Coupling Infusion and Gyration for the Nanoscale Assembly of Functional Polymer Nanofibers Integrated with Genetically Engineered Proteins

Nanofibers featuring functional nanoassemblies show great promise as enabling constituents for a diverse range of applications in areas such as tissue engineering, sensing, optoelectronics, and nanophotonics due to their controlled organization and architecture. An infusion gyration method is reported that enables the production of nanofibers with inherent biological functions by simply adjusting the flow rate of a polymer solution. Sufficient polymer chain entanglement is obtained at Berry number > 1.6 to make bead‐free fibers integrated with gold nanoparticles and proteins, in the diameter range of 117–216 nm. Integration of gold nanoparticles into the nanofiber assembly is followed using a gold‐binding peptide tag genetically conjugated to red fluorescence protein (DsRed). Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the engineered red fluorescence protein with the nanofibers. The gold nanoparticle decorated nanofibers having red fluorescence protein as an integral part keep their biological functionality including copper‐induced fluorescence quenching of the DsRed protein due to its selective Cu+2 binding. Thus, coupling the infusion gyration method in this way offers a simple nanoscale assembly approach to integrate a diverse repertoire of protein functionalities into nanofibers to generate biohybrid materials for imaging, sensing, and biomaterial applications.


Introduction
Inorganic-binding peptides have attracted tremendous interest in the last decade, ranging from assembly of nanoparticles [ 1,2 ] to oriented immobilization of proteins, [3][4][5] synthesis of inorganics, [ 6,7 ] and biofunctionalization of surfaces. [8][9][10][11][12] Solid-binding peptides have been shown to control the organic-inorganic materials interface in different applications, but their integration to design tunable, functional nanofi bers has not been investigated. This peptide-based nanoassembly process can be regulated at the molecular level to construct a self-organized architecture and establish an ordered nanostructure.
Nanofi bers featuring functional nanoassemblies show great promise as enabling constituents for a diverse range of applications in areas such as tissue engineering, sensing, optoelectronics, and nanophotonics due to their controlled organization and architecture. An infusion gyration method is reported that enables the production of nanofi bers with inherent biological functions by simply adjusting the fl ow rate of a polymer solution. Suffi cient polymer chain entanglement is obtained at Berry number > 1.6 to make bead-free fi bers integrated with gold nanoparticles and proteins, in the diameter range of 117-216 nm. Integration of gold nanoparticles into the nanofi ber assembly is followed using a gold-binding peptide tag genetically conjugated to red fl uorescence protein (DsRed). Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confi rms the integration of the engineered red fl uorescence protein with the nanofi bers. The gold nanoparticle decorated nanofi bers having red fl uorescence protein as an integral part keep their biological functionality including copper-induced fl uorescence quenching of the DsRed protein due to its selective Cu +2 binding. Thus, coupling the infusion gyration method in this way offers a simple nanoscale assembly approach to integrate a diverse repertoire of protein functionalities into nanofi bers to generate biohybrid materials for imaging, sensing, and biomaterial applications.
Over the past decade, a variety of techniques have been used to produce nanofi bers and among these pressurized gyration process, invented in 2013, has generated exceptional interest for the mass production of functionalized products including fi bers and microbubbles. [ 13,14 ] However, one drawback is that it does not allow control of fl uid fl ow through the fi ber generating orifi ces where the infusion rate of polymer solution infl uences fi ber size and distribution, and the morphology of the spun fi ber. Here, we have created a novel method, infusion gyration, which does not require external pressure, but rather, relies on controlling the infusion rate of the polymer solution. This allows to generate nanofi bers that are integrated with nanoassemblies, which in this work feature gold nanoparticles and inorganic-binding peptides. Namely, we utilized the wellcharacterized gold-binding dodecapeptide, Au-BP2, [ 15 ] for the fi ber formation process. As a means to easily trace the integration of Au-BP2 into the nanofi bers, we genetically conjugated the gold-binding peptide to a biomarker protein, i.e., red fl uorescence (DsRed). Additionally, the protein goes through reversible fl uorescence quenching upon copper ion binding. Thus, we show that the biohybrid nanofi bers could be further exploited to advance proteinintegrated functional materials for biofabrication.

Materials
Polyethylene oxide (PEO, powder, molecular weight ≈ 200 000 g mol −1 ) and gold nanoparticle solution (analytical grade, average particle size ≈10 nm) were purchased from Sigma-Aldrich (Poole, UK). All reagents were used without further purifi cation. Isopropylthiogalactopyranoside (IPTG) was purchased from Sigma-Aldrich (Milwaukee, WI, USA). Amylose resin for column chromatography was purchased from New England Biolabs (Ipswich, MA, USA). The Instant Blue coomassie based staining solution was procured from Expedeon Inc. (San Diego, CA, USA). All buffers were fi ltered and degassed before using. Details on expression and purifi cation of red fl uorescent gold-binding fusion proteins are given in Supporting Information and the genetic construction of the plasmid was described in our previous work. [ 16 ]

Preparation of Polymer and Engineered Protein Solution Mixture
The polymer solution was prepared in an air-tight bottle using deionized water as solvent to dissolve the PEO powder under magnetic stirring for 24 h at ambient temperature (20 °C). Solutions with various concentrations of PEO were prepared, however, as explained below, only the 10 wt% of PEO solution was chosen to integrate with the protein-nanoparticle assemblies. Phosphate buffer saline (PBS) solution with a pH ≈ 7.4 was prepared at ambient temperature and added to the purifi ed DsRed-AuBP2-engineered protein (molecular weight ≈ 30 kDa) using a micropipette to achieve a working stock solution of 50 × 10 −6 M . The gold nanoparticle solution was added to the gently shaken protein solution. 40 g of PEO solution was taken in an air-tight bottle and 0.4 mL of the gold nanoparticle-protein mixture was added while sonicating in a water bath using an ultrasound sonifi er (Branson sonifi er 250) at a power output of 60% for 15 min. This prevented aggregation of the functionalized gold nanoparticles in the polymer solution.

Infusion Gyration
The system consists of a rotary aluminum cylindrical vessel containing 20 small round orifi ces on the face ( Figure 1 a) much like in our work with pressurized gyration. [ 13,14 ] The dimensions of vessel and orifi ces are 60 mm in diameter with a height of 25 mm, and 0.5 mm in diameter located at the same vessel height, respectively. One end of the vessel was joined to a syringe  pump through a rotary joint, which can control fl ow into the vessel. The bottom end of the vessel was connected to a DC motor, which can produce variable speeds up to 36 000 rpm. In order to investigate the nanofi ber size and size distribution under different conditions, the polymer solution was spun at six different fl ow rates (5000, 4000, 3000, 2000, 1000, and 500 μL min −1 ) under a constant rotating speed (36 000 rpm) at the ambient temperature and relative humidity (≈42%). Protected by a transparent plastic container, the system allows convenient collection of the polymer fi bers on stationary aluminum foil sheets within the container. A schematic illustration of the different steps in the entire process is shown in Figure 1 b.

Fiber Characterization
Nanofi bers produced were studied using fi eld-emission scanning electron microscopy (FE-SEM). Samples were coated with gold using a sputtering machine (sputter time ≈75 s) before loading to the microscope. High-and low-magnifi cation images were acquired at randomly selected positions (>20) within a sample. About 150 measurements were made at random locations to plot the fi ber diameter distribution using ImageJ software. Additionally, the as-produced fi bers were characterized using a fl uorescence microscope to verify the integration of the DsRed fl uorescence proteins expressed with AuBP peptides in the nanofi bers.

Fourier Transform Infrared Spectroscopy
The infrared spectra of fi bers were recorded on a PerkinElmer Spectrum-400 Fourier transform infrared spectroscopy (FTIR) spectrometer at the ambient temperature between 4000 and 650 cm −1 with a resolution of 4 cm −1 . To obtain reasonable signal-tonoise ratio, the average of 20 scans was taken. Samples were analyzed directly by single-bounce diamond ATR.

Localized Surface Plasmon Resonance Spectroscopy
The effect of proteins on the plasmon excitation wavelength for gold nanoparticles (AuNPs) was analyzed by measuring the light absorbance of AuNPs in the absence and the presence of proteinconjugated AuNPs in the nanofi ber using a Cytation 3 Imaging Multi-Mode Plate Reader (BioTek Instruments, Inc., Vermont, USA). Each spectrum is an average of three individual samples recorded twice.

Copper-Binding Assay
Different concentrations of copper were added to the DsRed-AuBP2c protein fi ber solution. The fl uorescence intensity was measured (Varian Cary Eclipse Fluorescence Reader) by excitation of the samples at 556 nm and emissions were recorded at 590 nm.

Results and Discussion
Detailed information on protein production and their gold-binding characteristics are given in Supporting Information. Figure 2 provides details regarding the fi ber size (diameter) and size distribution of the protein-integrated nanofi bers produced. These ranged from 117 to 216 nm in average diameter for the six different fl ow rates studied. At the lowest fl ow rate, 500 μL min −1 , the mean fi ber size diameter was 117 nm. When the fl ow rate was doubled to 1000 μL min −1 the mean fi ber size was 161 nm. Surprisingly, the fl ow rate of 3000 μL min −1 resulted in a reduced mean fi ber size of 132 nm and this is explained below. The initial size increasing trend continued at higher fl ow rates, and, at 4000 μL min −1 size was 190 nm, and at 5000 μL min size was 216 nm. The polydispersities of the fi ber size distributions were 47%, 32%, 19%, 26%, 25%, and 23% for fl ow rate values of 500, 1000, 2000, 3000, 4000, and 5000 μL min −1 , respectively, with the rotating speed remaining constant at 36 000 rpm. The morphology of nanofi bers revealed bead-free, continuous, uniform structures. Single-strand pore-free fi bers were bundled together and it was also possible to form well-aligned structures due to the high stretching force experienced during gyration.
Compared to pressurized gyration, [ 13 ] the ability to use higher fl ow rates allows increased hydrostatic pressure, which was kept constant for fl ow rates at a fi xed rotating speed by ensuring continuous fl ow. But the hydrostatic pressure is much lower than the centrifugal force at the orifi ce. [ 17 ] This allows the destabilizing centrifugal force and the withholding surface tension force to determine the fi nal size and size distribution of the fi bers. Moreover, the fl ow rate regulates the volume of material and the mass transfer across the orifi ce. The overall increase of fi ber diameter with increasing fl ow rate is attributed to this phenomenon. The drop in fi ber diameter observed at 3000 μL min −1 , and subsequent increase at higher fl ow rates may be attributed to the balance of solvent evaporation and change in the volume of material at the orifi ce. At lower fl ow rates, the solvent has enough time to evaporate, allowing the polymer jet more time to stretch, which results in the formation of fi ner fi bers. At higher fl ow rates, the solvent does not have suffi cient time to evaporate before reaching collection, therefore, a coarser fi ber is formed. [ 18,19 ] Additionally, in infusion gyration, the volume and shape of the polymer droplets vary for different fl ow rates at the orifi ce, which could cause differences in fi ber size and size distribution. Finally, during spinning, the traveling polymer jet experiences aerodynamic forces that may impact the stretching of the jet and, thereby further alter the fi nal size and size distribution. [ 20 ] In fi ber-forming processes, viscosity and concentration of the polymer solution infl uence the resulting fi ber size and fi ber morphology. [ 13,21,22 ] In a typical pressurized gyration process, these properties also affect polymer chain entanglement, a prerequisite to the formation of nanofi bers. [ 13 ] To verify that this is also the case in infusion gyration, we adopted analysis using the Berry number (Be), a dimensionless index used to control and indicate the fi ber size. Be = [η] C , and [η] represents the intrinsic viscosity (0.32 dL g −1 for PEO), and C is the concentration.
A clear-cut relationship is evident (Table 1 ) where, as the concentration was increased, viscosity (η) increased gradually until a specifi c value was reached, after which the viscosity increased dramatically, giving, η = 0.92 C 2.73 . In dilute polymer solutions, Be < 1.6, and nanofi bers could not be formed due to insuffi cient polymer chain entanglement. When 1.6 < Be < 3.2 nanofi bers formed only at a rotating speed of 36 000 rpm, primarily due to the increased time constant of forces acting upon the polymer solution and the increased viscosity of the polymer solution. Thus, a minimum rotating speed of 36 000 rpm was required to initiate fi ber formation. For Be numbers between 3.2 and 4.8, a suffi cient amount of chain overlap and entanglement allowed the formation of nanofi bers. However, when Be > 4.8, a much thicker fi ber resulted. Therefore, a 10 wt% PEO polymer solution was selected for integration of AuNPs functionalized with DsRed-AuBP2 to the nanofi bers. Protein-integrated PEO nanofi ber yield steadily increases on increasing the infusion rate from 500 μL min −1 (0.02 kg h −1 ) until 3000 μL min −1 (0.18 kg h −1 ), after which the yield increased dramatically by nearly an order of magnitude to 1.45 kg h −1 at an infusion rate of 5000 μL min −1 . However, yields obtained for the infusion gyration method was lower than that achieved from pressurized gyration. This may be due to the absence of blowing in the former, but it should be noted that the yields are still greater than those achieved through conventional centrifugal spinning or electrospinning. [ 13 ] Concerning the images provided in Figure 2 , the left panels are bright-fi eld and the right panels are the corresponding fl uorescence images. These reveal the smooth structure of the nanofi bers and regardless of the fl ow rate variation, they indicate that the integration of engineered protein with gold-binding peptides within the nanofi bers is possible. FTIR analysis conducted on PEO and biohybrid (PEO/protein) fi bers confi rmed the presence of the protein. Figure 3 depicts that characteristic peaks of PEO observed at 2900 cm −1 (methylene group CH 2 molecular stretching), and at 1100 cm −1 and 960 cm −1 (C O C group stretching). [23][24][25] A change in bandwidth for the absorption centered around 2880 cm −1 also occurred and changed further with protein integration. Engineered proteins in the PEO/protein nanofi bers resulted in an FTIR peak at 1720 cm −1 , representing the characteristic amide bonds of protein. [ 25 ] After washing the PEO/protein nanofi bers with PBS, the same FTIR peak was still observed. [ 26 ] The maintained carbonyl peak suggests that the protein remained bound to the surface of the AuNPs, which were integrated in the PEO nanofi bers, even following a washing step. This is remarkable protection of the water-soluble PEO polymer and is further discussed below.
Many efforts have been made to decorate nanofi bers with metallic nanoparticles. [ 27 ] Here, genetically engineered proteins were used to stabilize AuNPs and direct their spatial order into 1D particle assemblies ( Figures S3  and S4, Supporting Information). Integrating discrete AuNPs into the hybrid nanofi bers may signifi cantly alter their inherent optical properties, which were evaluated by UV-Vis and fl uorescence spectrophotometry. We observed that integration of AuNPs onto the surface of the fi bers had a signifi cant effect, when compared to non-protein-integrated fi bers. Hybrid nanofi bers in PBS buffer exhibited a strong fl uorescence emission band at wavelength 570 nm ( Figure 4 a). Interestingly, proteinbased nanofi bers did not dissolve in PBS buffer and did not show any leakage of protein to the buffer. When nanofi bers were removed from the PBS buffer, the fl uorescence intensity of PBS buffer alone was completely mitigated confi rming that the red fl uorescent protein did not leak into PBS buffer (Figure 4 b). This might be related to peptide-enabled selforganization of fusion protein onto nanoparticles, which may be stabilizing the polymer complex. The resulting protein with goldbinding peptide tag (DsRed-AuBP2) is hypothesized to blend into fi ber formation by providing self-organized biomolecular-inorganic surface interactions to decorate gold nanoparticles along the nanofi bers. The functionalized-gold nanoparticles with inorganic-binding peptide potentially provide both electrostatic and steric  interactions to stabilize nanofi ber formed by the polymer PEO and consequently this may result in different phase behavior of the hybrid polymer.
Additionally, biohybrid nanomats composed of nanofi bers were analyzed in both dry and wet conditions using a cell-imaging multi-mode reader and indi-cates that protein-integrated samples did not dissolve in aqueous environments and that similar fl uorescent images were obtained in both dry and wet forms ( Figure S5, Supporting Information). Thus, protein-based nanofi bers did not show any leakage of protein into the PBS buffer, which was verifi ed through a fl uorescence analysis of the buffer solution after the nanofi bers were removed. Interestingly, as expected, PEO nanofi bers that were not integrated with proteins dissolved completely when exposed to an aqueous environment, suggesting a stabilizing role of the protein nanoassemblies on the nanofi bers. Here, engineered DsRed-AuBP2 proteins were utilized due to their low autofl uorescent potential given that their excitation and emission wavelengths in the near-far-red region of the spectrum allow for a higher signal-to-noise ratio. However, the protection of the water-soluble PEO polymer and the intact DsRed-AuBP2 proteins in the nanofi brous structures may be correlated to phases present in the complex polymer system and this requires a more in-depth study. It has been shown that phase separation can occur in the radial direction of fi bers due to rapid solvent evaporation during spinning, thus a poly mer-rich phase occurs in the inner regions of the spun hybrid fi bers whereas a secondary phase can be locked onto the outer surface of the drying liquid jet. [ 28 ] Moreover, PEO can crystalize at the ambient temperature thus preventing the rearrangement of amorphous chains that might infl uence the formation of secondary structures on the surface of the nanofi bers. [ 29 ] Our previous studies [ 16 ] found that signifi cant quenching of the red fl uorescence activity occurred in the presence of copper ions. Thus, we explored the potential of using the nanofi bers for rapid monitoring for the presence of copper ions, a major component in heavy metal pollution. Different concentrations of copper were added to nanofi ber samples in 100 μL of MOPS buffer and fl uorescence readings were measured at 558 and 590 nm excitation and emission wavelengths, respectively. The nanofi bers demonstrated quenching at different copper concentrations (Figure 4 c). This promising result not only demonstrates the potential of protein-integrated nanofi bers to monitor the presence of copper ions in solution, but also establishes the potential impact that may be achieved through further tuning of the system to detect biological and chemical changes in the environment. Our results provide evidence that the red-fl uorescence activity of the engineered proteins embedded in the nanofi bers maintain full functionally and can respond to various dynamic conditions.

Conclusion
An infusion gyration method that enabled the production of engineered protein-integrated nanofi bers has b) The removal of the PEO/protein nanofi bers from PBS buffer diminishes fl uorescence intensity demonstrating no signifi cant protein leakage into the solution. c)Titration of DsRed-AuBP2integrated nanofi ber with Cu 2+ . Emission spectra were obtained by excitation at 558 nm in the presence of 10 × 10 −6 , 20 × 10 −6 , 50 × 10 −6 M Cu 2+ . been invented. By adjusting the fl ow rate of the polymer solution, well-aligned, smooth-and bead-free-nanofi bers were generated. FTIR analysis confi rmed the presence of the protein, by revealing the characteristic peak of the amide bond in the protein structure in the nanofi bers. The assembly of the engineered proteins bound to the gold nanoparticles along the fi bers was easily tracked by exploiting the fl uorescent activity of the coupled proteins in the nanoassembly. Gold nanoparticles chaperoned the bifunctional protein integration into the nanofi bers. The nanofi bers produced also become much less soluble in water, due to the incorporation of the protein-coated gold nanoparticles. Copper-induced fl uorescence quenching of red fl uorescence protein was observed when the fi bers were exposed to an increased level of copper concentration. As the protein-infused fi bers were able to maintain their biological functionality, even in response to changes in buffer conditions, they can offer a fundamental platform in the design and fabrication of novel biomaterials.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.