phases of cerium at elevated temperatures (Vinothkumar et al., 2018).

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Fig. 3

UV–Vis DRS spectra of NCs. Inset: Band gap energy calculation using Tauc plot.

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In all these spectra, the broad peaks around 355 nm are typical of CeO₂. Ce(IV) is a 4f⁰ system, where 4f-4f transition is not possible. The intense yellow colour of Ce(IV) compounds is due to charge transfer to metal (O^2–(2p) to Ce⁴⁺(4f) in the 300–450 nm region (Choudhury et al., 2014). The peaks observed at 212, 256, 288 and 304 nm in CeNC@T3 are due to Ce(III) of CePO₄ and originate from ²F_5/2 (GS) to the 5d (ES) (200–320 nm range) (Vinothkumar et al., 2018; Dezfuli et al., 2014).

For comparison, CePO₄ NC was prepared using Ce(III) precursor (section 2.5) and air-dried (CePO₄NC@T1). It has a major peak at 286 nm and a small peak at 208 nm, revealing the presence of Ce³⁺. Most of the peaks due to Ce³⁺ are masked by LMCT in Ce⁴⁺ (Fang et al., 2003). The broad peak observed at 352 nm in CeNC@T3 is blue shifted at 336 nm in Ag-CeNC@T3 with a hump at 254 nm and cut-off wavelength of 600 nm. The hump around 300 nm due to Ce(III) is not observed in this spectrum.

The band gaps of synthesised CeNC@T1, T2, T3 and Ag-CeNC@T3 have been calculated by applying Tauc equation (Jubu et al., 2022). UV–Vis DRS spectra of the CeNC@T1, T2 & T3, Ag-CeNC@T3 and CePO₄NC@T1 have distinct absorption bands around 354 nm and the optical band gaps have been calculated as 2.77, 2.72, 2.85. 3.19 and 3.71 eV respectively (Fig. 1 (inset)). Kubelka-Munk function has also been used to determine the optical band gap of the prepared nanocomposites (Noor et al., 2021). The optical band gaps of CeNC@T1, CeNC@T2, CeNC@T3, Ag-CeNC@T3 and CePO₄NC@T1 using Kubelka-Munk function are 3.03, 2.95, 3.08, 3.34 and 3.82 eV. A band gap of 2.9–3.3 eV is observed for bulk CeO₂ and band gap of 3.2–3.6 eV for both direct and indirect band transitions. CePO₄ NPs have been reported to have a direct band gap of around 3.0 eV. The reduction in both direct and indirect band gaps in this study in comparison to the band gap values of pristine CeO₂ and CePO₄ nanoparticles is reported to be due to the synergy that exists between the two phases in the NCs. The same trend is observed in CeO₂-InVO₄ and CeO₂-BiVO₄ nanocomposites (Noor et al., 2021).

3.3 FT-IR spectral analysis

The FT-IR spectral data of P. semisulcatus extract, CeNC@T1, CeNC@T2, CeNC@T3 and Ag-CeNC@T3 are delineated in Fig. 4. The FT-IR spectrum of the extract (Fig. 4a) displays a broad peak centered at 3475 cm⁻¹, which is attributed to –OH/NH groups from the protein. The peaks at 2972 and 2933 cm⁻¹ indicate –CH stretching, while the band at 1637 cm⁻¹ is attributed to the C=O of the peptide group. The peak at 1044 cm⁻¹ corresponds to C-O stretch (Cozzolino and Murray, 2012). A specific feature of secondary amides is represented by the peak at 1456 cm⁻¹ (Power and Cozzolino, 2020). The peak at 981 cm⁻¹ confirms the presence of phosphorylated proteins (Jeyachandran et al., 2020), while the antisymmetric stretching of the C-O-C skeleton occurs at 1116 cm⁻¹ and the band at 2338 cm⁻¹, observed in all spectra, is indicative of trapped CO₂ (Cheng et al., 2021).

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Fig. 4

FT-IR spectra of (a) P. semisulcatus extract, (b) CeNC@T1, (c) CeNC@T2, (d) CeNC@T3 and (e) Ag-CeNC@T3.

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In the FT-IR data of CeNC@T1, Ce-O stretching is at 560 cm⁻¹ (Patil et al., 2016). The broad overlapping bands at 3447 cm⁻¹ and 3303 cm⁻¹ are due to –NH/OH of the protein or traces of H₂O molecules. The peak at 1656 cm⁻¹ is attributed to C=O of the peptide group. The peak located at 983 cm⁻¹ confirms P-O of distorted PO₄^3- tetrahedra. The FT-IR spectrum of CeNC@T2 shows a broad band centered around 3421 cm⁻¹ and a peak at 1624 cm⁻¹ corresponding to the stretching and bending modes of H₂O. The presence of bands due to H₂O molecules even after drying at 550 °C indicates that CePO₄ is crystallized in hexagonal form and contains water of hydration, CePO₄·nH₂O (Lucas et al., 2004). The formation of CeO₂ nanoparticles is corroborated by the Ce-O stretching observed at 558 cm⁻¹ (Patil et al., 2016). The broad intense band at 1008 cm⁻¹ and the peak observed at 711 cm⁻¹ represent PO₄^3- groups (Parimi et al., 2019).

FT-IR studies of CeNC@T3 show Ce-O stretching at 562 cm⁻¹ and the peak due to PO₄^3- (broad signal located at 1008 cm⁻¹ in the case of CeNC@T2, is structured into many peaks. The peaks at 952, 992, 1050, 1107 cm⁻¹ correspond to P-O stretching modes of PO₄^3- and the signals at 544, 583, 633 cm⁻¹ are due to bending vibrations of O-P-O and O=P-O, which confirm the presence of distorted tetrahedron of PO₄^3- in monoclinic phase (Bouddouch et al., 2021; Cheng et al., 2021). The peaks associated with H₂O molecules (3421 cm⁻¹ and 1624 cm⁻¹) observed for CeNC@T2 disappear in the spectrum of CeNC@T3 (dried at 800 °C) signifying the formation of monoclinic CePO₄ from hexagonal CePO₄·nH₂O.

Almost similar peaks are identified in the FT-IR profile of Ag-CeNC@T3. Ce-O stretching is observed as a minor peak at 562 cm⁻¹ which is masked by strong P-O bending vibrations. The P-O stretching modes of distorted tetrahedra are observed at 953 cm⁻¹, 992 cm⁻¹, 1061 cm⁻¹ and 1095 cm⁻¹. O-P-O bending and O-P bending corresponding to CePO₄ nanoparticles are located at 541, 581 and 619 cm⁻¹ (Bouddouch et al., 2021).

3.4 Raman spectral analysis

Raman spectrum of CeNC@T3 (annealed at 800 °C) was recorded from 3000 cm⁻¹ to 450 cm⁻¹ and the results are presented in Fig. 5. The inset represents a section of Raman spectrum in the range 1110 cm⁻¹ to 389 cm⁻¹. The most intense signal observed at 468 cm⁻¹ is associated with the F_2g band of CeO₂ (Aslam et al., 2016). A slight shift from 465 cm⁻¹ corresponding to single crystal ceria is related to the polycrystallinity of the nanocomposite (Schilling et al., 2017) or to the presence of defects, phonon confinement and strain effects. The broad nature of this peak indicates the smaller crystallite size of CeO₂ NPs, the presence of defects and strain effects (Gouadec and Colomban, 2007).

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Fig. 5

Raman spectrum of CeNC@T3. Inset: Expanded view from 1110 cm⁻¹ to 389 cm⁻¹.

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Combined E_g + A_1g stretching modes are observed as a small hump at 409 cm⁻¹ (Schilling et al., 2017). The intense peak observed at 976 cm⁻¹ ascertains PO₄^3- tetrahedron present in the nanocomposite. The minor peaks at 998, 1028, 1060, 1072 cm⁻¹ are also due to monoclinic CePO₄ (Allen et al., 2021). The peak at 1072 cm⁻¹ is associated with antisymmetric stretching of the P-O bond (Vinothkumar et al., 2018). The weak bands at 573 cm⁻¹ and 622 cm⁻¹ confirm the antisymmetric bending modes of PO₄^3- group (Vinothkumar et al., 2018; Cheng et al., 2021). The bands of weak intensity resulting from the different phonon symmetry modes corresponding to second order shattering are also observed. Oxygen vacancies present in the cubic fluorite structure are further confirmed by the signal at 1060 cm⁻¹ (Jayakumar et al., 2019). The ‘D band’ corresponding to the defects in nanocomposite is observed at 573 cm⁻¹ (Calvache et al., 2019).

3.5 X-ray photoelectron spectral analysis

The chemical states of Ce and O and the Ce³⁺/Ce⁴⁺ ratio at the surface were assessed using XPS between 0 and 1000 eV. The deconvoluted 3d XPS spectrum of CeNC@T3 is given in Fig. 6b, which consists of 6 doublets (spin–orbit) (Vinothkumar et al., 2018). The peaks positioned at 882.27, 888.43, 898.16, 902.92, 911.39 and 916.46 eV are assigned to v, v′′, v′′′, u, u′′ and u′′′ of 3d_5/2 and 3d_3/2 states Ce⁴⁺ respectively. In addition, it shows four components at 880.75 (v_o), 884.68 (v′), 900.67 (u_o) and 907.03 eV (u′) associated with the main and satellite peaks of Ce³⁺ state of CePO₄ (Beche et al., 2008; Konysheva and Francis, 2013). Ce(IV) present in cerium compounds is characterized by the satellite peak (u) associated with Ce 3d_3/2. The highest binding energy peaks, u and v respectively are at 916.46 (3d_3/2) and 898.16 eV (3d_5/2) and the spin–orbit splitting is 18.3 eV (Matussin et al., 2023). The [Ce³⁺] and [Ce⁴⁺] in CeNC@T3 are 31.7 % and 68.31 %, respectively (Vinothkumar et al., 2018).

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Fig. 6

XPS of CeNC@T3 (a) Survey spectrum, Core level spectra of (b) Ce 3d, (c) P 2p and (d) O 1 s.

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P 2p core level spectrum has an asymmetric peak centered at 133.1 eV which is deconvoluted further into two peaks identified at 132.85 eV (P 2p_3/2) and 133.69 eV (P 2p_1/2) (Fig. 5c), representing phosphorus in CePO₄ (Vinothkumar et al., 2018; Fu et al., 2022). The O 1s core level spectrum of CeNC@T3 shown in Fig. 5d has two peaks at binding energies 529.32 and 530.99 eV. The presence of oxygen in Ce-O lattice is confirmed by the prominent peak at 529.32 eV. P-O bonding from PO₄^3- octahedron corresponding to higher binding energy is located at 530.99 eV (Vinothkumar et al., 2018; Vinothkumar et al., 2018).

Chemical bonding nature of Ag-CeNC@T3 has also been analyzed applying XPS technique. The survey spectrum in Fig. 7a demonstrates that the sample comprises of Ce, O and Ag. The deconvoluted Ce 3d XPS spectrum of Ag-CeNC@T3 has peaks at 882.53, 886.22, 898.08, 900.63, 904.32, 907.95, 911.65 and 916.56 eV (Chen et al., 2022). The concentration of Ce³⁺ and Ce⁴⁺ species in Ag-CeNC@T3 are found to be 22.44 % and 77.56 %, respectively. One notable feature of this spectrum is that the peaks corresponding to Ce³⁺ ions are not located. This is in agreement with DRS data (section 3.1) in which LMCT peak of CeNC@T3 observed at 352 nm is shifted to 336 nm with narrowing of the peak and the band gap is increased to 3.19 eV from 2.85 eV. All these data suggest that on adding AgNPs to CeNC@T3, the defects associated with Ce³⁺ are reduced and some of the Ag⁰ are oxidized to Ag⁺ owing to the smaller work function of Ag compared to CeO₂ (4.2 Vs 4.7 eV) (Matussin et al., 2023; Chen et al., 2022) and both species coexist on the surface of Ag-CeNC@T3. All these data suggest that surface oxygen vacancies of ceria are affected by silver loading and silver predominantly exists as Ag⁰ and oxidation to Ag⁺ occurs to a smaller extent (Chen et al., 2022; Qu et al., 2013).

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Fig. 7

XPS of Ag-CeNC@T3 (a) Survey spectrum, Core level spectra of (b) Ce 3d, (c) Ag^+/0, (d) P 2p and (e) O 1 s.

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The deconvoluted 3d XPS spectrum of Ag has peaks at 367.15 eV due to Ag⁺ and the peaks at 368.87 eV, 373.15 eV and 374.87 eV correspond to Ag⁰. The peaks located at 368.87 and 374.87 eV correspond to Ag 3d_5/2 and Ag 3d_3/2 of metallic silver with spin–orbit coupling of 6 eV (Murugadoss et al., 2021). Further, in the case of Ag-CeNC@T3, the binding energy of Ag 3d_5/2 shows a relative increase compared to that of pure metallic Ag (∼368.2 eV) signaling the loading of AgNPs to the surface of CeO₂. This confirms that a new Fermi energy level is formed in Ag-CeNC@T3, and there is an interaction between Ag and CeO₂ in the nanocomposite (Firet et al., 2019).

In addition, the P 2p peaks are shifted to lower energies 132.52 eV (132.85 eV) and 133.36 eV (133.69 eV), in comparison with CeNC@T3. The deconvoluted O 1s XPS spectrum of Ag-CeNC@T3 has two peaks. While comparing with CeNC@T3, a slight shift to higher energy is observed, 529.73 eV (529.32 eV), 531.32 eV (530.99 eV). The peak at 529.73 eV is due to O₂ in the CeO₂ lattice, whereas the peak observed at 531.32 eV may be correlated O/OH of oxygen adsorbed on the surface or P-O bonding from PO₄^3- (Vinothkumar et al., 2018; Vinothkumar et al., 2018; Qu et al., 2013). No observable OH^– peaks in the CeO₂ nanoparticles adorned with Ag (Murugadoss et al., 2021).

3.6 EDX analysis

The EDX spectra of NCs is summarized in S3 and it features the signatures of cerium, oxygen, phosphorus and silver. In all NCs, a major peak is observed at 4.82 keV corresponding to cerium, and other minor peaks observed around 0.67, 0.87, 5.26, 5.63 and 6.01 keV are also attributed to cerium. In all the NCs, the presence of oxygen is confirmed from the prominent peak obtained at 0.52 keV. Similarly, a significant peak at 2.02 keV is attributed to the presence of phosphorus in all the four NCs (Vinothkumar et al., 2018). Additionally, in Ag-CeNC@T3, silver is confirmed by a salient peak observed at 2.96 keV along with minor peaks at 0.36 and 3.15 keV (Devi et al., 2022).

3.7 HR-TEM analysis

The surface morphologies of the synthesized nanocomposites were analyzed using HR-TEM and the representative images are presented in Figs. 8–10. The particles are found to be spherical and the SAED patterns reveal that the nanocomposites are highly crystalline. The average particle size of CeNC@T1, CeNC@T2 and CeNC@T3 are 2.36 nm, 3.30 nm and 29.64 nm correspondingly. The SAED pattern of CeNC@T2 has (1 1 1), (2 2 0), (3 1 1), (2 2 2), (4 2 0) and (3 3 1) planes of CeO₂ and (1 0 2) planes corresponding to the monoclinic phase of CePO₄. The crystalline nature of the nanocomposites is confirmed by the lattice fringes, and the SAED pattern, showing a ring-like pattern, further proves that the nanocomposites are crystalline, which corroborates with XRD data.

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Fig. 8

HR-TEM analysis: (a)-(d) images of CeNC@T1 at different magnifications, (e) SAED pattern and (f) Histogram representing particle size distribution.

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Fig. 9

HR-TEM analysis: (a)-(d) images of CeNC@T2 at different magnifications, (e) SAED pattern and (f) Histogram representing particle size distribution.

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Fig. 10

HR-TEM analysis: (a)-(d) images of CeNC@T3 at different magnifications, (e) SAED pattern and (f) Histogram representing particle size distribution.

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3.8 Fluorescence study

The colour of fluorescence emission of CeNC@T3 and Ag-CeNC@T3 are found out applying Commission Internationale de l'Eclairage (CIE) chromaticity diagram. CeNC@T3 emitted almost a white light and Ag-CeNC@T3 emitted light close to the blue light region, which are shown in Fig. S2 a & b. The room-temperature fluorescence spectra of CeNC@T3, Ag-CeNC@T3 and CePO₄NC@T1 were recorded as solid. The solid fluorescence spectra of NCs at different excitation wavelengths are given in Fig. 11.

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Fig. 11

Fluorescence spectra of solid CeNC@T3, Ag-CeNC@T3 and CePO₄NC@T1 at different excitation wavelengths.

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CeO₂ fluoresces in UV region due to emission from 4f energy levels (Krishnan et al., 2013) but owing to the defects attributed to Ce³⁺ it can emit light in visible region (Mochizuki and Fujishiro, 2009). The 220 nm and 240 nm excited emission spectrum of CeNC@T3 has peaks at 470, 486, 542, 616 and 824 nm and similar less intensity emission peaks are observed in 330 nm and 350 nm excitations. Ag-CeNC@T3 nanocomposite has major peaks at 396, 468, 486, 538 and 618 nm. The highest intensity emission signal observed around 470 nm in all the spectra is due to emission from 4f energy level. The less intense bands peaking at 542 and 616 nm could be attributed to emissions originating from oxygen defects. The emission bands spanning from 400 to 500 nm in the CeO₂ samples are ascribed to transitions between various defect levels and the valence band (Aslam et al., 2016). The 5 excitation peaks corresponding to the 4f → 5d transition, induced by Ce³⁺ ions, and the peak at 300 nm is one of them (Qin and Niu, 2016). The emission spectrum of CePO₄ nanocomposite formed using Ce³⁺ precursor (section 2.5) is compared with those of CeNC@T3 and Ag-CeNC@T3. CePO₄NC@T1 (air dried) contains only CePO₄ in hexagonal structure. The emission peaks are almost the same, suggesting that the emission is mainly due to CePO₄.

4.1 Sensing of Pb²⁺, Cd²⁺ and Hg²⁺

As the CeO₂-CePO₄ NCs display an intense fluorescence emission, their application to sense various metal ions is explored. The presence of various metal ions, especially Pb²⁺, Cd²⁺ and Hg²⁺ leads to an augmentation in the emission intensity (Fig. S3 & S4). Hence the potential of aqueous dispersions of CeNC@T3 to sense these heavy metals in the wide range of concentrations (10^-9 M to 10^-5 M) is investigated.

On mixing Pb²⁺ ions to the aqueous dispersion of CeNC@T3, the emission intensity proportionally enhanced with [Pb²⁺] (Fig. S5). When altering the concentration of Pb²⁺ from 5.0x10^-9 M to 4.0x10^-5 M, the fitting plot exhibits three clearly defined linear ranges (Fig. 12). The corresponding linear fitting equations are Y₁ = 6.4675X + 269.7334; Y₂ = 0.8546X + 2863.75; Y₃ = 0.04052X + 6574.321 with a correlation coefficient of 0.9934, 0.9978 and 0.9895 respectively, where X is the concentration of Pb²⁺ in nM. The LOD is found to be 2.41x10^-8 M, 1.83x10^-7 M, 3.85 x10^-6 M in three regions respectively. The inset of Fig. 12 illustrates the magnified view of the low concentration range of Pb²⁺. This linear region is highly sensitive to the concentration of analytes. Hence it is worth developing CeNC@T3 as an inorganic fluorescent probe which can be easily prepared and is compatible with green chemistry for sensing heavy metals.

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Fig. 12

Plot of fluorescence intensity (F-F₀) Vs [M²⁺], nM for CeNC@T3, Inset: Expanded view of 5 nM to 350 nM region.

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Similarly, the Cd²⁺ and Hg²⁺ ions also increase the emission intensity to a greater extent (Fig. S6 & S7). In these cases also, there are three linear regions in which the emission intensity is directly proportional while increasing the concentration of these ions 10,000 folds between 5.0x10^-9 M to 4.0x10^-5 M (Fig. 12). The regression equations for Cd²⁺ are Y₁ = 6.1004X + 323.5808; Y₂ = 0.5861X + 2468.1944 and Y₃ = 0.06173X + 5091.25 with a correlation coefficient of 0.9890, 0.9993 and 0.9915 respectively, where X is the concentration of Cd²⁺ (nM). The LOD is calculated to be 2.56x10^-8 M, 2.66x10^-7 M, 2.53x10^-6 M. The regression equations for Hg²⁺ are: Y₁ = 5.02132X + 324.3137; Y₂ = 0.4211X + 1932.3333; Y₃ = 0.06484X + 3889.4643 with a correlation coefficient of 0.9834, 0.9946 and 0.9917 respectively, where X is the concentration of Hg²⁺ (nM). The LOD is 3.11x10^-8, 3.71x10^-7, 2.41 x10^-6 M.

The fluorescence intensity of Ag-CeNC@T3 is also greatly enhanced in the presence of Hg²⁺, Pb²⁺ and Cd²⁺ and the increase is much higher than CeNC@T3 (Fig. 13). On mixing the Hg²⁺ ions to an aqueous dispersion of Ag-CeNC@T3, the emission intensity is directly proportional to [Pb²⁺]. Three linear regions are observed on varying the concentration of Hg²⁺ from 5.0x10^-9 M to 4.0x10^-5 M. The corresponding regression equations are Y₁ = 9.4062X + 1363.4360; Y₂ = 0.8473X + 4118.3333; Y₃ = 0.1366X + 7664.2857 with a correlation coefficient of 0.9823, 0.9883 and 0.9953 respectively, where X represents the concentration of Hg²⁺ (nM). The LOD is 10.13x10^-8 M, 11.24x10^-7 M, 6.97 x10^-6 M.

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Fig. 13

Plot of fluorescence intensity (F-F₀) Vs [M²⁺], nM for Ag-CeNC@T3, Inset: Expanded view of 5 nM to 350 nM region.

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Pb²⁺ and Cd²⁺ ions also increase the emission intensity to a greater extent. In these cases also, there are three linear regions in which the emission intensity is directly proportional while increasing the concentration of these ions 10,000 folds between 5.0x10^-9 M to 4.0x10^-5 M. The regression equations for Pb²⁺ are Y₁ = 8.9245X + 1293.9777; Y₂ = 0.7873X + 4206.1111; Y₃ = 0.1091X + 7683.9286 with a correlation coefficient of 0.9750, 0.9979 and 0.9990 respectively, where X is the concentration of Pb²⁺ (nM). The Limit of Detection in the three linear regions is found to be 10.68x10^-8, 1.21x10^-6, 8.73x10^-6 M. The regression equations for Cd²⁺ are Y₁ = 9.02267X + 1185.5860; Y₂ = 0.4893X + 4543.333; Y₃ = 0.05636X + 6455.7143 with a correlation coefficient of 0.9819, 0.9975 and 0.9807 respectively, where X is the concentration of Cd²⁺ (nM). The LOD is estimated as 10.56x10^-8 M, 1.95x10^-6 M, 1.69x10^-5 M in the three different regions.

Various mechanisms have been reported in literature for the sensing of heavy metals by CePO₄ based nanocomposites. The coordination of Pb²⁺ ions with phosphate group forming an insoluble complex is reported to be the reason for the quenching of fluorescence of cerium phosphate (Dezfuli et al., 2014); (Khandare et al., 2014); (Song et al., 2019). The presence of Hg(II) leads to enhanced fluorescence in biofunctionalized Ag nanoparticles. This is a result of fluorescence resonance energy transfer, facilitated by the aggregation of nanoparticles brought about by the complexation of Hg(II) with Ag nanoparticles (Sebastian et al., 2018). A similar pattern has been documented in the instance of Ru-loaded CeO₂ NCs for electrochemical detection of Hg(II), attributed to an abundance of oxygen vacancies (Sun et al., 2020). In this case, it appears that the heavy metals are adsorbed over the oxygen vacancies created in the NCs, enhancing fluorescence. As the availability of vacancies decreases, the intensity of fluorescence also decreases, leading to different linear regions.

4.2 Evaluation of SPF of CeNC@T1 and CeNC@T2

Commonly, sunscreens are protectants that either absorb or block the harmful UV rays from sunlight and show immunosuppressive effects. In summer, using sunscreen is an efficient way for skin protection, which lessens UV- induced ROS-mediated photoaging. SPF is defined as the ratio of the required UV energy to generate a Minimum Erythema Dose (MED) on protected skin to the UV energy essential to produce MED that is not protected. MED is the shortest span of time or amount of UV light exposure required to cause the most perceptible and minimal erythema on exposed skin. SPF number determines the efficacy of a sunscreen-based product. Sunscreens are more effective at preventing sunburns if their SPF value is higher.

The results of SPF at different concentrations of CeNC@T1 are given in Fig. 14. SPF values exhibit a marked increase with increasing concentration of NC. The possible mechanism for the good UV shielding property of NC can be due to their smaller size (2.36 nm), larger band gap (E_g = 3.03 eV) and photon energy consumption. The SPF of CeNC@T2 is also given in the figure for comparison. CeNC@T2 demonstrates less SPF in comparison to CeNC@T1, this may be due to their larger size and less band gap (E_g = 2.95 eV) compared to CeNC@T1 and photon energy consumption (Miri et al., 2020). High SPF values are observed even with low concentrations (100 µg/mL), making this system more significant than those reported in literature (Table 1).

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Fig. 14

SPF of CeNC@T1 and CeNC@T2.

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4.3 Evaluation of the cytotoxic effect of CeNC@T1 and CeNC@T2

The variation of percentage cell viability with different concentrations of CeNC@T1 and CeNC@T2 are shown in Fig. 15. It is found that a concentration of 6.25–25 µg/mL yields a viability rate of 99 %. At 50 and 100 µg/mL concentration, it is 98.62 % and 97.93 % for CeNC@T1 and 98.15 % and 97.70 % for CeNC@T2 respectively, as given in Figs. 16 & 17. According to the findings of cytotoxicity studies, L6 cell line is not significantly harmed by the marine shrimp produced CeNC@T1 and CeNC@T2 and shows no notable toxicity (Miri et al., 2021).

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Fig. 15

Cytotoxic findings for CeNC@T1 and CeNC@T2.

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Fig. 16

Cytotoxic results for CeNC@T1: (a) Control, (b)-(f) Different concentrations of CeNC@T1 (6.25–100 µg/mL) incubated with L6 cell line.

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Fig. 17

Cytotoxic results for CeNC@T2: (a) Control, (b)-(f) Different concentrations of CeNC@T2 (6.25–100 µg/mL) incubated with L6 cell line.

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