Low cost and eco-friendly nanoparticles from cockle shells as a potential matrix for the immobilisation of urease enzyme

2.1 Chemicals

Acrylic acid N-Hydroxysuccinimide ester (NAS) was supplied by Sigma Aldrich. Phenolphthalein (PP) and the Bradford reagent were purchased from R&M Chemicals. The Urs enzyme (RN 9002-13-5; 490 units/mg from jack beans) and bovine serum albumin (BSA) with 98.0% purity were purchased from Tokyo Chemical Industry Co. and Vivantis Technologies Sdn Bhd, respectively. Phosphate-buffered solution (PBS) was prepared using di-potassium hydrogen phosphate anhydrous (K₂HPO₄) and potassium dihydrogen phosphate (KH₂PO₄) supplied by HmbG Chemicals. All chemicals were of analytical grade and used without any further purifications. All solutions and standard buffer solutions were prepared with deionised water.

2.2 Preparation of the CaCO₃ NPs from wasted cockle shells

The production of micro-sized particles was performed according to Islam et al. (2012) with slight modifications. Cockle shell waste obtained from seafood restaurants were cleaned and scrubbed to remove any dirt, then boiled for 10 min before being left to cool down at room temperature. Following this, the shells were washed thoroughly using distilled water and dried in an oven at 50 °C for five days before finely grounded using a blender (Seiketsu Cosmos Soylove II) and sieved using a stainless steel laboratory sieve with an aperture size of 63 μm to obtain micron-sized (10–63 μm diameter) powders.

The following procedures were carried out according to the method by Karthick et al. (2014), with some modifications, to obtain nano-sized CaCO₃. Micron-sized cockle shell powders were sonicated in distilled water using the QSONICA Q125 sonicator. For the sonication process, approximately 5 g of the sample was mixed in 50 mL distilled water and the total process duration was 15 min, with 3 s OFF and ON pulse simultaneously using 30% of amplitude. The products were then filtered using a filter paper and dried at 50 °C for two days in an oven.

The dry particles were milled using a planetary ball milling machine. First, a stainless steel jar (volume of 50 mL) and a stainless steel ball (diameter: 4.0 ± 0.01 mm, weight: 0.51 ± 0.005 g/ball) were cleaned using distilled water to remove any contaminants. A quarter of the jar was filled with a 10:1 ball to sample ratio to generate a high energy impact. For milling, a rotational speed of 500 rpm was used for 3 h before the balls were removed and cleaned as the powder attached to the surface of the balls would reduce the collision energy. The same step was repeated twice with another 3 and 2 h of milling. Finally, the CaCO₃ NPs of cockle shells were collected and dried at 50 °C for 3 h to remove the moisture before further use.

2.3 Functionalisation of CaCO₃-NPs

The functionalisation of CaCO3-NPs was carried out according to a previous method (Ulianas et al., 2011). A total of 15 g NPs was suspended in 50 mL deionised water, then approximately 0.3 g of NAS was added and the mixture was stirred for 24 h at room temperature. The functionalised NPs were repeatedly washed, centrifuged, and sonicated with deionised water to reduce the pH level to pH 7. Finally, the modified NPs were dried at room temperature for 2 days and labelled as F-NPs.

2.4 Immobilisation and activity of the Urs enzyme

A combination of 94 mg NPs and 6 mg NAS was stirred in distilled water for 24 h to produce F-NPs. The F-NPs were then centrifuged and washed multiple times with PBS to remove the unbound NAS and reduce the pH level. The Urs enzyme (0.2 mL, 2.0 mg/mL of PBS) was then added to the dried F-NPs and kept at 4 °C for 24 h to allow the Urs enzyme to attach onto the surface of the F-NPs. The resulting F-NPs suspension immobilised with Urs enzyme was then centrifuged at 3000 rpm for 30 min and washed three times with 0.05 M PBS (pH 7) to remove the loosely bound Urs enzyme. The immobilised Urs was then dried and kept at 4 °C until further use.

The percentage of immobilised Urs on the surface of F-NPs was determined according to the Bradford protein assay protocol as previously described (Ulianas et al., 2011). In brief, a series of BSA concentrations ranging from 0.006 to 0.06 mg/mL were prepared to form a linear calibration graph of the BSA-protein standard. For the determination of immobilised urease adhering to the F-NPs surfaces, 100 mg F-NPs were added to 200 μL Urs (2.0 mg/mL) in a centrifuge tube and kept at 4 °C for 24 h. After 24 h, the Urs/F-NPs mixture was centrifuged and the supernatant was decanted. Following this, 60 μL of the decanted solution was then mixed with 940 μL Bradford reagent and 1000 μL deionised water then the absorbance of the solutions was measured at λ = 595 nm using the UV–Visible Spectrophotometer Cary 50. The amount of immobilised Urs was calculated according to the following equation (Alqasaimeh et al., 2014; Ulianas et al., 2011): $$\%\mathrm{U}\mathrm{r}\mathrm{s}\mathrm{i}\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{d}=[(\mathrm{A}-\mathrm{B})/\mathrm{A}]\times 100\%$$ where A is the amount of Urs used and B is the concentration of the Urs left after immobilisation.

2.5 Sample characterisation

The prepared NPs were identified using FTIR in the range of 400–4000 cm⁻¹ and Energy Dispersive X-Ray Analyser (EDX). The morphology and structure of the samples were analysed using a Field-Emission Scanning Electron Microscopy (FESEM, ZEISS). After the sonication and milling processes, the sizes of the NPs produced were analysed using the Particle Size Analyser (PSA, Malvern Instruments). The chemical bonds between NPs, NAS, and the Urs enzyme were determined using FTIR and XPS (Shimadzu).

3.1 Particle size of CaCO₃-NPs

Table 1 shows the particle size distribution of cockle shell powders which was determined by several processes including grinding, sieving, sonication, and milling. The cockle shells were grounded and sieved using an aperture size of 63 μm siever and the sieved powders with sizes from 10 to 63 μm underwent a sonication process to reduce the particle size. However, a long sonication period can cause agglomeration of the particles (Franco et al., 2004). In this study, a 15 min of sonication process reduced the size of the powders to within the range of 0.62–1.99 μm, thereby reducing the duration of the milling process. The average size (hydrodynamic diameter) of CaCO₃-NPs as measured by the PSA was 78.8 ± 10.8 nm after 8 h of milling. The particle size distribution of NPs is shown in Fig. 1.

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Fig. 1

The particle size distribution of NPs after 8 h of the milling process.

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3.2 EDX analysis of NPs

The elemental compositions of the NPs from the cockle shells were analysed by single point EDX mapping, revealing each element that was present in the sample as shown in Fig. 2. Findings showed that the NPs contained 51.3% oxygen, 30.2% calcium, 18.0% carbon, and 0.5% sodium, and had greater proportions of carbon and oxygen compared to the ratio of calcium as shown in Fig. 2. The two peaks at 2 keV and 4 keV were due to the iridium that was used to coat the surface of the NPs to prevent charging of the sample surface. This analysis has shown that the composition of NPs from cockle shells was approximately 99.5% of CaCO₃.

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Fig. 2

Spectral peak data of CaCO₃ NPs from EDX spectroscopy.

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3.3 Morphological characteristics of NPs, F-NPs, and F-NPs immobilised with Urs

The morphological characteristics obtained using FESEM revealed the structure of aragonite CaCO₃-NPs, which were smooth and irregular in shape, as shown in Fig. 3 (a). A comparison of the images of F-NPs and F-NPs immobilised with Urs displayed in Fig. 3 (b) and (c) confirmed that F-NPs were successfully immobilised with Urs. While F-NPs (Fig. 3 (b)) typically have a smooth surface morphology, the surface structure of F-NPs changed and appeared sticky, as shown in Fig. 3 (c), after immobilisation with Urs. This indicated that the enzyme immobilisation process did not significantly agglomerate the particles and alter the structure of F-NPs. Furthermore, this also indicated that the reaction occurred only on the surface of the F-NPs through interactions between the succinimide group of NAS and the amino groups of Urs (Migliorini et al., 2018).

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Fig. 3

FESEM images of (a) NPs, (b) F-NPs, (c) Urs/F-NPs.

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3.4 FTIR analysis

In Fig. 4, the absorption peaks at 1780.74 cm⁻¹, 1444.34 cm⁻¹, 1080.93 cm⁻¹, 857.29 cm⁻¹, and 711.92 cm⁻¹ from the spectral data of NPs were common characteristic features for CO₃²⁻ in CaCO₃ of cockle shells, as previously reported (Islam et al., 2012). The characteristic peaks representing CO₃²⁻ of aragonite were observed at 1080.93 cm⁻¹ and 857.29 cm⁻¹, due to the symmetrical stretching and out-of-plane bending modes of the structure (Kamba et al., 2013). Following the addition of NAS to CaCO₃-NPs, the characteristic absorption of N-H stretching vibration appeared at 3368.38 cm⁻¹ and peaked at 1646.55 cm⁻¹ (corresponding to N-H stretching in amide II). The increased characteristic peak at 1435.96 cm⁻¹ confirmed the formation of C-O bonds between CaCO₃ and the acrylic of NAS. Data from the FTIR spectrum after immobilisation of Urs over F-NPs showed additional bands at 1120 cm⁻¹ and 1016.63 cm⁻¹ due to the presence of C-N of the aliphatic amine and the amide bond, revealing the immobilisation of Urs to F-NPs (Souza et al., 2013).

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Fig. 4

FTIR spectra of the NPs, F-NPs, and Urs/F-NPs.

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3.5 XPS analysis

Analysis of two samples, one before and one after immobilisation of Urs, was performed using XPS to measure the presence of the enzyme on the functionalised CaCO₃. As seen in Fig. 5 (a), additional peaks were identified between 100 and 200 eV, which indicated the presence of S and P elements. The immobilisation of Urs was confirmed by the presence of sulphur through XPS analysis at around 170 eV after immobilisation, as all enzymes contain amino acids with sulphur bonds (Adeloju et al., 1993). In addition, the P peak at 131 eV demonstrated the presence of phosphate groups from the active sites of jack bean Urs, and there was no peak before immobilisation (Balasubramanian & Ponnuraj, 2010). Based on Fig. 5 (b), a peak at 284.3 eV in the spectrum after the immobilisation of Urs indicated the presence of C—C bonds, while the peak at 287 eV was related to C—O bonds, and the last peak at 287.6 eV was related to amide (C⚌O) bonds (Barreca & Gentile, 2018; Kahoush, 2019). The relative carbon content on the surface decreased with increased oxygen due to oxidation following Urs immobilisation (Kahoush, 2019). The increase in oxygen atoms (529 eV) and nitrogen atoms (around 400 eV) in Fig. 5 (c) and (d) after Urs immobilisation on the surface of functionalised NPs was due to increased C⚌O and C—N bonds respectively. Fig. 5 (d) displays a typical Ca 2p high-resolution XPS spectrum for both before and after the immobilisation of Urs on the surface of the NPs corresponding to Ca (II) ions. The immobilisation of Urs resulted in a Ca 2p peak shift to lower binding energy, indicating interactions between functionalised NPs and Urs (Migliorini et al., 2018).

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Fig. 5

A comparison of XPS spectra of F-NPs before and after immobilisation with Urs. (a) XPS survey spectra, (b) C 1s scan, (c) O 1s scan, (d) N 1s scan, and (e) Ca 2p scan.

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3.6 Percentage of Urs immobilised onto the matrix

The amount and percentage of Urs enzyme were quantitatively determined based on the calibration graph of standard BSA in the range of 0.006–0.06 mg/mL with R² = 0.9943, as seen in Fig. 6. The amount of Urs immobilised on different amounts of F-NPs, as calculated in Section 2.4, is shown in Fig. 7. The weight of F-NPs as a matrix affected the amount of enzyme immobilised on the F-NPs. Increased matrix weight, which provided a higher succinimide ester group, resulted in an increased amount of immobilised enzyme, demonstrating that the binding of F-NPs with Urs occurred through covalent bonding which was supported by FTIR and XPS analysis.

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Fig. 6

Calibration graph of standard BSA.

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Fig. 7

The amount of Urs immobilised on the F-NPs at various sample weights.

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