Synthesis and Characterization of the Conducting Polymer Micro-Helix Based on the Spirulina Template

As one of the most interesting naturally-occurring geometries, micro-helical structures have attracted attention due to their potential applications in fabricating biomedical and microelectronic devices. Conventional processing techniques for manufacturing micro-helices are likely to be limited in cost and mass-productivity, while Spirulina, which shows natural fine micro-helical forms, can be easily mass-reproduced at an extremely low cost. Furthermore, considering the extensive utility of conducting polymers, it is intriguing to synthesize conducting polymer micro-helices. In this study, PPy (polypyrrole), PANI (polyaniline), and PEDOT (poly(3,4-ethylenedioxythiophene)) micro-helices were fabricated using Spirulina platensis as a bio-template. The successful formations of the conducting polymer micro-helix were confirmed using scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FTIR) and Raman and X-ray diffraction (XRD) were employed to characterize the molecular structures of the conducting polymer in micro-helical forms. In the electrochemical characterization, the optimized specific capacitances for the PPy micro-helix, the PANI micro-helix, and the PEDOT micro-helix were found to be 234 F/g, 238 F/g at the scan rate of 5 mV/s, and 106.4 F/g at the scan rate of 10 mV/s, respectively. Therefore, it could be expected that other conducting polymer micro-helices with Spirulina as a bio-template could be also easily synthesized for various applications.

In order to generate helical micro/nano-structures, various techniques, including molecular self-assembly, electrospinning [21], photolithography [22], mechanical twisting [23], microfluidics [1,24], Polymers 2018, 10 and oblique angle-based deposition [25] have been developed.However, it is still difficult to realize the large-scale production of a uniform helix at a low cost since most of these techniques are based on delicate conditions and sophisticated equipment [26].Thus, the high requirements for these fabrication approaches are expected to limit their practical applications.On the one hand, there are plenty of highly-ordered biological structures in a wide range of dimensions and patterns that can be employed as templates [27].For micro-sized helix synthesis, two kinds of biological templates have mainly been used: one is from xylem vessels that are composed of chiral crystalline cellulose and widely distributed in almost every part of vascular plants [28], and the other is from Spirulina that grows naturally in a helical shape and has already been commercialized as food supplements [29].Though xylem-originated helical microstructures are abundant in a wide variety of plants, the difficulty in the isolation process of the spiral vessels through chemical methods still exist [28].In contrast, Spirulina that is in a perfect helical shape can be easily cultured and extracted in large quantities by simple filtration.Until now, Spirulina-based templates have successfully contributed to the synthesis of the magnetic helix [4,30], silica helix [31], copper helix [32][33][34], Ni-Fe-P alloy helix [35], and silver helix [36].
In this article, Spirulina-templated conducting polymer micro-helices, including PPy micro-helix, PANI micro-helix, and PEDOT micro-helix, were successfully synthesized by in situ polymerization using FeCl 3 as an oxidant.The morphologies, molecular structures, and electrical and electrochemical properties of the conducting polymer micro-helices were characterized by SEM, FTIR, Raman, XRD, four-point probes, CV, and EIS, respectively.

Cultivation and Preparations of Spirulina platensis
The cultivation of the Spirulina started with the initial inoculum density of 0.1 with magnetic stirring in an illumination incubator (Ningbo Yanghui instrument Co., Ltd., Ningbo, China) at 36 ± 1 °C in 12/12 h light-dark cycles.When the inoculum density was above 1.2 after 12 days of cultivation, the Spirulina was collected by a 350-mesh nylon filter and rinsed with 0.9% w/w sodium chloride solution three times.The tissue fixation of the Spirulina was carried out in 1:9 v/v glutaraldehyde aqueous solution for 6 h to maintain the bio-helix form.After the fixation, the unreacted glutaraldehyde was rinsed out with 0.01 M phosphate-buffered solution (PBS) and deionized water, successively.Fixed Spirulina template suspension was used throughout the experiments.

Fabrication of the Spirulina-Templated PPy Helical Microstructure
For the fabrication of Spirulina-templated PPy helical microstructure, 11 g of Spirulina template was suspended in a 50 mL solution containing 0.1 M HCl and 0.2 M pyrrole by magnetic stirring at 300 rpm for 0.5 h.For PPy polymerization, 50 mL of 0.6 M FeCl3 solution was slowly added into the suspension and reacted for 6 h.After the polymerization, the suspension was filtered by a 350-mesh nylon filter and rinsed with 0.1 M HCl to remove the loosely bound or uncoated PPy.Finally, the PPy micro-helix was dried in an oven at 50 °C for 6 h.

Fabrication of the Spirulina-Templated PANI Helical Microstructure
For the fabrication of Spirulina-templated PANI helical microstructure, 12 g of Spirulina template was suspended in 160 mL of water containing 0.5 M aniline by magnetic stirring at 300 rpm for 1 h.The aniline adsorbed Spirulina cells were added into 150 mL of 0.5 M FeCl3 solution and reacting overnight with magnetic stirring.After the polymerization, the suspension was filtered by a 350mesh nylon filter and sonicated in ethanol to dislodge loosely bound or free polymers.The suspension was then washed with 0.1 M HCl and deionized water, successively.Finally, the PANI micro-helix was dried in an oven at 50 °C for 6 h.

Cultivation and Preparations of Spirulina platensis
The cultivation of the Spirulina started with the initial inoculum density of 0.1 with magnetic stirring in an illumination incubator (Ningbo Yanghui instrument Co., Ltd., Ningbo, China) at 36 ± 1 • C in 12/12 h light-dark cycles.When the inoculum density was above 1.2 after 12 days of cultivation, the Spirulina was collected by a 350-mesh nylon filter and rinsed with 0.9% w/w sodium chloride solution three times.The tissue fixation of the Spirulina was carried out in 1:9 v/v glutaraldehyde aqueous solution for 6 h to maintain the bio-helix form.After the fixation, the unreacted glutaraldehyde was rinsed out with 0.01 M phosphate-buffered solution (PBS) and deionized water, successively.Fixed Spirulina template suspension was used throughout the experiments.

Fabrication of the Spirulina-Templated PPy Helical Microstructure
For the fabrication of Spirulina-templated PPy helical microstructure, 11 g of Spirulina template was suspended in a 50 mL solution containing 0.1 M HCl and 0.2 M pyrrole by magnetic stirring at 300 rpm for 0.5 h.For PPy polymerization, 50 mL of 0.6 M FeCl 3 solution was slowly added into the suspension and reacted for 6 h.After the polymerization, the suspension was filtered by a 350-mesh nylon filter and rinsed with 0.1 M HCl to remove the loosely bound or uncoated PPy.Finally, the PPy micro-helix was dried in an oven at 50 • C for 6 h.

Fabrication of the Spirulina-Templated PANI Helical Microstructure
For the fabrication of Spirulina-templated PANI helical microstructure, 12 g of Spirulina template was suspended in 160 mL of water containing 0.5 M aniline by magnetic stirring at 300 rpm for 1 h.The aniline adsorbed Spirulina cells were added into 150 mL of 0.5 M FeCl 3 solution and reacting overnight with magnetic stirring.After the polymerization, the suspension was filtered by a 350-mesh nylon filter and sonicated in ethanol to dislodge loosely bound or free polymers.The suspension was then washed with 0.1 M HCl and deionized water, successively.Finally, the PANI micro-helix was dried in an oven at 50 • C for 6 h.For the fabrication of Spirulina-templated PEDOT helical microstructure, 30 mL of a suspension containing 2.5 g of Spirulina template, 0.23 M 3,4-ethylenedioxythiophene (EDOT), 2.34 M FeCl 3 •6H 2 O, and 0.12 M p-toluenesulfonic acid monohydrate were mixed.For the PEDOT polymerization, the suspension was sonicated in an ultrasonic washer for 1 h.After the polymerization, the suspension was filtered by a 350-mesh nylon filter and washed with deionized water and pure ethanol successively to remove the loosely bound or free polymers.Finally, the PEDOT micro-helix was dried in an oven at 50 • C for 6 h.

Preparations of PPy, PANI, and PEDOT Helix Modified Electrodes
The PPy, PANI, and PEDOT micro-helix powders, acetylene black, and poly(tetrafluoroethylene) (PTFE) were mixed by the mass ratio of 8:1:1, and ground homogeneously with the addition of pure ethanol, respectively.For each conducting polymer micro-helix, about 0.01 g of the mixture was coated onto a nickel foam sheet in the size of 1 cm × 1 cm and oven-dried at 60 • C overnight.

Morphology Analysis
The morphology of Spirulina platensis in its natural form was observed using an optical microscope (SMARTR, Chongqing Ott optical instruments Co., Ltd., Chongqing, China).The morphologies of the PPy micro-helix, PANI micro-helix, and PEDOT micro-helix were observed using a field emission scanning electron microscope (FESEM) (Helios NanoLab G3, FEI Company, Brno, Czech Republic and Nova Nano SEM 450, FEI Company, Eindhoven, The Netherlands) with the accelerating voltages of 2 and 10 kV.

Fourier Transform Infrared Spectroscopy (FTIR)
A Fourier transform infrared spectrometer (Vertex 70, Bruker Corporation, Karlsruhe, Germany) was used to obtain the FTIR spectra of the PPy, PANI, and PEDOT micro-helices with a scanning range of 400-4000 cm −1 at a resolution of 1.93 cm −1 at room temperature.The sample tablets were prepared by mixing the sample powders with 5% (w/w) potassium bromide (KBr).

Raman Spectroscopy
A confocal laser Raman spectrometer (LabRAM HR800, Horiba Jobin Yvon, Paris, France and Renishaw inVia plus, Renishaw plc, Gloucestershire, United Kingdom) was employed to obtain a Raman Spectroscopy of the PPy, PANI, and PEDOT micro-helices.Raman spectra were acquired with a 532 nm excitation laser source with a power of 1.8 mW in the range of 500-2000 cm −1 .

X-ray Diffraction (XRD)
An X-ray diffraction meter (Empyrean XRD, Malvern Panalytical Ltd., Malvern, UK) was used to determine the atomic and molecular structure of the PPy, PANI, and PEDOT micro-helices.The XRDs were conducted with a Cu K-Alpha radiation source with a step size of 0.01313 • in the scanning range of 5 • -50 • and wavelength of 1.540598 Å.

Electrochemical Characterization
The sheet resistance of each conducting polymer micro-helix was measured in three different areas, three times by four-point probes (ST2263 digital four-point probes, Suzhou Jingge Electronic Co., Ltd., Suzhou, China).Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of the PPy, PANI, and PEDOT micro-helix-modified electrodes were conducted on a CS 1350 electrochemical workstation (Wuhan Corrtest Instrument Co., Ltd., Wuhan, China) with a three-electrode cell in which a Pt foil (1 cm × 1 cm) and Ag/AgCl electrode were used as a counter electrode and reference electrode, respectively.In CV measurements, ten cycles between −0.3 and 0.7 V in 0.1 M Na 2 SO 4 at scan rates of 5, 10, 20, 30, 50, and 100 mV/s were performed.EIS measurements were performed in the frequency range of 10 5 to 0.01 Hz at an open circuit potential with an AC perturbation of 10 mV, and the data were analyzed by ZView software (Wuhan Corrtest Instrument Co., Ltd., Wuhan, China).

Morphologies of PPy, PANI, and PEDOT Micro-Helices
In Figure 2a, the distinct display of the helix micro-structures without any metal sputtering coatings confirmed that conductive PPy has been successfully formed on the surface of Spirulina and the coating process showed no adverse effect on the initial helical shape of Spirulina.In Figure 2b, it can be observed that the cross-section of the PPy micro-helix has a solid and compact interior which has a diameter of about 7.3 µm and a relatively thin outer layer with about 0.3 µm thickness.The appearance of this solid interior is interesting because both the hollow and the solid helical structures have been observed on Cu-coated Spirulina and Ag-coated Spirulina by electroless plating [32,34,36].This solid interior of the helical structure should be composed of the fixed cytoplasmic components from glutaraldehyde treatment because it also existed in the Spirulina templates without PPy coating.This difference between the hollow and solid interiors may result from the Spirulina template preparation process.In the hollow helical structure formation, the drying process vaporized the moisture of Spirulina and the volume of Spirulina shrunk to form the hollow helical structure [34], while in our preparation, no drying process was included.PPy coating shows a typical granular raspberry morphology and a fully-encapsulated Spirulina surface [49].As no bare Spirulina can be observed and the PPy layers fully covered on all these helical template surfaces, it can be speculated that there exists a chemical bonding between PPy and the Spirulina surface.As one genre of cyanobacteria, Spirulina is also covered by the external surface layers that are composed of 2D crystalline arrays of glycoprotein (S-layers) and carbohydrate structures [50,51].Though the compositions of these carbohydrate structures may vary with the species of cyanobacteria, they mainly include monosaccharides and polysaccharides, such as cellulose-like homoglucan, xyloglucan, uronic acid, glucose, xylose, and ribose [52].Therefore, this bonding is likely to be hydrogen bonding that is from the interaction between the hydroxyl groups of the saccharides in the Spirulina cell wall and the H of the N and/or the N in the pyrrole ring [53].Figure 2c shows that the Spirulina templates were not only enwrapped, but also bounded with each other by PANI.Though aniline monomers are likely to initiate the polymerization on the hydroxyl groups of the saccharides on the Spirulina surface, the free PANI seems to be easily formed between the spaces of the Spirulina templates, as shown in Figure 2d.This result may be due to the relatively high aniline monomer concentration since the morphology of the PANI-coated cotton fiber was also monomer concentration-dependent [54].In Figure 2e, it can be seen that the Spirulina templates were coated with an uneven granular PEDOT and their cross-sections also showed solid and compact interiors since they had the same preparation process as that of the PPy micro-helix.In Figure 2f, a rougher surface of PEDOT micro-helix can be found in comparison with the PPy and PANI micro-helix surfaces.This morphology may suggest that there exists the weakest interaction between the PEDOT and Spirulina surfaces among these three conducting polymers.
FTIR spectra of free PPy and PPy micro-helix are shown in Figure 3b.A broad vibrational band between 3200-3500 cm −1 can be assigned to N-H stretching vibrations [56].The bands in the PPy micro-helix at about 2928 and 1647 cm −1 can be assigned to sp 3 C-H stretching and N-H bending vibration, respectively [60].Free PPy exhibits a distinct band at 1535 cm −1 , which is attributed to C-C stretching vibrations in the pyrrole ring, while a small blue shift to 1538 cm −1 of this band is observed in the PPy micro-helix [61].The bands at 1442 and 1451 cm −1 in the spectra of PPy and the PPy microhelix correspond to C-N stretching vibrations in the ring [61].PPy and the PPy micro-helix have broad bands in the range of 1400-1250 cm −1 which can be assigned to C-H or C-N in-plane deformation modes with the maximum at 1292 and 1309 cm −1 , respectively [62].The bands between 1250 to 1100 cm −1 correspond to the breathing vibration of the pyrrole ring with the maximum at 1161

Raman Spectroscopy Analysis
In Figure 4a, the band at 1577 cm −1 is assigned to the C=C backbone stretching of PPy, and the band at 1380 cm −1 is attributed to the ring stretching mode for both PPy and the PPy micro-helix [74].The band at 1240 cm −1 in the PPy micro-helix can be assigned to the anti-symmetrical C-H in-plane bending [75].In both PPy and the PPy micro-helix, the peaks at 1050 cm −1 represent the symmetrical C-H in-plane deformation and the peaks at 968 cm −1 are associated with the polaron structure [74,75].The peak at 938 cm −1 in PPy and the peak at 935 cm −1 in the PPy micro-helix are attributed to ring deformation induced by dication (dipolaron) [76].
In Figure 4b, the bands at 1617 cm −1 in both PANI and the PANI micro-helix spectra are attributed to C-C stretching [77].The bands at 1588 cm −1 in PANI and at 1513 cm −1 in the PANI microhelix may be associated with C=C stretching in the quinonoid ring [77,78], and the bands at 1562 cm −1 in PANI and at 1560 cm −1 in the PANI micro-helix can be attributed to the C-C stretching of the intermediate structure between quinoid and semiquinoid [79].The band at 1494 cm −1 in PANI represents benzenoid ring vibration (C=C stretching deformations) [80].The bands at 1345 cm −1 in PANI and 1342 cm −1 in the PANI micro-helix can be attributed to C-N + stretching of radical cations [81].The bands at 1187 cm −1 in the PANI micro-helix and at 1166 cm −1 in PANI can be assigned to C-H in-plane bending vibrations of bipolaronic forms and C-H bending quinoid, respectively [81,82].The bands at 814, 610, and 520 cm −1 in both the spectra may result from the C-N-C bending, ring deformation, and amine in-plane deformation, respectively [82,83].
In Figure 4c, the band of C=C symmetrical stretching in the thiophene ring is at 1444 cm −1 in PEDOT and shifts to 1426 cm −1 in the PEDOT micro-helix [84,85].The bands of asymmetrical C=C stretching are at 1506 cm −1 for both PEDOT and the PEDOT micro-helix [86,87].The peak at 1361 cm −1 that is related to C-C stretching is only observed in the PEDOT micro-helix [87].The peaks at 1265 cm −1 in the PEDOT micro-helix and 1274 cm −1 in PEDOT are assigned to the C-C inter-ring stretching mode [85].The peaks of the C-C anti-symmetrical stretching mode can be seen at 983 cm −1 in the PEDOT micro-helix and 994 cm −1 in PEDOT [85].FTIR spectra of free PPy and PPy micro-helix are shown in Figure 3b.A broad vibrational band between 3200-3500 cm −1 can be assigned to N-H stretching vibrations [56].The bands in the PPy micro-helix at about 2928 and 1647 cm −1 can be assigned to sp 3 C-H stretching and N-H bending vibration, respectively [60].Free PPy exhibits a distinct band at 1535 cm −1 , which is attributed to C-C stretching vibrations in the pyrrole ring, while a small blue shift to 1538 cm −1 of this band is observed in the PPy micro-helix [61].The bands at 1442 and 1451 cm −1 in the spectra of PPy and the PPy micro-helix correspond to C-N stretching vibrations in the ring [61].PPy and the PPy micro-helix have broad bands in the range of 1400-1250 cm −1 which can be assigned to C-H or C-N in-plane deformation modes with the maximum at 1292 and 1309 cm −1 , respectively [62].The bands between 1250 to 1100 cm −1 correspond to the breathing vibration of the pyrrole ring with the maximum at 1161 and 1195 cm −1 for PPy and the PPy micro-helix, respectively [62].The bands of aromatic C-H and N-H in the plane deformation vibration situate in the region from 1130 to 1000 cm −1 for the PPy micro-helix and PPy [62,63].The bands at 966 and 960 cm −1 can be assigned to C-C out-of-plane ring deformation vibrations of the PPy micro-helix and PPy, respectively [63].The bands of C-H out-of-plane deformation vibrations have maximums at 916 cm −1 and 888 cm −1 for the PPy micro-helix and PPy, respectively.The peaks at 788 and 773 cm −1 correspond to the C-H out-of-plane ring deformation of the PPy micro-helix and PPy, respectively [63].
FTIR spectra of free PANI and the PANI micro-helix are shown in Figure 3c.The peak at 3431 cm −1 for free PANI can be assigned to N-H stretching vibration [64].The bands at 3277 and 3058 cm −1 in the spectrum of the PANI micro-helix are due to hydrogen-bonded N-H stretching and aromatic C-H stretching, respectively [65].Free PANI exhibits two distinct bands at 2919 and 1643 cm −1 which are attributed to the asymmetrically stretching vibration of C-H and the O-H bending of the absorbed water, respectively [66], while these bands show a slight blue-shift to 2920 and 1652 cm −1 in the spectrum of the PANI micro-helix.The absorption bands at 1556 and 1467 cm −1 in the spectrum of PANI can be assigned to the stretching vibrations of the quinoid (N=Q=N) and Polymers 2018, 10, 882 8 of 18 benzoid (N-B-N) structure, respectively, while these bands have redshifts to 1532 and 1466 cm −1 in the PANI micro-helix, respectively.This result may suggest the existence of π stacking interaction between PANI and Spirulina [67].The band at 1298 cm −1 in the spectrum of the PANI micro-helix corresponds to π-electron delocalization which is caused by polymer protonation [63].The band at 1291 cm −1 can be assigned to the bending vibrations of the N-H mode of the polaron structure on the PANI micro-helix [68].The band at 1240 cm −1 corresponds to the asymmetric C-N + stretching vibration in the polaron structure on the PANI micro-helix and slightly shifts to 1232 cm −1 in the spectrum of PANI [63,69].A specific band at 1143 cm −1 observed in the PANI micro-helix can be attributed to a vibration mode of the N-H structure which is absent in PANI [69].
FTIR spectra of free PEDOT and the PEDOT micro-helix are shown in Figure 3d.The bands at 1517 and 1341 cm −1 are attributed to the C=C stretching modes or C-C stretching of the quinoid structure in the thiophene ring, while the bands shift to 1515 and 1332 cm −1 in the spectrum of PEDOT micro-helix [70,71].Peaks at 1195, 1141, and 1087 cm −1 that are due to C-O-C bond stretching in the ethylene dioxy group exist in both PEDOT and the PEDOT micro-helix [71].The bands at 980, 835, and 685 cm −1 in the spectrum of PEDOT can be attributed to the deformation modes of the C-S bond in the thiophene ring which shift to 979, 832, and 684 cm −1 in the spectrum of the PEDOT micro-helix, respectively [70].The bands around 3400 cm −1 in both PEDOT and the PEDOT micro-helix are attributed to the vibration of H 2 O and suggest water residue in the samples [72].In addition, the band at 1640 cm −1 is ascribed to the C=C stretching vibration which is strongly affected by the doping level of the conducting polymer [73] and suggests a higher doping level of the PEDOT micro-helix in comparison with that of PEDOT.

Raman Spectroscopy Analysis
In Figure 4a, the band at 1577 cm −1 is assigned to the C=C backbone stretching of PPy, and the band at 1380 cm −1 is attributed to the ring stretching mode for both PPy and the PPy micro-helix [74].The band at 1240 cm −1 in the PPy micro-helix can be assigned to the anti-symmetrical C-H in-plane bending [75].In both PPy and the PPy micro-helix, the peaks at 1050 cm −1 represent the symmetrical C-H in-plane deformation and the peaks at 968 cm −1 are associated with the polaron structure [74,75].The peak at 938 cm −1 in PPy and the peak at 935 cm −1 in the PPy micro-helix are attributed to ring deformation induced by dication (dipolaron) [76].
In Figure 4b, the bands at 1617 cm −1 in both PANI and the PANI micro-helix spectra are attributed to C-C stretching [77].The bands at 1588 cm −1 in PANI and at 1513 cm −1 in the PANI micro-helix may be associated with C=C stretching in the quinonoid ring [77,78], and the bands at 1562 cm −1 in PANI and at 1560 cm −1 in the PANI micro-helix can be attributed to the C-C stretching of the intermediate structure between quinoid and semiquinoid [79].The band at 1494 cm −1 in PANI represents benzenoid ring vibration (C=C stretching deformations) [80].The bands at 1345 cm −1 in PANI and 1342 cm −1 in the PANI micro-helix can be attributed to C-N + stretching of radical cations [81].The bands at 1187 cm −1 in the PANI micro-helix and at 1166 cm −1 in PANI can be assigned to C-H in-plane bending vibrations of bipolaronic forms and C-H bending quinoid, respectively [81,82].The bands at 814, 610, and 520 cm −1 in both the spectra may result from the C-N-C bending, ring deformation, and amine in-plane deformation, respectively [82,83].
In Figure 4c, the band of C=C symmetrical stretching in the thiophene ring is at 1444 cm −1 in PEDOT and shifts to 1426 cm −1 in the PEDOT micro-helix [84,85].The bands of asymmetrical C=C stretching are at 1506 cm −1 for both PEDOT and the PEDOT micro-helix [86,87].The peak at 1361 cm −1 that is related to C-C stretching is only observed in the PEDOT micro-helix [87].The peaks at 1265 cm −1 in the PEDOT micro-helix and 1274 cm −1 in PEDOT are assigned to the C-C inter-ring stretching mode [85].The peaks of the C-C anti-symmetrical stretching mode can be seen at 983 cm −1 in the PEDOT micro-helix and 994 cm −1 in PEDOT [85].

X-ray Diffraction Analysis
The crystal structures of PPy, the PPy micro-helix, PANI, the PANI micro-helix, PEDOT, and the PEDOT micro-helix were characterized by XRD analysis.Figure 5a shows the XRD pattern of PPy and the PPy micro-helix.The broad peaks in the region of 15-30° for both PPy and the PPy microhelix are mainly due to the scattering from PPy chains at the interplanar spacing and indicate a typical form of amorphous polymer [88].The peaks that shift from 23.2° in PPy to 21.6° in the PPy microhelix suggest the interaction between the Spirulina template surface and PPy coating [89].In Figure 5b, the peaks at 2θ = 14.8° and 25.0° in both PANI and the PANI micro-helix are caused by periodical perpendicularity and parallelism in the polymer chain, respectively.Additionally, the peak at 20.5° can be attributed to the distance of the polymer chain layers for both PANI and the PANI micro-helix [90,91].The broadened peaks at 2θ = 14.8° and 25.0° in the PANI micro-helix can be attributed to the intermolecular interaction between PANI and the Spirulina surface [92].In Figure 5c,

X-ray Diffraction Analysis
The crystal structures of PPy, the PPy micro-helix, PANI, the PANI micro-helix, PEDOT, and the PEDOT micro-helix were characterized by XRD analysis.Figure 5a shows the XRD pattern of PPy and the PPy micro-helix.The broad peaks in the region of 15-30 • for both PPy and the PPy micro-helix are mainly due to the scattering from PPy chains at the interplanar spacing and indicate a typical form of amorphous polymer [88].The peaks that shift from 23.2 • in PPy to 21.6 • in the PPy micro-helix suggest the interaction between the Spirulina template surface and PPy coating [89].In Figure 5b, the peaks at 2θ = 14.8 • and 25.0 • in both PANI and the PANI micro-helix are caused by periodical perpendicularity and parallelism in the polymer chain, respectively.Additionally, the peak at 20.5 • can be attributed to the alternating distance of the polymer chain layers for both PANI and the PANI micro-helix [90,91].The broadened peaks at 2θ = 14.8 • and 25.0 • in the PANI micro-helix can be attributed to the intermolecular interaction between PANI and the Spirulina surface [92].In Figure 5c, both PEDOT and the PEDOT helix have wide diffraction peaks in the region of 2θ = 22-28 • , which suggest their amorphous forms [93,94].The peak at 2θ = 25.4 • in PEDOT shifted to 2θ = 25.6 • in the PEDOT helix, which suggests the increase of the doping level [95,96].

Electrical and Electrochemical Properties
Four-point probes were used to determine the sheet resistance of the conducting polymer microhelices without any extra disposition.The sheet resistances of the PPy micro-helix, the PANI microhelix, and the PEDOT micro-helix are measured as 10.2 ± 0.60, 17.7 ± 2.22, and 42.2 ± 3.15 Ω/sq, respectively.The sheet resistance of the PPy micro-helix is close to that of the reported PPy/MWCNT/cotton (6.0 ± 0.4 Ω/sq) [97].The sheet resistance of the PANI micro-helix is at the same both PEDOT and the PEDOT helix have wide diffraction peaks in the region of 2θ = 22-28°, which suggest their amorphous forms [93,94].The peak at 2θ = 25.4° in PEDOT shifted to 2θ = 25.6° in the PEDOT helix, which suggests the increase of the doping level [95,96].

Electrical and Electrochemical Properties
Four-point probes were used to determine the sheet resistance of the conducting polymer microhelices without any extra disposition.The sheet resistances of the PPy micro-helix, the PANI microhelix, and the PEDOT micro-helix are measured as 10.2 ± 0.60, 17.7 ± 2.22, and 42.2 ± 3.15 Ω/sq, respectively.The sheet resistance of the PPy micro-helix is close to that of the reported PPy/MWCNT/cotton (6.0 ± 0.4 Ω/sq) [97].The sheet resistance of the PANI micro-helix is at the same both PEDOT and the PEDOT helix have wide diffraction peaks in the region of 2θ = 22-28°, which suggest their amorphous forms [93,94].The peak at 2θ = 25.4° in PEDOT shifted to 2θ = 25.6° in the PEDOT helix, which suggests the increase of the doping level [95,96].

Electrical and Electrochemical Properties
Four-point probes were used to determine the sheet resistance of the conducting polymer microhelices without any extra disposition.The sheet resistances of the PPy micro-helix, the PANI microhelix, and the PEDOT micro-helix are measured as 10.2 ± 0.60, 17.7 ± 2.22, and 42.2 ± 3.15 Ω/sq, respectively.The sheet resistance of the PPy micro-helix is close to that of the reported PPy/MWCNT/cotton (6.0 ± 0.4 Ω/sq) [97].The sheet resistance of the PANI micro-helix is at the same

Electrical and Electrochemical Properties
Four-point probes were used to determine the sheet resistance of the conducting polymer micro-helices without any extra disposition.The sheet resistances of the PPy micro-helix, the PANI micro-helix, and the PEDOT micro-helix are measured as 10.2 ± 0.60, 17.7 ± 2.22, and 42.2 ± 3.15 Ω/sq, respectively.The sheet resistance of the PPy micro-helix is close to that of the reported PPy/MWCNT/cotton (6.0 ± 0.4 Ω/sq) [97].The sheet resistance of the PANI micro-helix is at the same level as that of the reported pristine PANI (about 5 Ω/sq) [98,99].The sheet resistance of the PEDOT micro-helix is much better than that of the PEDOT/PET films (190 Ω/sq) [100].
For a single electrode, its specific capacitance, C S (F/g) can be calculated from CV curves: where ∆U is the potential window, ν is the scan rate, m is the mass of the active material electrode, I is the current, and V is the voltage.In Figure 6, the CV curves of the PPy micro-helix, the PANI micro-helix, and the PEDOT micro-helix with a series of scan rates exhibit a double-layer capacitance characteristic.In Figure 6a, the highest specific capacitance of the PPy micro-helix was found to be 234 F/g, which is almost the same as those of the reported PPy-coated cotton (268 F/g) and PPy-coated viscose rayon (244 F/g), and higher than those of the reported PMAS/PPy-coated Actitex carbon fiber (152.0F/g) and PPy-coated acrylonitrile butadiene rubber (125.8F/g) with the same scan rate [101][102][103].In Figure 6b, the highest specific capacitance of the PANI micro-helix was found to be 238 F/g, which is slightly higher than that of the reported PMAS/PANI-coated Actitex carbon fiber (212 F/g) [102,104].In Figure 6c, with the scan rate of 10 mV/s, the PEDOT micro-helix showed the highest specific capacitance of 106.4 F/g, which is higher than that of the reported CNTs/PEDOT composite (85/15, w/w) (70 F/g) [105].EIS measurements were also carried out to study the characteristics of charge transfer in conducting polymers.An electrical equivalent circuit model for the conducting polymer micro-helix which is composed of electrolyte resistance (R s ), double-layer capacitance (C d ), charge transfer resistance (R ct ), and Warburg impedance (Z W ) was introduced to fit the EIS result, as shown in Figure 7 inset [97,106].In the high-frequency region, the well-defined semicircles which represent charge transfer-dominated regimes and whose diameters represent the charge transfer resistance (R ct ) are observed for all the micro-helices.The R ct values of the PPy micro-helix, the PANI micro-helix, and the PEDOT micro-helix-modified electrodes are calculated as 1.666, 1.68, and 1.565 Ω, respectively, which is in the same level as those of the reported PPy/graphene electrode film (0.15-1.73 Ω) [107] and better than those of the reported PANI/MnWO 4 composite (22 Ω) [108] and PEDOT-coated Au microelectrode arrays (749 Ω) [109].This result suggests that the micro-sized Spirulina bio-template can effectively enlarge the specific surface of the conducting polymer, which facilitates counterion exchange on the electrode/electrolyte interface.The R s value of the PANI micro-helix and the PEDOT micro-helix are extracted as 3.737 and 4.108 Ω, respectively, which is much lower than that (8.201 Ω) of the PPy micro-helix, suggesting that the PANI micro-helix and the PEDOT micro-helix have low internal resistances for ion transfer in Na 2 SO 4 aqueous solution.

Conclusions
In this study, we proposed a new strategy to fabricate conducting polymers in micro-helical form by using naturally-occurring Spirulina as a helical bio-template.In comparison with the conventional techniques for manufacturing micro-helices, Spirulina-templated conducting polymer micro-helices can be easily mass produced with an extremely low cost.The PPy micro-helix, the PANI micro-helix, and the PEDOT micro-helix were successfully synthesized in fine helical form.The strong interactions between the conducting polymers and the surface groups of Spirulina can be inferred from the multiple molecular structure analysis.The sheet resistances of the PPy micro-helix, the PANI . Ltd. (Taiyuan, China).Deionized water (18.2MΩ•cm, Milli-Q Reference, Merck Millipore, Shanghai, China) was used throughout the experiments.Polymers 2018, 10, x FOR PEER REVIEW 3 of 18 Nickel foam (99.8% Ni) and acetylene black (battery grade) were purchased from the Taiyuan Liyuan Lithium Technology Center Co. Ltd. (Taiyuan, China).Deionized water (18.2MΩ•cm, Milli-Q Reference, Merck Millipore, Shanghai, China) was used throughout the experiments.

Figure 1 .
Figure 1.The optical microscope images of Spirulina cells.

Figure 1 .
Figure 1.The optical microscope images of Spirulina cells.

Figure 4 .
Figure 4.The Raman spectra of PPy and the PPy micro-helix (a); PANI and the PANI micro-helix (b); and PEDOT and the PEDOT micro-helix (c).

Figure 4 .
Figure 4.The Raman spectra of PPy and the PPy micro-helix (a); PANI and the PANI micro-helix (b); and PEDOT and the PEDOT micro-helix (c).

Figure 5 .
Figure 5.The X-ray diffraction (XRD) pattern of PPy and the PPy micro-helix (a); PANI and the PANI micro-helix (b); and PEDOT and the PEDOT micro-helix (c).

Figure 5 .
Figure 5.The X-ray diffraction (XRD) pattern of PPy and the PPy micro-helix (a); PANI and the PANI micro-helix (b); and PEDOT and the PEDOT micro-helix (c).

Figure 5 .
Figure 5.The X-ray diffraction (XRD) pattern of PPy and the PPy micro-helix (a); PANI and the PANI micro-helix (b); and PEDOT and the PEDOT micro-helix (c).

Figure 5 .
Figure 5.The X-ray diffraction (XRD) pattern of PPy and the PPy micro-helix (a); PANI and the PANI micro-helix (b); and PEDOT and the PEDOT micro-helix (c).

Figure 6 .
Figure 6.The cyclic voltammograms of the PPy micro-helix (a); the PANI micro-helix (b); and the PEDOT micro-helix (c) with different scan rates from 5 mV/s to 100 mV/s.

Figure 6 .
Figure 6.The cyclic voltammograms of the PPy micro-helix (a); the PANI micro-helix (b); and the PEDOT micro-helix (c) with different scan rates from 5 mV/s to 100 mV/s.

Figure 6 .
Figure 6.The cyclic voltammograms of the PPy micro-helix (a); the PANI micro-helix (b); and the PEDOT micro-helix (c) with different scan rates from 5 mV/s to 100 mV/s.Polymers 2018, 10, x FOR PEER REVIEW 13 of 18

Figure 7 .
Figure 7.The Nyquist plots for the PPy micro-helix, the PANI micro-helix, and the PEDOT microhelix in the frequency range of 10 5 Hz to 0.01 Hz at an open circuit potential with an alternating current (AC) perturbation of 10 mV; inset: the electrical equivalent circuit for PPy, PANI, and the PEDOT micro-helix.