3.4.3 Recognition selectivity of MTCFs@SIP@CBMA
Taking isoelectric point and molecular weight as investigating factors, OVA, HSA, Lyz, and Bhb were selected as protein analogues to investigate the recognition selectivity of MTCFs@SIP@CBMA to BSA. The adsorption capacities of MTCFs@SIP@CBMA and MTCFs@NIP@CBMA on BSA and other four proteins are illustrated in Figure 8. The IF values are listed above the histogram. Obviously, MTCFs@SIP@CBMA exhibited a higher adsorption capacity and IF for BSA than the other four proteins. This was explained by that BSA matched the imprinting sites of MTCFs@SIP@CBMA in three-dimensional space and functional group arrangement. HSA not only possessed an isoelectric point and molecular weight similar to BSA, but also their three-dimensional conformation and 60% amino acid sequence were also very similar. Thus, the adsorption capacity and IF of MTCFs@SIP@CBMA to HSA ranked second. However, the adsorption capacity and IF of HSA were much lower than those of BSA because the exposed functional groups were different from BSA and were affected by the anti-protein adsorption segments. The isoelectric point of OVA was similar to that of BSA, but the molecular weight was smaller. The molecular weight of Bhb was close to that of BSA, while Lyz had a smaller molecular weight. However, due to the mismatching of charge and the effect of anti-protein adsorption chain segments, the adsorption capacities and IF values of MTCFs@SIP@CBMA for Bhb and Lyz were not high. In view of above analysis, the order of adsorption capacities and IF values of MTCFs@SIP@CBMA for the other four proteins was HSA > OVA > Bhb > Lyz. The β values calculated by IF are listed in Table S1. The β values of MTCFs@SIP@CBMA for OVA, HSA, Lyz and Bhb were 5.81, 5.25, 6.67 and 6.03, indicating that MTCFs@SIP@CBMA could selectively identify BSA. From above results the conclusion can be inferred that the introduction of CBMA anti-protein adsorption chain segments can indeed improve the recognition selectivity of imprinted materials.
Figure 8 Selective adsorption of MTCFs@SIP@CBMA and MTCFs@NIP@CBMA toward BSA, OVA, HSA, Lyz and Bhb. Error bars represent ± standard deviations, n = 3.
3.4.4 Competitive adsorption
In order to further evaluate the recognition selectivity of MTCFs@SIP@CBMA fibers, adsorption experiments were carried out with binary mixed protein of Bhb and BSA as adsorption objects. The mixed proteins (CBhb=CBSA=0.1 mg/mL) were adsorbed with 1 mg MTCFs@SIP@CBMA or MTCFs@NIP@CBMA for 30 min. After magnet separation, the absorption spectrum of proteins in supernatants was scanned by UV-vis spectrophotometer with a scanning range of 250-550 nm. As can be seen from Figure 9a, the absorbance of the mixed proteins at 278 nm decreased significantly after adsorption by MTCFs@SIP@CBMA, while the absorbance at 409 nm remained almost unchanged. When MTCFs@NIP@CBMA was selected as the adsorbent, BSA was adsorbed and Bhb was simultaneously adsorbed (the absorbance at 278 nm and 409 nm were both reduced). The results showed that MTCFs@SIP@CBMA could selectively recognize BSA in binary mixed proteins while MTCFs@NIP@CBMA could not. Moreover, SDS-PAGE gel electrophoresis was used to analyze whether MTCFs@SIP@CBMA can specifically recognize BSA from the five-component mixed protein solutions of BSA, OVA, HSA, Lyz, and Bhb. The results are given in Figure 9b. Compared with lane 2, the intensity of the corresponding BSA band in channel 3 was weakened, while the intensity of other protein bands hardly changed. Compared lane 4 with lane 2, it was found that the band strength of each protein did not change significantly. In lane 5, only a BSA band was present in the primary eluent fractions of MTCFs@SIP@CBMA. The above results indicated that MTCFs@SIP@CBMA could also specifically recognize BSA from multicomponent mixed proteins. In summary, MTCFs@SIP@CBMA demonstrated outstanding recognition selectivity for BSA, which was ascribed to the reduction of non-specific adsorption by anti-protein adsorption chain CBMA grafted on the surface of the imprinted layer.