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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 6793−6798
Carbon-Nanotube-Wrapped Spider Silks for Directed Cardiomyocyte Growth and Electrophysiological Detection Junfeng Hou,†,‡ Yu Xie,§ Aiguo Ji,*,† Anyuan Cao,§ Ying Fang,‡ and Enzheng Shi*,§,⊥ †
Marine College, Shandong University, Weihai 264209, P. R. China National Center for Nanoscience and Technology, 11 Beiyitiao Street, Zhongguancun, Beijing 100190, P. R. China § Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, P. R. China ‡
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S Supporting Information *
ABSTRACT: The combination of nanostructures with biomaterials offers great opportunities in constructing innovative functional devices such as biosensors and actuators. Here, we create a multifunctional fiber by wrapping a thin film of carbon nanotubes (CNTs) on naturally found spider silks, which shows great flexibility and conductivity. The hybrid CNT−silk fiber demonstrates intimate contact with cardiomyocytes and can direct the cell growth and simultaneously record potential signals evoked from cell beating. Cell activities reflected in the form of potential signals have been monitored clearly and reliably through the CNT−silk fibers without degradation over the long term. KEYWORDS: carbon nanotube, spider silk, biosensors, electrochemical detection, cardiomyocytes
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more than 2000 years as a natural protein in folk medicine to prevent infection and heal wounds.10 Recent studies also demonstrated their potential applications in cell interfacing and tissue engineering, in which spider silks could enhance cell growth, proliferation, and nerve regeneration.11,12 On the basis of the investigation of the interaction between CNTs and excitable cells reported by several groups,13−16 the CNTs provided a soft artificial extracellular matrix that may ultimately facilitate cell adhesion by the formation of tight contacts between CNTs and cell membranes, which was clearly confirmed by transmission electron microscopy (TEM) or scanning electron microscopy (SEM) data. Coupled with the recent results, which showed that CNTs enhance disk assembly in cardiomyocytes by activating β1-integrin signaling at the cell membrane and the subsequent signaling of kinases,17 we speculate that the nanotopographical features and conductivity improvement of CNT substrates could induce and guide cell adhesion, and CNT-based scaffolds have the ability to improve cardiomyocyte proliferation, maturation, and electrical behavior by providing a desired artificial extracellular matrix and regulating protein expression, which is superior to other substrates like silicon and gold. Herein, we combine CNTs and spider silks to fabricate robust and conducting hybrid fibers that contact well with growing cells and enable in situ detection of cell activity. The hybrid fiber takes full advantage of these two materials,
iomedical applications of nanomaterials have stimulated tremendous interest in recent years.1 Among various nanoscale materials, carbon nanotubes (CNTs) are chemically stable and electrically conductive and have been studied in biological and medical areas either at the individual level or in macroscopic self-assembled structures.2 CNT films, fibers, coatings, and three-dimensional (3D) electrodes could serve as structural supports for cell culture and tissue engineering, and, in general, they were found to create a favorable environment and promote cell attachment, subsequent growth, differentiation, and long-term survival.3,4 For example, it was observed that CNTs formed tight contacts with rat ventricular myocytes and increased syncytia development and the electrical activity of cardiomyocytes.5 Also, a CNT coating on metal electrodes could boost electrophysiological signals from neurons and cardiac myocytes, suggesting effective coupling between CNTs and excitable cells.6−8 The versatility of CNTbased materials (including films, fibers, and 3D structures), together with their high conductivity, allows the configuration of many different biosensors and actuators with high performance. Despite such great promise, it remains challenging to develop functional CNT devices that are fully compatible with biological systems. Conventional substrates (e.g., metals, ceramics) are too rigid or hard-to-soft tissues; CNTs must be anchored in the appropriate substrates that offer high flexibility and compliance to surrounding media. In this regard, spider silks could be appropriate choices because they are lightweight, mechanically strong and stretchable in tension (with the tensile strength and strain up to 4.8 GPa and 35%, respectively), and extremely soft as well.9 In fact, spider silks have been used for © 2018 American Chemical Society
Received: September 29, 2017 Accepted: February 9, 2018 Published: February 9, 2018 6793
DOI: 10.1021/acsami.7b14793 ACS Appl. Mater. Interfaces 2018, 10, 6793−6798
Letter
ACS Applied Materials & Interfaces
Figure 1. Morphology of the spider silk, CNT film, and CNT−silk fiber. (a) A 0.5-m-long spider dragline twisted around a glass rod, together with a thin CNT film. (b) Raman spectrum of a CNT−silk fiber. (c) Optical images of a single spider silk before and after coating of a CNT film. The scale bars are 50 μm. (d) SEM images of a single CNT−silk fiber. The scale bars are 5 and 500 nm.
Figure 2. Electromechanical properties of a single CNT−silk. (a) Cyclic stress−strain curves with 1% and 2% strain for the same CNT−silk, respectively. (b) Simultaneously recorded electrical resistance change (ΔR/R) of CNT−silk during loading and unloading cycles in part a. (c) Realtime capture of cyclic gastrocnemius muscle contraction, corresponding to the electrical stimulation pulse transferred by CNT−silk and the length changes of CNT−silk corresponding to the contraction of the gastrocnemius muscle.
Figure 3. SEM image of a cardiomyocyte growing along the CNT−silk fiber, where the interface and the strong contact between the cardiomyocyte and CNT−silk are clearly shown.
dragline silk could be separated into several single silks (Figure S1). These spider silks have diameters ranging from 5 to 10 μm and lengths of up to a few meters. Several factors related to our fabrication process are important and would facilitate subsequent applications. First, we used freestanding CNT films to directly wrap each individual spider silks of over centimeter lengths without interruption (Figure 1a). The CNT films synthesized by chemical vapor deposition are highly conductive with a few defects, as indicated by the high G/D
particularly the high conductivity of CNTs and the biocompatibility of spider silks, to enable high-performance multifunctional devices. With this combination, we demonstrate fiber-shaped biosensors that can reliably detect electrophysiological signals from cultured cardiomyocytes and with high sensitivity over a long period. Our fabrication of CNT-wrapped spider silks was based on a dry-coating and wet-collapsing method. Nephila clavata spider dragline silks were collected on the campus. Each bundle of 6794
DOI: 10.1021/acsami.7b14793 ACS Appl. Mater. Interfaces 2018, 10, 6793−6798
Letter
ACS Applied Materials & Interfaces
showed stable loops with small hysteresis. Within the strain ranges of 1% and 2%, a linear stress−strain relationship was observed, yielding a Young’s modulus of about 70−72 GPa, and there was no stress degradation over the cycles (Figure 2a). Simultaneously, the electrical resistance (R) of the hybrid CNT−silk fiber during the tensile tests shows repeated fluctuations, indicating that repeated stretching of the spider silk leads to periodic changes of the resistance, which is a consequence of deformation of the CNT film wrapped onto the silk (Figure 2b). Specifically, the resistance increased by 2.2% at 1% tensile strain and by 5.3% at 2% strain, respectively, and it would recover to the initial value after stress unloading. A conductivity of about 4300 S·cm−1 (initial R = 1170 Ω) can be deduced based on the testing silk diameter (5 μm) and distance (1 cm) between the two electrodes wired at the silk ends, which is much higher than those of previously reported spider silks coated with multiwalled nanotubes (12−15 S·cm−1).18 The above results imply that the CNT film could follow deformation of the spider silk (due to strong adhesion) without detachment from the silk surface after plenty of strain cycles. This property also promises future applications in stretchable conducting wires and sensors. For example, a bundle of CNT−silk fibers was connected to a frog gastrocnemius muscle for conducting the artificial electronic stimulation signals. The frog sciatic nerve−gastrocnemius muscle is a common model for examining nerve conduction and muscle activity in vitro.19 In this case, the CNT−silk fibers serve as a conducting wire to pass a stimulation pulse train (0.2 ms, 6.0 mA square pulses with an interval 0.5 s) to stimulate the gastrocnemius muscle (Figure S3). As a result, the frog muscle contracted and the CNT−silk fibers were elongated by 17% strain (Figure 2c). Both the muscle and hybrid CNT−silk recover to their original relaxed states after removal of the electrical stimulation. During the repeated relaxation and contraction process of the gastrocnemius muscle, the CNT− silk could be reversibly stretched to a strain of 17% without obvious plastic deformation. To investigate the biocompatibility of CNT−silk, cardiomyocytes were seeded and cultured on the CNT−silk. The primary cultured cardiomyocytes isolated from 11-day-old chicken embryos were spontaneously excitable cells. A monolayer of cardiomyocytes was formed after 3 days of culturing, and the CNT−silk laid in the cell layer. SEM images (Figure 3) show that cardiomyocytes tended to adhere to as well as grow on the surface of CNT−silk. Interestingly, cell elongation was aligned with the fiber, which indicates that the hybrid fiber can be applied to guide the growth of cardiomyocytes. Further enlarged SEM images in Figure 3 show a hybrid fiber tightly wrapped by a cardiomyocyte cell. The filopodia extending from the cell indicate strong contact between the cardiomyocyte and hybrid fiber, which is consistent with the former results of neonatal rat ventricular myocytes seeded on CNT-coated glass slides.15 These results reveal that the spider silk covered partially by the CNT film on the surface has good biocompatibility and can favor the adhesion of cardiomyocyte.17 The uniform coating of conductive CNTs on the spider silk and the tight interaction between the cultured cardiomyocytes with CNT−silk allow sensitive electrophysiological recording from cardiomyocytes by the CNT−silk. A poly(dimethylsiloxane) (PDMS) substrate was used to fabricate the cell culture chamber, which was nontoxic, optically transparent, and biologically compatible.20 The CNT−silk
Figure 4. Electrophysiological recordings of cardiomyocytes by CNT− silk. (a) Schematic illustration of the fabrication of a transistor array based on CNT−silk, including connecting the CNT−silk with silver electrodes, insulating with PDMS, and fabricating the PDMS chamber for cell culturing and recording. (b) Photograph of the device chip for culturing cardiomyocytes and recording their electric signals by electrodes made with CNT−silk. (c) Electrical resistance change (ΔR/ R) of CNT−silk with the soaking time in a culture solution. (d) Current signals recorded from cultured cardiomyocytes by three devices in the CNT−silk transistor array. The right panels are the zoom-in views of a single current signal at the time indicated by the stars on the current traces of the corresponding left panels.
ratio in the Raman spectrum (Figure 1b), creating a continuously conducting path along the spider silks. Second, the CNT film consisting of interconnected CNT bundles is very thin (thickness
