As one of the most potential functional polymer materials, polyaniline (PANI) has been intensively studied due to the high environmental stability, simple synthesis, and reversible redox behavior [1-3]. In recent years chiral PANI has attracted much attention of researchers because it can be widely applied in catalysis, enantioselective separation, chemical and biological sensors etc. [4, 5]. In view of the potential and broad application of chiral PANI, many efforts have been made on the synthesis of chiral PANI by chemical, electrochemical or enzymatic in situ polymerization methods [6-8]. By comparison, the template-mediated polymerization of PANI catalyzed by enzyme has been developed and attracts great attention due to the simple and environmental friendly characteristics [9-12]. The enzymes including horseradish peroxidase (HRP), palmtree peroxidase, soybean peroxidase, and even the mimic enzyme, hemoglobin (Hb) have been investigated in the synthesis of conductive polyaniline as the catalysts in the past few years [13-17]. Among these, the Hb-catalyzed synthesis of PANI has been intensively studied by Hu et al. [18-20]. Furthermore, these biological catalysts were recently found to be capable of inducing the production of chiral PANI. Huang et al. reported that horseradish peroxidase could induce the formation of chiral PANI without any other foreign chiral dopants . Meanwhile, we have also found that Hb had the capacity to direct enantio specificity of PANI . These evidences suggest that protein has the ability of inducing the synthesis of chiral PANI which opens new routes to develop artificial helical polymers and renders the natural molecules useful beyond their typical biological function.
However the synthesis of PANI was usually performed under harsh conditions including the relatively low pH . As known to all of us, most of the enzymes can preserve their activities in the mild environment which profoundly blocks the application of proteins in the synthesis of PANI due to the expensiveness of the enzymes. Duan et al. reported the polymerization of aniline by using HRP immobilized on chitosan powder which was found to be stable and remain active after being stored in pH=6.0 buffer solutions for more than 72 h . Mecerreyes et al. utilized the ionic liquid immobilized HRP for the biocatalytic synthesis of PANI to reduce the cost .
To explore the possibility of biological molecules applied in the hard polymerization reaction system, in this work modified Hb by separation, inactivation, and immobilization methods were used in the synthesis of chiral PANI. Interestingly, it was found that chiral PANI was acquired by using the modified Hb as the chiral inducers. The details of the comparative study were discussed.Ⅱ. EXPERIMENTS A. Materials
Bovine Hb was purchased from Shanghai Kayon Biochemistry Company (Shanghai, China). Dodecylbenzenesulfonic acid (DBSA) were obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). Ammonium persulfate (APS) and aniline monomer was purchased from Shanghai Chemical Agent (Shanghai, China). Aniline was distilled twice under reduced pressure before being used. All other chemicals and solvents were of analytical grade, and used as received.B. Separation of Hb
The solution of Hb was prepared by dissolving 50 mg Hb in 5 mL distilled water. Then 25 mL hydrochloric acid solution containing 4% acetone was added into the Hb solution with vigorous stirring for 30 min at 4 ℃. After centrifugation for 5 min at 8000 r/min and freeze drying for 12 h, the white precipitation of globin was obtained. The solution after centrifugation was collected and adjusted by 2 mol/L NaOH to pH=4.0, followed by the addition of 1% (
10 mg Hb dissolved in 2 mL distilled water was mixed with 1 mL 0.1 mol/L glutaraldehyde and stored at 4 ℃ for 12 h. After that, 2 mL 0.05 mol/L sodium alginate was added into the mixed solution followed by the dropwise addition of 20 mL 0.2 mol/L calcium chloride under vigorous stirring within 30 min. The resulted small balls of calcium alginate containing Hb were then washed by distilled water twice. And the immobilized Hb was obtained by drying at room temperature for 24 h.D. Preparation of PANI
The polymerization of aniline was typically carried out at room temperature in 10 mL Na
The polymer was characterized by UV-visible absorption spectroscopy, circular dichroism (CD) spectra, Fourier transform infrared (FTIR) spectra, field emitted scanning electron microscopy (FESEM), and X-ray diffraction (XRD).
Ultraviolet-visible (UV-Vis) spectra of the products were recorded on a UV-Vis spectrophotometer (TU-1901, China). Before measurement of UV-Vis spectra, the samples were diluted 100-fold with water. In each measurement, distilled water was used as a control. JASCO 815 CD spectrometer measures the CD signal of the product. Before recording the CD spectra, the samples were diluted 30-fold with water. The reaction solution without APS was used as a control. Fourier transform infrared (FTIR) spectra were obtained using KBr pellets on a FTIR spectrophotometer (Nicolet Avatr 370 DTGS, America). The morphology of the obtained PANI was determined by field emitted scanning electron microscopy (FESEM) (S-4800, Hitachi Co., Japan). Crystallinity of the polymer was carried out by X-ray diffractometer (Rigaku D/Max 2000, Japan).Ⅲ. RESULTS AND DISCUSSION
Before being introduced into the polymerization of aniline, Hb was dealed with different methods. The inactivated Hb was acquired by denatured in boiling water for 15 min, whereas the immobilized hemoglobin was obtained by entrapped in calcium alginate. The separation of Hb was performed by using the above mentioned method, resulting in two components, hematin and globin.
The UV-Vis spectra of PANI were recorded in the presence of inactivated and immobilized Hb firstly. It can be seen from FIG. 1 that three characteristic absorption bands of the polymer at about 340-360, 430-440, and 750-800 nm were observed respectively. The first absorption band attributed to
Hb is a common mimic enzyme which is made up of hematin and globin. In order to examine the possibility of Hb used under extreme conditions, hematin and globin were separated from Hb first of all and then utilized to polymerize aniline, respectively. As shown in FIG. 2(a), results indicated that conductive PANI can be obtained with the addition of globin or hematin. Our results thus proved that the conditions of proteins had few effects on the production of conductive PANI.
Based on our previous experiments, Hb had the capacity to direct enantio specificity of PANI . In the following experiments, the CD spectra of PANI synthesized in the presence of modified Hb were examined. Interestingly, the globin-PANI was observed to exhibit a characteristic peak at around 500 nm in the CD spectra as shown in FIG. 2(b), which is a signature of the optical active PANI [28, 29]. What's more, it was observed the CD peak of globin-PANI showed positive signal which meant the acquired PANI had the controlled one-handedness [30, 31]. In contrast, it was found that the PANI obtained in the presence of hematin gave no CD signal, suggesting that hematin cannot induce the production of chiral PANI. This result was in consistent with the report of Cholli et al. who had proven that the hematin had no capacity to direct enantio specificity for the PANI chains . Unexpectedly, the chiral signal was found to appear in the determination of Hb(inactivated)-PANI which can be seen from the characteristic peak at around 460 nm in the CD spectra as shown in FIG. 3. It is well known that the boiled protein will be deactivated and lose its native steric structure. However our experiment demonstrated that the synthesis of chiral PANI was not interfered by the inactivation of protein. Similarly it was observed that the CD peak of Hb(immobilized)-PANI exhibited positive signal which can be seen from the characteristic peak at around 500 nm, indicating that chiral PANI was produced in the presence of immobilized Hb. As known to us, the structure of immobilized protein which has been entrapped in calcium alginate is relatively rigid compared to that of the free protein [33, 34]. Results implied that the structure of protein may have no close relationship with the formation of chiral PANI.
The stereochemical selectivity (enantiomeric purity) of the polymerization of aniline was provided by the dissymmetry
Subsequently, the stability of the chiral PANI prepared in the presence of inactivated Hb was performed by doping and dedoping process. FIG. 4 shows the UV-Vis and CD spectra of chiral Hb(inactivated)-PANI under various pH conditions respectively. It was found from the UV-Vis spectra of dedoped PANI that an absorbance band at around 600 nm appeared which was ascribed to the excition transition of quinoid ring, suggesting the presence of emeraldine base of PANI at pH=10.0. Nevertheless after the pH was adjusted to 2, the peak at about 800 nm reappeared in UV-Vis spectra, indicating that PANI had been redoped. Results showed that the redox state of chiral PANI was reversible which was consistent with the previous reports [35-37]. Furthermore, it was observed that the CD spectra of the as-prepared, dedoped and redoped PANI exhibited similar peaks at around 460 nm, which demonstrated that the Hb(inactivated)-PANI preserved good chirality under different conditions.
The morphologies of chiral PANI in the presence of modified Hb and globin were investigated by SEM. As shown in FIG. 5, the nanometered, spherical conformation of chiral PANI produced by Hb (FIG. 5(b)) was observed, whereas the globin-PANI presented irregular and amorphous granules (FIG. 5(a)). The particles of Hb(immobilized)-PANI (FIG. 5(c)) was nanoscaled, short rod-like in shape. Nevertheless, Hb(inactivated)-PANI (FIG. 5(d)) exhibited nanofibers in shape with an average diameter of about 20 nm. Results indicated that the type of protein, i.e., the steric structure of protein may have effects on the morphologies of PANI. Further studies on the morphologies of protein-induced chiral polyaniline have still been in progress.
FIG. 6 presents the XRD patterns of chiral PANI synthesized in the presence of different inducers. It can be found that PANI exhibited an identical peak at 2
In summary, the optical active polyaniline induced by modified Hb was deeply investigated to survey the possibility for that protein can be applied in the production of chiral PANI as the chiral inducer. Results indicated that the inducing ability of protein was still preserved even after the protein was separated, inactivated or immobilized by entrapment. Among the three types of modified Hb, the inactivated Hb exhibited similar or even better abilities than natural Hb in the polymerization process. It was speculated that the production of chiral PANI can be ascribed to the inducing of the chiral amino acids among the protein. Because of the inactivated process, more amino acids of the inactivated Hb were exposed to reaction system which resulted in higher chirality in PANI. The separation of Hb may have no obvious effects on the formation of chiral PANI. Nevertheless the immobilization of Hb by entrapment was not beneficial to the polymerization reaction compared to natural Hb. In addition, our experiments indicated that the chirality of PANI did not have close relationship with the steric structure of protein. However the steric structure of protein was found to have significant effects on the morphology and crystalline structure of chiral PANI.Ⅴ. ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (No.21303105), the Scientific Research Foundation for the Returned Overseas Chinese Scholars and State Education Ministry (No.ZX2012-05).
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