Analysis of conditions for the preparation of BSA/MMT composites

2.1 Experimental materials

Na-MMT was obtained from Jilin Liufangzi Bentonite Tech Co., Ltd, Changchun City, China. The ideal formula of MMT is (M_x⁺nH₂O)(Al_2-xMg_x)^VI[Si₄^IVO₁₀](OH)₂ and M indicates the interlayer cation such as Ca²⁺, Na⁺ and Mg²⁺[10]. BSA (analytically pure) was purchased from Beijing Solarbio Science & Technology Co., Ltd (China), imported from Roche Group (Switzerland), which is a simple protein whose molecular weight is 68 KD. It comprises 581 amino acid residue and its isoelectric point is 4.8. KH570 (purity >98.0%) was purchased from Nanjing Xinhuai Chemical Co., Ltd, Nanjing City, China. Other chemicals and reagents were analytically pure.

2.2 Modification of MMT and BSA adsorption

The MMT was purified by sedimentation and then sieved through a 200 mesh. The concentration of KH570 was chosen to be 2%, 6% and 10% by diluting with 80%, 90% or 100% of ethyl alcohol, respectively. A certain amount of Na-MMT was mixed with different concentrations of KH570 for 12 h. The treated MMT was then washed by pure ethanol solution twice and DI water twice to remove KH570. The MMT modified by KH570 was added into centrifugation tubes. A total of 1.875 ml of 0.12 g ml/l BSA solution was poured into a centrifugation tube and diluted with buffer solutions to 5 ml. After 12 h, centrifugalized under 8900 g for 15 min at 10°C, the precipitation was washed with corresponding buffer solutions two or three times. The supernatants were combined and the concentration of BSA was determined by Folin-phenol method. The amount of protein that intercalated into MMT was calculated by subtracting the content in the supernatant from one in the original protein solution. The results of different washing time (twice or three times) of the protein composite precipitate were compared in the experiment.

2.3 Orthogonal test of BSA adsorption

Based on single factor experiments, the level of L8 (2⁷) orthogonal table was applied for experimental design. Experimental factors include pH value, buffer, KH570 concentration, and the amount of BSA. The pH values was 4.8 or 5.8; KH570 concentration was 2% or 10%; buffer solution was selected as phosphoric acid or acetic acid, the volume of BSA solution was 375 or 1125 μl. 0.05 g MMT was treated by KH570 solution overnight in the tube, washed, and added with BSA solution. The solution was mixed for 10 h, centrifuged for 15 min at 8900 g at 10°C. The precipitate was washed three times, supernatants were combined and BSA was determined as previously described.

2.4 Desorption experiment

In the experiment, 0.1 g MMT was treated separately with 2% KH570 according to the previously described methods. Then, after adding 3.75 ml 0.12 g/ml BSA solutions, the mixtures were left statically for 10 h. The complexes were washed after the precipitation three times, dried and ground into powder and then dissolved into buffer (pH=7.0). After shaking for a period of time, they were placed at 4°C to release. The concentration of protein was measured every three days to calculate protein desorption content. The determination lasted for 21 days.

2.5 Material characterization

X-ray diffraction spectra were recorded by a TD-3000 diffract meter made by Dandong Tongda Science and Technology Co., Ltd, Dandong City, Liaoning province (China), using a CuKα X-ray source. The voltage and current were kept at 30 KV and 20 mA, respectively. Scans were collected at 2° per min from 1.5 to 40° 2θ.

Fourier transform infrared (FTIR) spectra tests were carried out on a Nicolet 510P infrared spectrometer. The samples were mixed with spectroscopic grade KBr. The ratio of composite and KBr was 1:10, and the resolution was 4 cm^-1; Scanning spectrum was 4000–400 cm^-1.

3.1 Effect of pH values on adsorption

As can be seen from Figure 1, the pH values of the buffer solution affected the content of adsorption. When the pH values of the solution declined from the isoelectric point (pH=4.8), the adsorption quality of BSA increased. On the contrary, with the increase of pH values, the adsorption content of BSA decreased and the least adsorption quality was at pH value 7.6. This is because the BSA molecules were positively charged and easily intercalated into the layers of MMT. This was through ion exchange when the pH values of the solution were lower than the isoelectric point, while there was intercalated difficultly with the pH values higher than the isoelectric point.

Figure 1

Effect of ionic strength and pH values on adsorption.

3.2 Effect of ionic strength on adsorption

As shown in Figure 1, when the ionic strength is 0.3125 mol/l, the adsorption quantity reaches maximum value of 1.028 g/g. Subsequently, the adsorption quantity falls with the increase of ionic strength. The main reason for these effects is that ionic strength affects the exchange of BSA and Na⁺ iron. Higher ionic strength will prevent the charged protein molecules from exchanging into the interlayer of MMT.

3.3 Effect of buffers, KH570 and ethyl alcohol concentrations

The effect of buffers on adsorption is shown in Figure 2. The adsorption content is different at the same concentration of KH570, the adsorption content in the buffers is significantly more than that in the water. Comparing the two buffers, the phosphate buffer is better than the acetate buffer. The concentrations of KH570 also have an effect on the adsorption quantity and the 10% is better than the 2%. This is due to the higher concentration being more helpful for surface organic modification than the lower one. Comparing the effect of the two different concentrations of ethanol that are used as the solvent of KH570 shows that 80% of the ethanol contributes to adsorption of BSA on the MMT in the water or acetic acid buffer system, however, it has the completely opposite result in the phosphate buffer.

Figure 2

Effect of buffers, KH570 and ethyl alcohol concentrations.

W, water solution; P, sodium phosphate buffer solution; A, sodium acetate-acetic acid buffer solution.

3.4 Effect of washing times on adsorption

Washing times also affect the adsorption content as the adsorption quantity reduces as the washing time increases. The decreasing amplitude of content in the acetic acid buffer is more than that in the phosphate buffer. The mechanisms of phosphate and acetic acid buffers are different with the changing of the pH value. It can be concluded that the phosphate buffer contributes to the adsorption of BSA on the MMT. A possible reason is that acetic acid molecules contribute to the spin-off of the MMT layers. The phosphate is possible a catalyst which catalyzes the reactions of the chemical groups on the MMT lamellae with the functional groups of KH570.

3.5 Analysis of orthogonal test

The result of the orthogonal test is shown in Table 1. The pH values, concentration of KH570 and the dosage of BSA impact the results significantly. According to the analysis of variance, the third column can stand for the interaction between the pH values and the buffers and its effect is obvious. These results can also be seen from the analysis of extreme deviation. As can be seen from these data, the influence of various factors follows the order: interaction of the buffer and pH, the coupling agent, pH effect, and the dosage of BSA.

3.6 Protein desorption experiment on the MMT

The result of the BSA desorption content is shown in Figure 3. The three solvents are water, acetic acid buffer and phosphate buffer, respectively. Acetic acid buffer is the most conducive to the release of intercalation protein. This may due to the acetic acid molecules contribute to the spin-off of the MMT layers. The release content in the phosphate buffer is also more than that in the water environment. The MMT treated with KH570 can reduce the structure change of the BSA and contribute to the slow releasing of the BSA. In the water environment, the BSA integrates with the layer of the MMT and the released content is the least. In most cases, KH570 dissolved in 100% ethanol is more helpful to protein release.

Figure 3

The amount of slow-release in different experimental conditions.

P, sodium phosphate buffer solution; A, sodium acetate-acetic acid buffer solution; W, water solution.

3.7 X-ray diffraction (XRD) analysis

The XRD spectra of BSA intercalated into MMT treated with 2% KH570 is shown in Figure 4. The value of d₀₀₁ for untreated MMT is 1.14 nm. After 2% KH570 treatment which was dissolved in 100% ethanol solution, the KH570-MMT was intercalated by BSA. The d₀₀₁ peak of the BSA/KH570-MMT in the acetic acid buffer can not be detected in the range of the detection. It can be concluded that the MMT layer is being stripped while others can not. The d₀₀₁ peak is 2.67 nm in the phosphate buffer system. When the KH570 is dissolved in 80% ethanol solution the d₀₀₁ peak is scattered, indicating that the existence of water molecules can play an important role in the hydrolysis of KH570.This makes the interaction between MMT surface and KH570 more complicated as more bonds may be produced [11]. The 0.310 nm peak which is the d₀₀₅ peak of MMT disappeared after the treatment with KH570. This indicates that the structure of MMT changes to a certain extent. XRD spectra also contain peaks (0.329 nm) of quartz which is produced by the impurities in MMT.

Figure 4

X-ray diffraction (XRD) patterns of BSA/KH570-MMT, MMT.

P, sodium phosphate buffer solution; A, sodium acetate-acetic acid buffer solution.

3.8 FTIR analysis

The FTIR spectra of BSA intercalated MMT composite treated with 2% KH570 is shown in Figure 5.

Figure 5

Fourier transform infrared (FTIR) spectra of BSA/KH570-MMT.

P, sodium phosphate buffer solution; A, sodium acetate-acetic acid buffer solution.

The characteristic peak of 1530–1550 cm^-1 is the stretching vibration of amide II, which is illustrated in the BSA/MMT composite but it is significantly strengthened in the acetic acid buffer with the KH570 dissolved in 80% ethanol solution. The peak of 1410 cm^-1 which corresponds to the stretching vibration of C–N and the peak of the asymmetric stretching vibration of -CH₃ (2959 cm^-1) are both significantly strengthened with the same treatment. The characteristic peak of 3610–3640 cm^-1 is the stretching vibration of free -OH which becomes weaker with the intercalation of BSA. While the peak of 3200–3550 cm^-1 corresponds to the stretching vibration of intermolecular hydrogen bonding intensifies. This may be caused by the interaction between BSA molecules and MMT layers.