Volume 11 Issue 10 - December 18, 2009 PDF
Via zinc(II) protoporphyrin to the synthesis of poly(ZnPP-MAA-EGDMA) for the imprinting and selective binding of bilirubin
Shih-Kai Chou, Mei-Jywan Syu*

Department of Chemical Engineering, College of Engineering, National Cheng Kung University

Biomaterials 30 (2009) 1255–1262

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Poly(zinc protoporphyrin-methacrylic acid-ethyl glycol dimethylacrylate) (poly(ZnPP-MAA- EGDMA)) imprinted with α-bilirubin can cause spectroscopic change in wavelength and absorption intensity due to the metal ion coordination between ZnPP and bilirubin. The fluorescent imprinted polymer was able to selectively bind α-bilirubin. The corresponding imprinted polymer monolith was synthesized by using the functional monomer, methacrylic acid and the fluorescent monomer, zinc(II) protoporphyrin. It revealed that via the combined utilization of ZnPP and MAA for the fluorescent and functional effect, the MIPs thus prepared were then able to create the highly selective cavities. The imprinting factor of 3.069 could be achieved from the fluorescent imprinted polymer by comparing the results from the MIP and the NIP (non-imprinted polymer). The imprinting factor from bilirubin/biliverdin mixture was reduced to 2.111 because of the presence of biliverdin. The selectivity toward bilirubin of 2.269 from the bilirubin/biliverdin mixture was obtained.

Bilirubin occurs in mammals and is abundant in blood plasma. It is oxidized from biliverdin. In the metabolism, only a small fraction of bilirubin is present in free molecules (direct bilirubin) in blood, while the rest is the albumin complex (indirect bilirubin). Indirect bilirubin is transported to the liver, where it liberates albumin and free bilirubin, which is then conjugated with glucuronic acid, and is finally excreted to the bile. Consequently, the level of serum bilirubin can be an index of liver function. The normal levels of serum bilirubin in adults range from 0.1 to 1.2 mg/dl (10-5 M~10-6 M). It is of particularly clinical concern such as cirrhosis or hepatitis, jaundice, brain damage or even death in newborns. Additionally, a low level of bilirubin concentration is associated with iron deficiency and coronary artery disease. Owing to the above mentioned, the rapid and accurate detection of bilirubin is required.

The concept of molecular imprinting was originally from “formation of antibodies” proposed by Pauling in 1940. They defined molecular imprinting as “Construction of the ligand-selective recognition sites in synthetic polymers where a template is employed to facilitate recognition-site formation during the covalent assembly of the bulk phase by a polymerization or poly-condensation process, with subsequent removal of the template being necessary for recognition to occur in the spaces vacated by the templating species. The functional monomers are mixed with the template molecules so they could bind covalently or non-covalently. In this study, MAA and EGDMA were used during the preparation of the MIP complex. However, another monomer, zinc(II) protoporphyrin, was used as the monomer for the fluorescent effect. For supramolecular photochemistry, appropriate assembly of molecular components (supramolecular systems) can be designed to synthesize typical biological systems or to fabricate artificial electronic devices at the molecular level. When a metalloporphyrin is used as a receptor, the metal ion in the center of the porphyrin molecule can serve as a Lewis acidic site to bind Lewis bases. Concluded from the above, zinc(II) protoporphyrin was chosen in this work. Metal ions can bind functional groups through the donation of electrons from the atoms of templates to the unfilled orbitals of the outer coordination sphere of the metal. The metalloporphyrins can also provide hydrogen bonds with the template molecules.

In recent years, our research work was aimed at the specific binding and sensing of bilirubin by the molecular imprinting technique. In this work, a fluorescent MIP based on zinc(II) protoporphyrin (ZnPP) for the imprinting of bilirubin was synthesized and able to specifically recognize bilirubin. The preparation scheme is illustrated in Fig. 1. The imprinted poly(zinc protoporphyrin-methacrylic acid-ethyl glycol dimethylacrylate) (poly(ZnPP-MAA-EGDMA)) achieved superior binding as well as imprinting result toward bilirubin. Additionally, the shift of the peak wavelength and the absorbance intensity were both used to identify the formation of the bilirubin-imprinted receptor by complexation with bilirubin solution. The complex formation in the pre-polymerization solution was further confirmed by the compleximetric titration.
Fig. 1 Preparation scheme of bilirubin imprinted poly(ZnPP-MAA-EGDMA.

Protoporphyrin IX contains a tetrapyrrole structure and is similar to bilirubin. The four-pyrrole rings of PP could form π-π stacking interaction with the four-pyrrole rings of bilirubin. The zinc in the ZnPP structure could further coordinate with bilirubin to form much rigid recognition sites in the MIP matrix. The recognition cavities formed within the imprinted polymer containing ZnPP were supposed to be maintained much firmly in shape and complementary interaction compared to those sites from the polymer prepared with PP. Therefore, the binding capacity was higher to be achieved from the ZnPP-contained imprinted polymer than that in the PP (porphyrin)-contained polymers. The combined utilization of two functional monomers for the preparation of the imprinted polymer already indicated the enhanced imprinting effect of the porphyrin-based and carboxyl residues in the polymer matrix rather than the individual monomer for the specific uptake of bilirubin. Consequently, the iPZME (imprinted poly(ZnPP-MAA-EGDMA)) was applied in this work.

Evidence for the formation of the metal complex by ZnPP with bilirubin could be observed by the compleximetric titration of ZnPP in a pre-polymerization mixture with bilirubin. Interaction of ZnPP and bilirubin was examined by measuring the absorption bands at the visible region of a spectrophotometer. The UV-Vis absorption spectra of the MIP in DMSO solution and NaOH solution of 50 mM without bilirubin are shown in Fig. 2. The profile of MIP/DMSO appeared a Soret band (S0/S2 transition) at 416 nm and the Q-bands (S0/S1 transition) at 543 nm and 579 nm. Another profile was obtained from MIP suspended in 50 mM NaOH solution (pH 12). The Soret band at 412 nm and the Q-bands at 542 and 578 nm were observed. These two profiles were very similar. In the Soret region, the relative intensity at 412 nm in aqueous NaOH solution was significantly smaller when compared to the 416 nm observed in DMSO. However, compared to the much sharper absorption peak observed from the MIP/DMSO, more broad bandwidths were obtained from the MIP suspended in NaOH solution, which indicated stronger or more binding of the MIP with other species in the solution. It could be sensed that aqueous NaOH solution contained ions to be bound with MIP while DMSO had only little chance. The deprotonation of an axial ligand of ZnPP contained in the imprinted polymer matrix both in DMSO and in NaOH solution caused a slightly red shift of the visible absorption bands. In Fig. 2, the bandwidths read at half-height of the Soret bands (Δλ1/2) were 26 nm and 24 nm for MIP in DMSO and NaOH solutions, respectively. Therefore, Δλ1/2s for both cases were almost identical.

Fig. 2 UV-VIS absorption spectra of MIP in DMSO and in 50 mM NaOH solution.
Zinc(II) protoporphyrin could form the metal complex through the coordination of Zn with bilirubin during the imprinted polymerization and the binding effect could be revealed by the spectroscopic profile. The porphyrin could cause a red shift in UV-Vis absorbance spectra because of complexation. As ZnPP in the MIP binds to any other chemical species, the absorption bands become broad. It indicated that the ZnPP content in the MIP matrix might bind more bilirubin molecules or might bind them more firmly in the NaOH solution. Additionally, the stabilization of the system via these bindings caused a red shift of the visible absorption band. Visible spectra of the MIP suspended in bilirubin samples, in which bilirubin concentrations ranged from 10-5 to 10 mM, were measured. The changes in the Soret band and all the Q-band peaks are shown in Fig. 3(a), (b). Bilirubin can be dissolved in aqueous solution at a pH of higher than 11. The higher the pH, it would become more unstable. In this study, the NaOH solution with the concentration of 50 mM was used to dissolve bilirubin. The UV-Vis spectrum obtained from the bilirubin dissolved in aqueous solution was compared with the result obtained in DMSO. The visible absorbance spectra gave a significant red shift as the bilirubin concentration was increased. It can be observed from the zoom-in profiles of peak wavelength versus bilirubin concentration shown in Fig. 3(a), (b). The red shift of the absorption bands along the increased bilirubin concentration could be inspected from MIP in both DMSO and NaOH solutions. Although the MIP particles in these solvents could both shift the UV-Vis spectra, the degree of shift was obviously larger in the aqueous bilirubin/NaOH system even at lower bilirubin concentration. Being different from the MIP in bilirubin/NaOH solution, the red shift did not occur in the DMSO system below the bilirubin concentration of 1 mM. Therefore, the results further suggested that other than the fluorescent detection, the red shift of the peak wavelengths in the visible region of the absorption band could be an alternative approach for the detection of bilirubin by using the MIP in NaOH environment.
Fig. 3 Shift of λmax of the bilirubin-imprinted receptor by complexation with bilirubin (a) in DMSO; (b) in 50 mM NaOH solution. (●) Soret band; (○) Q1 band; (▲) Q2 band.

Fig. 4 Spectrometric observation from the titration of bilirubin with ZnPP/DMSO solution.
Bilirubin was added into ZnPP suspended DMSO solution and the spectrum changes of the Q-bands of ZnPP at the visible region (around 548 nm and 586 nm) were monitored as shown in Fig. 4. Additionally, via the formation of the metal complex with other chemical species, porphyrin or ZnPP would cause the broadening of all the absorption spectra as well as the red shift in UV-Vis absorption spectra. As bilirubin concentration was increased, the absorption bands of ZnPP were getting broadened in this range, which confirmed qualitatively the complexation of ZnPP with bilirubin.

Biliverdin was used for the investigation of the selectivity of MIPs because biliverdin has similar chemical structure as bilirubin. It is an analogue of bilirubin. The results are showed in Table 1. As inspected from the table, the binding capacity of MIP in bilirubin solution was 1.052 ± 0.102 mg/g, which was comparably higher than the one, 0.735 ± 0.055 mg/g obtained from the MIP in biliverdin solution. MIP had an intension to adsorb biliverdin via the non-specific interaction. It implied that the MIP did not create specific cavities for biliverdin. Additionally, the adsorption ability of MIP toward bilirubin was stronger than that toward biliverdin. Therefore, the imprinting effect of the polymer was significant for bilirubin. The binding capacities of MIP and NIP for bilirubin in mixture solution were 1.483 ± 0.050 and 0.770 ± 0.17 mg/g, respectively, which were larger than that from bilirubin solution. In fact, the binding capacities of bilirubin in mixture by MIP and NIP were enhanced because of the presence of biliverdin. However, still, a worse imprinting result regarding bilirubin was obtained from the mixture when compared to the solution of only bilirubin. On the contrary, the binding capacities of biliverdin in mixture were reduced compared to the solution of only biliverdin and the imprinting result regarding biliverdin in mixture solution was slightly enhanced. Nevertheless, it revealed that even in the presence of biliverdin the imprinted polymer was still able to discriminate bilirubin from biliverdin.
Table 1 Influence of specific binding toward bilirubin in the presence of biliverdin.

Based on the utilization of the fluorescent monomer, ZnPP, another monomer, MAA, was added to create the functionality with the imprinted template molecule, bilirubin. In the presence of the cross-linker, EGDMA, the polymerization was initiated by AIBN at 60 ℃ for 6 h. The imprinting factor of 3.069 was achieved from this fluorescent imprinted polymer, namely, the imprinted poly(ZnPP-MAA-EGDMA). Consequently, selectivity of 2.269 could be achieved from the mixture of bilirubin/biliverdin. The difference in the shape of the spectrometric bands from the titration of different concentrations of bilirubin into ZnPP solution was observed and discussed. Investigation of the red shift of peak wavelengths in the visible absorption band from the fluorescent MIP suspended in DMSO and aqueous NaOH, respectively was also made. The feasibility of using ZnPP for the preparation of fluorescent polymer for the imprinting and selective binding of bilirubin was investigated and thus confirmed.
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