Plant extracted natural fluorescent protein C-phycocyanin doped in PVA nanofibers for advanced apparel application

Natural dyes are gaining a great deal of attention due to their eco-friendly and sustainable properties for advanced apparel applications. However, the reproducibility and accessibility of various colors using natural dyes remain challenging. In this study, plant-extracted fluorescent protein C-phycocyanin (CP) is used as a natural dye source and doped in polyvinyl alcohol (PVA) nanofibers via electrospinning for advanced apparel applications. The prepared nanofibers show a smooth and bead-free surface morphology. The FTIR results confirmed the formation of PVA nanofibers followed by a major peak at 3304 cm−1 due to the stretching of hydroxyl groups. Subsequently, CP-doping in PVA nanofibers is observed by the N–H deformation peaks at 1541 cm−1; C–N stretching vibrations at 1250 cm−1 and 1092 cm−1; and the C=O stretching vibrations of the carboxyl group at 1722 cm−1, respectively. Thus, CP-doped PVA nanofibers exhibit a good color strength (K/S) of 0.2 having a blue color tune and good color fastness properties. The mechanical strength of PVA nanofibers increased from 6 MPa to 18 MPa, due to crystalline characteristics endowed by the dope dyeing technique. Further, CP-doped PVA nanofibers exhibit homogeneous bright red fluorescence in individual nanofibers. Therefore, the proposed CP-doped PVA nanofibers can be used for flexible advanced apparel and biosensor applications.


Introduction
Natural dyes are of interest for physical investigations and reproducible dyeing processes [1,2]. However, advanced properties such as bright fluorescence in apparel using natural dyes are still challenging [3][4][5]. Natural fluorescent proteins (NFPs), such as phycobiliprotein, are preferred due to their abundance and stability for ecofriendly and reproducible dyeing processes [6,7]. NFPs have made impressive advances in biological and medical research, and can monitor dynamic changes in environments in a simple and effective way [8][9][10].
Spirulina maxima CP is a water-soluble accessory phycobiliprotein colorant with a strong blue color and can exhibit a strong red fluorescence in its native and concentrated forms [11,12]. It has a dimensional structure of two subunits and is extensively studied for immunological analysis due to its broad excitation spectrum, high Stokes shift, and high quantum yield [13][14][15]. CP is commonly used as a natural dye, nutrient ingredient, and natural color in foods and cosmetics. Additionally, CP can be used as a fluorescent marker in biomedical research and as a therapeutic agent for oxidative stress-induced diseases [16]. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Conventional dyeing generates dye pollutants during the dyeing process and requires high temperature and time, whereas dope dyeing is a more effective alternative that prevents dye loss into effluents, provides excellent color fastness, and achieves mass coloration with economical time and energy consumption [17][18][19][20][21]. Nanofibers have diverse applications including biosensor [22], filtration [23], tissue engineering scaffolds [24,25] protective clothing [26], wound dressings [27], drug delivery systems [28], and nanofibrous composites [29]. Researchers have mainly focused on the aesthetic properties of nanofibers for apparel applications [30]. To the best of our knowledge, Polyvinyl alcohol (PVA) nanofibers have not yet been dyed and colored with doped dyeing, which is an area of potential exploration. PVA nanofibers have several hydroxyl groups appearing in the structure [31]. It is believed that the NFPs performance is also anticipated by the existence of OH groups in the polymer [32] due to the creation of hydrogen bonding between the dye (CP) and polymer, leading to the uniform dispersion of the CP within the nanofibers. Therefore, we anticipated that PVA nanofibers to be used with CP.
Herein, we have aimed to develop low-cost, efficient, and repeatable colored dope-dyed PVA nanofibers via the electrospinning technique. The dope-dyed PVA nanofibers incur no dye loss compared to the conventional method and prevent environmental pollution. We introduce colored PVA nanofibers with strong fluorescence using CP which makes our material highly promising material for advanced apparel and biosensors applications. The surface morphology, fluorescence, color strength, chemical, and mechanical properties of nanofibers were analyzed using SEM, CLSM, XRD, FTIR, and universal tensile tester respectively. In contrast, color strength was determined through the use of a data color spectrophotometer and color-fastness tests such as light, washing, and hot pressing.

Materials
Polyvinyl alcohol (PVA, 88% hydrolyzed, Mw 125000) and. C-phycocyanin (purified lyophilized powder having a chemical structure shown in figure 1) were purchased from Sigma-Aldrich (USA). The glutaraldehyde (GA) and hydrochloric acid (HCL) were purchased from Wako Pure Chemical Industries (Japan). All the chemicals were used without further purification. Deionized (DI) water was used as a solvent for preparing PVA solution.

Electrospinning of PVA nanofibers and dope dyeing
PVA nanofibers were fabricated via electrospinning technique and a high voltage power supply. Har-100 * 13, Matsusada, Tokyo, Japan was used for electrospinning. Different PVA polymer compositions were fabricated i.e. 8%, 10%, 12%, 14%, and 16% w/w and subsequently dissolved in DI water. For complete dissolution and homogenous solution, PVA solution was stirred at 80°C temperature for 3.0 h. To observe the dye's effect on polymer composition, 10% CP (i.e. 0.01g) of 10% PVA polymer composition was added after complete dissolution of PVA polymer. Furthermore, the CP concentration of 3%, 5%, 7%, 10%, 12% and 15% was added to a 10% PVA composition to examine its effect on the composition. After adding CP individual dye in polymer composition, the solution was further stirred for one hour to obtain complete dissolution of dye in polymer solution to obtain blue color or some indication of complete dissolution. Once solution homogenized, the solution was poured in a 5 mL syringe fitted with a capillary tip measuring 0.6 mm internally. The solution was supplied with a voltage of 12 kV and fibers were collected at aluminum foil at a distance of 12 cm. After electrospinning, crosslinking of nanofibers was carried modifying the reported method [33]. The pure PVA and CP-doped PVA nanofibers were placed under the fumes of glutaraldehyde and HCL (3:1) for 36 h. After proper crosslinking of nanofibers, the resultant nanofibers were peeled off carefully from aluminum foil and dried overnight in the ambient environment.
Here 'R' represents reflectance decimal fraction of DP-doped PVA nanofibers and 'K' presents the co-efficient of absorption, and 'S' is scattering co-efficient.

Fluorescence imaging
Confocal laser scanning microscopy (CLSM, Zeiss LSM 510) images were recorded at fixed excitation wavelength of 580 nm. The CP-doped PVA nanofibers emit significant red fluorescence having emission wavelength at 650 nm, while the excitation and emission slit width is 10.0 nm. The CLSM images were taken at 20X magnification. Original doped PVA nanofibers fluorescence images were taken under UV-light (λ ext −366 nm).

Characterizations
Surface morphology of the PVA and doped PVA nanofibers were observed using field emission scanning electron microscopy (FE-SEM, S-4800; Hitachi Ltd Japan). FE-SEM samples were coated with gold and examined at an accelerating voltage of 15 kV. Chemical structure of PVA and doped PVA nanofibers were analyzed using fourier transform infrared (FTIR) spectroscopy (Thermo Nicolet 5700, Thermo Fisher Scientific Inc., USA). The crystallinity of PVA and doped PVA nanofibers were analyzed using x-ray diffraction (XRD, model D/max-IIB, Rigaku, Japan). The mechanical strength of nanofibers were examined using universal testing machine (Titan universal Tester 3-910, Germany) according to ASTM D-638 method. The crosshead speed on universal testing machine was set at 10.0 mm min −1 .

Influence of dye on polymer concentrations
It is crucial to systematically study the influence of CP dye on different PVA concentration and composition for dope dyeing method. Initially, the effect of different polymer concentrations (8%-16%) on their surface morphology was analyzed by FE-SEM as shown in figure 2. Based on the results, the nanofibers obtained are smooth and beads-free. Figure 2 also shows the diameter distribution of all above polymer compositions. PVA nanofibers produced at 8% concentration have a smaller average diameter than electrospun nanofibers produced at higher concentrations. In PVA nanofibers, the average diameter increases directly with increasing polymer concentration, as measured by the average diameter [19]. In comparison to same pure PVA nanofibers, the diameter decreases when CP (dye) is added to the polymer solution. We reason that the water-soluble nature of C-phycocyanin might be responsible to reduce the polymer concentration [34]. Hence, the results confirm the presence of the dye protein in part with PVA dope dyed nanofibers. Furthermore, the effect of dye on polymer concentration was analyzed at constant dye concentration i.e. 10% (0.01g) in weight of dye on 10% PVA composition. Figure 2 shows the effect of C-phycocyanin on color strength (K/S) on PVA nanofibers during dope dyeing. The results showed that higher color strength (K/S) value of 0.17 was obtained at 8% polymer concentration of CP-doped PVA nanofibers and it was observed that the K/S value decreases with increasing the polymer concentration. This was due to lower polymer concentration (8% CP-doped PVA nanofibers) has lower polymer mass and give higher color strength at fixed CP amount in all other polymer concentrations. Similarly, higher polymer concentration (16% CP-doped PVA nanofibers) gives low color strength due to high polymer mass [35]. While the CP-doped PVA nanofibers show blue color due to the presence of natural blue pigment in CP [12]. Figure 3 showed the surface morphology of PVA nanofibers after adding dye concentrations (3%-15%) and the diameter distribution of different dye concentrations doped nanofibers. It was observed that the diameter of nanofibers is slightly lower while adding dye into nanofibers. We hypothesize water-soluble nature of CP could be responsible for the diameter decrease. It may be due to water-soluble nature of CP which ultimately reduces the polymer concentration [34,36]. These results attest to strong interaction between dye and the nanofibers displaying blue color gradient with increasing concentration.

Influence of dye concentration on polymer composition
To explore the color strength of CP on PVA nanofibers, different dye concentrations i.e., 3%, 5%, 7%, 10%, 12% and 15% in weight of polymer were doped into 10% PVA solution to electrospun the nanofibers. Figure 3 shows that K/S is linearly related to initial dye concentration. PVA nanofibers exhibit an excellent capability to color strength properties and 15% CP concentration displayed the highest color strength (K/S) value of 0.2 on doped PVA nanofibers. While increasing dye concentration might saturate polymer deposition or cause perturbations in the polymer chains and ultimately block the further formation of nanofibers [37].

Fluorescence properties of nanofibers
CP-doped PVA nanofibers exhibit very bright red fluorescence under UV light (365 nm), as shown in figure 4(a), which is the characteristic emission of CP. To further verify the fluorescent nature of the dye, the enlarged fluorescent emission of the CP [13] in the PVA nanofibers membrane, was observed from the CLSM images ( figure 4(a)). The observed uniform fluorescence to entire nanofibers suggested a homogeneous distribution of CP in nanofibers. Doping the nanofibers with different concentrations of CP distorted their uniformity and homogeneity. Accordingly, with increasing concentrations of CP, the nanofiber membrane showed minute color discrepancies (in terms of fluorescence) ranging from light red to dark red. Thus, by simply adjusting the CP concentration, the structural features and functional properties of nanofiber membranes can be effectively controlled.
In order to develop practical sensors, we evaluated the fluorescence stability of doped PVA nanofibers since CP stability was limited to a cold environment (4°C). In the present case, the doped PVA nanofibers membranes  were left at room temperature for 3 months, and then CLSM images were recorded as shown in figure 4. The experimental results confirmed that there is no substantial decrease in the emission intensity and the dope-dyed PVA nanofibers continue to maintain red fluorescence. Polymer chains around fluorescent CPs provide a protective environment, which enhances the stability of the fluorescent CPs, as reported earlier [38].

Mechanical properties of nanofibers
The mechanical properties of pure and CP-doped PVA nanofibers are shown in figure 4(i). The pure PVA nanofibers showed 6 MPa tensile strength which significantly increased to18 MPa in CP-doped PVA nanofibers. It could be due to the enhanced crystalline characteristics conferred by dope dyeing compared to pure PVA nanofibers [39].

XRD measurement on nanofibers
X-ray diffraction patterns of nanofibers are shown in figure 5(a). The pure PVA and CP-doped PVA nanofibers show main peaks at 19.7°and 19.8°respectively. The CP-doped PVA nanofibers exhibit a more intensive peak, indicating a better crystallinity than the pure PVA nanofibers. The possible reason for this may be due to the proper CP diffusion with the polymer chains during the electrospinning process and as noted earlier the tensile strength increased is well supported by these results [40,41].

FTIR measurement of nanofibers
To ascertain the presence of CP and successful dope in the nanofibers structure, FTIR spectroscopy was performed on pure PVA and CP-doped PVA nanofibers ( figure 5(b)). The major peaks appeared on nanofibers in the range of 800-3500 cm −1 . Pure PVA nanofibers showed a major peak at 3304 cm −1 due to the stretching of hydroxyl groups [31]. While peaks related to the chemical structure of PVA appeared as C-H stretching at 2942 cm −1 , C-H bending at 1431 cm −1 and C-O stretching at 1092 cm −1 [42,43]. Moreover, the peak at 1730 cm −1 is due to the C-O group of residual acetate groups of polyvinyl acetate [44]. In the spectrum of CPdoped PVA nanofibers, the N-H deformation peaks at 1541 cm −1 ; C-N stretching vibrations at 1250 cm −1 and 1092 cm −1 ; and the C=O stretching vibrations of the carboxyl group at 1722 cm −1 . Furthermore, the CP-doped PVA nanofibers have peaks of C-H stretch of alkyl chain at 2950 and 2885 cm −1 ; and C-O stretching in hydroxyl groups at 1021 cm −1 [45]. The results showed that the C-phycocyanin was successfully doped with PVA nanofibers.

Color fastness
Color fastness to washing, hot pressing, and light of doped PVA nanofibers are shown in table 1. Based on the blue wool reference scale, the CP scored a 7 in light fastness on doped PVA nanofibers. The C-phycocyanin showed good results in hot pressing on doped PVA nanofibers. The washing fastness test was taken to analyze the colorfastness properties of doped PVA nanofibers. Grayscale grading showed good results for doped PVA nanofibers [30].
According to table 2, dope dyeing of PVA nanofibers has comparatively good results. PVA nanofibers dyed using this method were found to be more efficient and economical than conventional dyeing under exhaust dyeing methods. Our method is based on completely natural dye CP which is environment friendly. The high fluorescence nature of CP is a strong candidate for flexible biosensors.

Conclusion
The colorful and bright fluorescent PVA nanofibers were successfully fabricated via a simple electrospinning technique and doping a natural CP protein. FE-SEM study shows that PVA nanofiber diameter increases with increasing polymer concentration, and that PVA nanofiber diameter for doped nanofibers is significantly smaller than that for pure nanofibers. Furthermore, dope dyeing confirmed that K/S values increased with the concentration of CP. The CP-doped PVA nanofibers showed good color fastness properties. CLSM images showed that CP has doped homogenously on nanofibers with bright red fluorescent. Furthermore, FTIR results revealed that CP was successfully doped in PVA nanofibers and good affinity towards PVA nanofibers. The XRD results indicates that CP-doped PVA nanofibers exhibit a more intensive peak, indicating a better crystallinity than the pure PVA nanofibers and increased tensile strength observed. The purposed colorful PVA nanofibers with unique fluorescent properties make a suitable product for advanced apparel, smart identity tags, and biosensors applications.

Data availability statement
All data that support the findings of this study are included within the article. a dyeing conditions: Dope dyeing directly collected from electrospinning, no need of time and temperature for dyeing. b Blue wool scale 1-9 rating, 1 is poor and 9 is excellent. c Gray scale 1-5 rating, 1 is poor and 5 is excellent. d CTA cellulose triacetate, CT cotton, PA polyamide, PET polyester, PA polyacrylic, Wo wool.