Combined Chemical and Physical Encoding with Silk Fibroin‐Embedded Nanostructures

Nanostructures spun into regenerated silk fibroin fibers encode physical and chemical information. These materials are composed of Raman enhancing nanoscale structures whose location in a linear array and chemical functionality endow them with a tunable identity or code. These structures remain functional after being electrospun into fibroin fibers and report a unique spectroscopic signature to prove the authenticity of a material through their code and by uniquely linking it to the composition of the host material. Material-embedded chemical or physical tags are becoming increasingly important for applications such as brand verification, anti-counterfeiting, and tracing the origin of controlled goods (e.g. explosives, medicine, or currency). Because of their small size, nanoscale tags are particularly attractive for anti-counterfeiting because they can be integrated into existing materials with a minimal influence on the properties of the host material. One important technique for the rapid synthesis of nanoscale tags is electrodeposition using nanoporous membranes.1 This technique can be used to synthesize multisegmented nanowires where each segment is of prescribed composition and size. Such nanowires can function as physical tags where the structure of the nanomaterial encodes information.2 This electrodeposition approach also forms the foundation for on-wire lithography (OWL), a procedure in which segmented nanowires are functionalized with a rigid backing layer that allows them to maintain their synthesized structure even when some segments are chemically removed.3 Not only can such freestanding structures be used as physical tags,4 but dimers can be created with nanoscale gaps that are useful for surface-enhanced Raman spectroscopy (SERS).5 Therefore, by incorporating specified Raman chromophores into a gap, the structure is endowed with an easily interrogated chemical identity.6 To translate these techniques into an encoding strategy, a prescribed linear array of chemically functionalized dimers can be fabricated. Such tags, known as a nanodisk codes (NDCs), combine the physical location of a dimer and the chemical identity of the SERS chromophore to achieve a multiplexed scheme that dramatically increases the available number of codes and the difficulty of counterfeiting. While nanomaterials such as NDCs form ideal tags, incorporating them into bulk materials is not straightforward. Utilizing NDCs as a platform for anti-counterfitting or brand validation has been proposed, yet proving this concept requires integrating NDCs into a macroscale material, determining their location within the material, and reading their code. An especially difficult challenge is integrating rigid nanomaterials into flexible or deformable materials in such a way that they retain their function, are not removed through normal use, and can still be interrogated.7 Here, we explore the potential for incorporating NDCs into silk fibroin fibers electrospun from protein collected from the cocoons of Bombyx mori (Scheme 1). Silk fibroin represents an ideal proof-of-concept material as methods for processing it are well established;8 furthermore silk fibers are widely used in clothing and represent a functional analogue to materials used for paper currency. In addition, we explore how the enhancing properties of the NDC can be used to not only enhance the Raman signal from the dyes that reside near the disk pairs that comprise them, but also the host regenerated silk fibroin material. Interestingly, while the fibroin by itself produces no observable Raman signatures over the 800 to 1800 cm−1 range, unique signatures can be observed in the presence of NDCs, resulting in a well-defined spectroscopic fingerprint of the composite NDC-silk material. In a typical experiment, NDCs were synthesized using literature methods[4],9 and characterized by scanning electron microscopy (SEM, Figure 1A−C, i−ii). Briefly, 360 nm diameter multi-segmented nanowires were synthesized electrochemically within the pores of an anodic aluminum oxide template.1 These multi-segmented nanostructures were converted into linear arrays of plasmonically coupled disk pair structures using OWL.3, 9 By tuning the length of each disk and the gap between disk pairs, these structures were optimized to produce significant SERS enhancement.5, 10 Because the morphology of NDCs is essential to their function, it is necessary to incorporate them into a host material without altering their structure. We hypothesized that electrospinning could be used to gently incorporate the NDCs into regenerated silk fibroin fibers. To explore this, equal volumes of a suspension of NDCs in 2,2,2-trifluoroethanol (TFE) and a solution of fibroin and polyethylene oxide (PEO) dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were mixed to form an electrospinning precursor. PEO has been found to promote nanofiber generation in electrospinning experiments.[8] Fibers were spun by pumping the precursor through an 18 gauge blunt-tip needle, serving as a spinneret, at a flow rate of 35 mL/h utilizing a syringe pump. A positive DC voltage of 15 kV was applied between the spinneret needle and a grounded collection substrate held 25 cm apart. Glass slides, silicon chips, or TEM grids were then placed in the path between the tip and the collector, to prepare electrospun samples for additional characterization. In principle, this approach could be used to tailor the concentration of multiple NDC species in a given fiber. Here, we utilize a homogenous solution of precursor to ensure that there was a uniform density of NDCs in the fibers, however, one could dynamically vary the NDC concentration in the precursor and synthesize fibers with a prescribed density profile of multiple NDC species. To test the viability of NDCs as nanoscale tags, we sought to show that multiple codes could be integrated into regenerated silk fibroin fibers through this method. To convert structure to information, we used a convention wherein ‘1’ indicates the presence of a dimer and ‘0’ indicates the absence of a dimer. By changing the synthetic parameters during electrodeposition, NDCs encoding 111 (Figure 1A, iii), 10101 (Figure 1B, iii), and 11001 (Figure 1C, iii) were synthesized. Because 111 is a symmetric code, the trailing or leading 0's should be avoided for clarity when using a 5-bit naming convention. Importantly, electron microscopy of the NDCs within the fibers did not reveal appreciable mechanical damage to the NDCs after electrospinning. Once the method for incorporating NDCs had been established, we performed confocal Raman microscopy to evaluate their function as SERS active nanostructures while embedded within the regenerated silk fibroin fibers and thus confirm their viability as chemically encoded nanoscale tags. In this experiment, NDCs were functionalized with the Raman chromophore para-mercaptoaniline (PMA) prior to electrospinning following previously established procedures.9 Because the gold features of the NDC were reflective under bright-field optical microscopy, it was possible to locate individual NDCs within the fibroin fibers. Once individual structures had been located, a Raman map of the area was collected using a 633 nm laser excitation. Because each dimer was separated by 1 μm, it was easily possible to resolve the peaks associated with each disk pair within a given NDC.[4] These maps show that Raman readout of the codes is feasible (Figure 1A−C, iv), and by extension, that the electrospinning method preserves the enhancement and addressability of the NDC hotspots, a result that is not obvious given the nanoscale gaps between the dimers. Additional electron microscopy showed that NDCs were successfully incorporated into the fibrous network with multiple copies of the code visible in a single field of view (Figure 2A, red boxes). By simply controlling the concentration of NDCs in the electrospinning precursor, their density within the nanofibers also can be tuned. The ability of the NDCs to remain inside the fiber is particularly important should these structures be utilized as tags in textiles. As a model for the typical care of silk fabric, substrates were laundered in a detergent solution (Figure 2B) or with tetrachloroethylene (a typical dry cleaning solvent) (Figure 2C) and characterized by electron microscopy. It was observed that these treatments had no effect on the structure of the NDCs or the morphology of the nanofibers. Conceptually, through the use of SERS, the NDC provides spectroscopic information about the material in its vicinity. We have shown that pre-functionalized Raman chromophores can be used to assign a given NDC a chemical identity. However, being embedded in silk fibroin, it is interesting to consider whether these structures can also be used to report information regarding the chemical composition of the host material. To explore this, a single batch of NDCs was divided into two parts; one remaining chemically unfunctionalized, and one that was functionalized with PMA. Suspensions of these NDCs were electrospun into regenerated silk fibroin fibers as before. We then performed Raman spectroscopy to establish a baseline by probing an area of regenerated fibroin fiber that contained no NDC structure (Figure 3a, black box). Without metallic nanostructures to provide enhancement, no chemical information could be collected from the resulting featureless Raman spectrum (Figure 3c, black trace). However, this is not a property of silk as in a different system, chemical information could be obtained via Raman spectroscopy, but only at considerably higher laser power.11 We then probed an area that contained a NDC that had not been functionalized with PMA (Figure 3a, blue box), and recorded a Raman spectrum of the fibroin fiber (Figure 3c, blue trace). This result implies that by using the NDC as a SERS enhancer, it was possible to collect spectroscopic information from the silk itself. Interestingly, performing the same measurement on PMA functionalized NDCs in silk fibers (Figure 3b, red box) revealed a Raman spectrum containing peaks from both PMA and silk fibroin (Figure 3c, red trace).12 These results provide a compelling example of how these nanoscale tags can prove the authenticity of a material through their code and by uniquely linking it to probe the composition of the host material. Finally, the optical signal was measured after the cleaning described in Figure 2. From the spectra, we observe that the detergent washing procedure damages the NDC function (Figure 3d, black trace), as may be expected for a silk textile, but the solvent washing procedure does not (Figure 3d, gray trace). By measuring the optical signal before and after this cleaning, we have provided evidence that these embedded tags could be consistently used in textiles. Earlier examples of NDCs relied heavily on physical encoding through the presence or absence of disk pairs functionalized with a Raman chromophore of interest. This physical structure-based approach limits the encoding capacity because it creates only a small number of possible codes. However, the present work demonstrates that it is possible to greatly expand the encoding complexity of NDC/fiber hybrid materials by forming new codes that combine the spectral characteristics of a chromophore with that of the host matrix itself. Because of this, NDCs are no longer restricted to a digital encoding platform; because of the enormous number of available chromophores and unlimited mixtures of them, this approach potentially allows for a massive increase in encoding complexity. Given the low cost nature and possibility of batch fabrication of these structures, fiber-embedded NDCs may have potential application in the anti-counterfeiting of currency and high-value designer merchandise. Nanodisk Code Synthesis: Nanowires (360 nm diameter) were synthesized electrochemically within the pores of Whatman Anodisc membranes. Utilizing this approach afforded near nanometer control over the length of each segment and resulting nanogap dimension because segment length is directly proportional to the quantity of charge passed during the electrochemical synthesis.13 Following deposition, the nanowires were liberated by dissolving the AAO template in 3M NaOH overnight. After being rinsed several times with water, the nanowires were subjected to the OWL process.3 Specifically, wires were drop cast onto glass slides and dried overnight. A thin SiO2 film (∼50 nm) was then deposited onto the slides by plasma enhanced chemical vapor deposition (PECVD) to serve as a backing layer for the NDCs. The nanowires were then removed from the slides by sonication in water and the sacrificial Ni segments were subseqeuntly dissolved by resuspending the wires in a 50% (v:v) solution of HCl for 2 hours. Following chemical etching, wires were resuspended in water. Funcationalization with Raman Chromophore: The NDCs were centrifuged down (1500 rpm for 2 min) and resuspended in an 5 mM ethanolic solution of p-mercaptoaniline (PMA, Alfa Aesar) for 2 hours. Following functionalization, the NDCs were washed again several times and resuspended in 2,2,2-trifluoroethanol (TFE). Silk Protein Preparation: Bombyx mori cocoons were purchased from Mulberry Farms (Fallbrook, CA), fibroin was purified from the cocoons utilizing a protocol detailed in the literature.[8] Aqueous silk fibroin solutions resulting from this procedure were frozen in liquid N2 and lyophilized for 16 h to produce a fibroin powder. Electrospinning: Unless otherwise noted, all chemicals were purchased from Sigma Aldrich and used without further purification. Electrospinning of fibroin/polyethylene oxide (PEO) fibers was conducted by modifying previously established spinning protocols.[8], Purified silk fibroin powders and polyethylene oxide (900 kDa MW) were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at concentrations of 5 and 0.5 wt%, respectively. The electrospinning precursor was produced by combining the fibroin/PEO HFIP solution with an equal volume of TFE containing a complete synthetic batch of NDCs. The silk/PEO/NDC solution was pumped through an 18 gauge blunt-tip needle at a flow rate of 35 mL/h utilizing a syringe pump. A positive DC voltage of 15 kV was applied between the spinneret needle and the grounded collection substrate (25 cm spinning gap). The regenerated fibroin-based electrospun fibers and mats were crystallized in a 90 vol% methanol bath for 10 min, in order to render the fibers water-insoluble. The PEO portion of the fibers was selectively removed by incubating the electrospun materials in an 18.2 MΩ water bath held at 37 °C for 16 h.[8] Laundering Experiments: A substrate containing encoded fibers was washed at room temperature in a 120 mg/200 mL aqueous solution of commercially available detergent (Tide, Free and Clear) and stirred vigorously for 2 hours. Following laundering, the substrate was rinsed several times with tap water, and blown dry. A second substrate was added to a 30 °C solution of tetrachloroethylene (Aldrich) and stirred vigoroously for 15 minutes. Following treatment, the substrate was blown dry. SERS Characterization: Raman spectroscopy was performed using a WITec confocal Raman spectrophotometer system. In a typical mapping acquisition, a ∼4 × 4 um2 area was chosen which encompassed an embedded NDC within a regenerated silk fibroin nanofiber. This was scanned by acquiring 500–1000 unique spectra each acquired with a 0.08 s acquisition time, an excitation wavelength of 633 nm, and a laser power of 1.4 mW. Samples were then probed with 50× and 100× objectives in a backscattering configuration. A 785 nm excitation source was used to collect the individual Raman spectra of the silk fibers containing the NDCs. For these experiments, it was found that using a 785 nm laser excitation provided more consistent spectra than those taken at 633 nm exciation. The backscattered Raman signals were collected on a thermoelectrically cooled (–60 °C) CCD detector. This material is based upon work supported by the AFOSR under Award No. FA9550-09-1-0294. This material is based upon work supported by the Office of the Asst. Secr. of Defense for Research and Engineering, DoD/NSSEFF Program/Naval Postgraduate School under Award Nos. N00244-09-1-0012 and N00244-09-1-0071. AFRL Bio-X STT is acknowledged for financial support. K.A.B. gratefully acknowledges support from Northwestern University's International Institute for Nanotechnology. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the sponsors. Electron microscopy was performed at the EPIC facility of the NU Atomic and Nanoscale Characterization Experimental Center (NUANCE) Center at Northwestern University.