1. Introduction
There are increasing interests in biomedical electronics such as electronic skins, wearable healthcare sensors, and wearable human-device interfaces. The base materials for the next-generation wearable electronic systems should possess biocompatibility, antibacterial activity, surface electrical conductivity, and appropriate flexibility and toughness.
Recently, with the aforementioned concerns of the base materials for human healthcare device, cellulose has emerged as a base material because it is cheap, abundant, and has no cytotoxicity. In particular, a single thread of nanofibrous cellulose not only exhibits great mechanical property of ~120 (GPa) [
1,
2], it is also light in weight [
3] compared to Kevlar or steel [
4]. However, at a nanoscale point of view, cellulose disintegration is an energy-consuming process, due to intra- and interchain hydrogen bonds between the cellulose chains [
5,
6]. Carboxylation of cellulose fibrils by (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO), a catalyst for selective oxidization, is a facile chemical pretreatment method for physical disintegration of cellulose fibrils [
7]. Individual carboxylated cellulose nanofibers (CCNF) generated by TEMPO-mediated carboxylation have a high aspect ratio with an average width of about 3–5 nm and length of a few µm [
6]. CCNF could generate transparent and flexible films with abundant carboxyl and polar hydroxyl functional groups, rigid and self-standing hydrogels, and aerogels with high surface area [
6]. However, poor mechanical properties due to weak inter-fibril interaction, low bacterial resistance, and low electrical conductivity have impeded the application of CCNF to biomedical or wearable devices. Thus, many studies have focused on chemical modification to enhance inter-fibrillar interaction [
8], biological activities [
9] and electrical conductivity [
10] of CCNF.
Dopamine (DA), a mussel-inspired building block that contains the key adhesive chemistry of
l-3,4-dihydroxyphenylalanine (
l-DOPA) and lysine, is known to form a material-independent adhesive coating via oxidative self-polymerization under wet conditions [
11]. The catechol moiety of DA can also induce mineralization of metal nanoparticles as a green reducing agent [
12,
13,
14,
15]. DA can be easily conjugated onto a CCNF surface through amidaition reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and
N-hydroxysuccinimide (NHS) coupling catalysts over the carboxyl groups of CCNF. The catechol moiety of DA can lead to the reinforcement of binding between CCNFs by reducing and overcoming electrostatic repulsive forces. In addition, the catechol moiety can reduce Ag
+ and form silver nanoparticles (AgNPs) on the cellulose nanofiber without additional chemical treatments or heating. AgNPs have been incorporated into various cellulose-based materials such as bacterial cellulose, filter paper, cotton fabric, and cellulose gels to create remarkable electrical conductivity and antibacterial activity [
16,
17,
18]. However, additional chemical treatments and/or heating are generally required for the Ag
+ reduction in the cellulose materials. In addition, 3-dimensional architecture of cellulose nanostructure due to AgNPs incorporation has been poorly studied.
In this study, we developed AgNP-containing cellulose nanofiber composites with improved mechanical property, antimicrobial activity, and electrical conductivity by exploiting mussel’s catechol chemistry. Interestingly, the composite of AgNPs and DA-conjugated CCNF (CCNF-DA) via
in situ reduction of silver ions in silver nitrate (AgNO
3) solution (
Figure 1A) was anisotropically aligned due to the strong interaction between catechol moieties and AgNPs. The anisotropically aligned CCNF-DA/AgNPs composite can be applied to electrically conductive biomaterials with beneficial features including flexibility, electronic conductivity, and antibacterial activity (
Figure 1B).
2. Materials and Methods
2.1. Materials
Commercial coffee filter papers (Kalita, Seongnam, Korea) from wood pulp were used to produce cellulose nanofibrils. TEMPO was purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), sodium bromide (NaBr), AgNO3 solution, hydrochloride (HCl) solution, sodium hydroxide (NaOH), l-DOPA, dopamine hydrochloride (DA·HCl), and sodium nitrate (NaNO3) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were used as received.
2.2. Preparation of Carboxylated Cellulose Nanofibers (CCNF)
CCNFs were prepared by TEMPO mediated oxidation as described elsewhere [
19]. In brief, cellulose from coffee filters (20 g) was mixed with TEMPO (0.312 g), and NaBr (2.058 g) in 2 L of distilled water (DW), and then NaClO (200 mmol) was added to the solution. The reaction was maintained at pH ~10 for several hours by adding 0.5 M NaOH or HCl. When the reaction is complete, the surface oxidized cellulose fibers were filtered and then washed 3 times with DW. The obtained slurry was dispersed in 2 L of DW and ground with a super masscolloider (MKCA6-2J; Masuko Sangyo Co., Ltd., Tokyo, Japan) at 15,000 rpm. The carboxyl group content (mmol·g
−1, dry mass cellulose) of CCNF was analyzed through conductometric titration (Orion, Thermo Scientific, Waltham, MA, USA), and it was ~1.5 mmol·g
−1. The weight concentration of CCNF (wt %) was measured with a moisture analyzer (MB 35, Ohaus, Parsippany, NJ, USA).
2.3. Preparation of CCNF-Dopamine (DA)
CCNF-DA was prepared following the previous study with minor modification [
20]. In brief, EDC·HCl (1 equivalent) was directly added to the CCNF hydrogel, followed by addition of NHS (1 equivalent) and DA·HCl (2 equivalent) with stirring at 4 °C. The molar ratio of EDC·HCl, NHS, and DA·HCl was based on the carboxyl group molar content of CCNF. The mixture was stirred for 24 h and dialyzed with DW at pH 4–5 at room temperature. The suspension was stored in argon purged vials until further use. The catechol content of the CCNF-DA was quantified using a colorimetric assay developed by Arnow [
21]. The CCNF-DA was added with 100 μL of DW, 300 μL of 0.5 M HCl, 300 μL of nitrite molybdate reagent (1.45 M sodium nitrite and 0.41 M sodium molybdate), and 300 μL of 1 M NaOH. The absorbance was measured at 500 nm using UV–Vis spectroscopy (PerkinElmer, Waltham, MA, USA) and the concentration of catechol was determined by Arnow’s standard curve of
l-DOPA. To cast films, the CCNF-DA was poured into plastic petri dishes covered with adhesive Teflon tape and dried in an oven at 40 °C–50 °C for at least one day. The films were stored in a vacuum package prior to use.
2.4. Preparation of CCNF-DA/Silver Nanoparticles (AgNPs)
The solution of AgNO3 (3.25 mM) in acetate buffer (pH 4) was added to the CCNF-DA suspension. The mixture was sealed and left at room temperature for one day for reduction of Ag+ ions via oxidation of catechol groups. The film of CCNF-DA/AgNPs was fabricated following the method described above.
2.5. Characterization of CCNF-DA/AgNPs Film
CCNF, CCNF-DA, and CCNF-DA/AgNPs hydrogels with the concentration of 1 (w/v) % were analyzed by a cryogenic transmission electron microscopy (cryo-TEM; JEOL JEM 1011, Tokyo, Japan) and a polarized optical microscopy (POM; Nikon LV100 Pol) before the film casting. The samples were covered with the flat side of another B-type planchette and were rapidly frozen in a Bal-Tec HPM 010 high-pressure freezer (Boeckeler Instruments, Tucson, AZ, USA). After freeze substitution for 5 days at −80 °C in anhydrous acetone containing 2% OsO4, the samples were warmed up to room temperature over 2 days (24 h from −80 °C to −20 °C, 20 h from −20 °C to 4 °C, 4 h from 4 °C to 20 °C). After washing 3 times with anhydrous acetone, the samples were embedded in a graduated Epon resin (Ted Pella Inc., Redding, CA, USA) and diluted in acetone (5%, 15%, 25%, 50%, 75% and 100% (v/v)) over 3 days. After polymerization in a 60 °C oven for 24 h, the samples were sectioned and post-stained with aqueous 2% (v/v) uranyl acetate (UA) and Reynolds lead citrate (LC) solution. The resin was cut into 200 nm-sections using Leica EM UC7 (Wetzlar, Germany) and applied onto copper grids. The TEM images of samples were recorded. The optical textures were characterized by depolarized transmitted light microscopy (DTLM).
2.6. Characterization of AgNPs
For UV–Visible spectroscopy analysis, CCNF-DA/AgNPs films were immersed with ultra-pure water in a PCR tube, followed by ultrasonication to obtain the AgNPs from films. The absorbance of the soluble extract was measured using a UV–Visible spectrophotometer (OPTIZEN POP BIO, Mecasys, Daejeon, Korea) in a wavelength range of 350–600 nm. A few drops of NaNO
3 were added to the extract to prevent aggregation of AgNPs [
22].
For transmission electron microscopy (TEM) analysis, the CCNF-DA/AgNPs suspension was centrifuged at 9000 rpm for 10 min, followed by removal of the supernatant [
23]. The aliquots of sample pellet (5–20 µL) were applied onto a copper microgrid, and the excess liquid was removed using filter paper. Subsequently, the sample was dried in a vacuum oven for 2 days. TEM images, selected area electron diffraction (SAED) pattern and energy-dispersive X-ray spectroscopy (EDS) data were obtained through high-resolution transmission electron microscopy (HRTEM; JEOL JEM-2100F, Tokyo, Japan). The results were further analyzed using image analysis software (ImageJ v.148; National Institutes of Health, Bethesda, MD, USA).
An inductively coupled plasma spectrometer (ICP; SHIMADZU ICPE-9000, Tokyo, Japan) was used to characterize the AgNPs content in the CCNF-DA/AgNPs.
The AgNPs release of the CCNF-DA/AgNPs composite in aqueous medium was investigated as follows. The composite film was incubated in phosphate-buffered saline (PBS) buffer for 2 days. The composite film was washed with deionized water and fully dried in a convection oven for 1 day. The AgNPs weight % in the incubated CCNF-DA/AgNPs composite film was measured using ICP.
2.7. Mechanical Properties and Conductivity Test
The CCNF-DA and CCNF-DA/AgNPs films were cut into circles with a width of ~5 mm and a thickness of ~40 µm. The mechanical properties of the composite films were measured on a universal tensile tester (UTS, Instron, Norwood, MA, USA). The composite films were loaded to failure at a strain rate of 0.01 s−1. Conductivities of the composite films were measured with a four-point probe. All measurements were replicated 5 times.
2.8. Antibacterial Activity Assay
The antibacterial activity of the CCNF-DA/AgNPs film was measured using Kirby-Bauer method for Gram-negative (Escherichia coli, ATCC 25922) and Gram-positive (Staphylococcus aureus, ATCC 6538) bacteria. The CCNF film was used as a negative control. The sample films were placed on Luria–Bertani (LB) agar plates containing bacterial cells in log-phase, and incubated overnight at 37 °C. The area of the inhibition zone was measured in triplicate using image analysis software (ImageJ v.148; National Institutes of Health, Bethesda, MD, USA).
To generate growth curves on the CCNF-DA/AgNPs, the sample films were incubated in LB medium inoculated with the bacterial cells in log-phase at a ratio of 1:100 (v/v). In addition, to determine a death curve, the films were incubated with 1 × 109 cells·mL−1 of E. coli in 500 μL of LB medium in 24-well culture plate (SPL Life Science, Pocheon, Korea). For the growth/death curves, each bacterial strain was incubated at 37 °C and 300 rpm, and bacterial concentrations were monitored by measuring optical density at 600 nm (OD600) for 24 h.
2.9. Sustainability Test for Antibacterial Activity
The repeated growth-inhibiting and bactericidal activity of the CCNF-DA/AgNPs film was evaluated for up to 5 days, following a method described previously [
24]. To determine growth-inhibiting efficacy, the CCNF-DA/AgNPs film was incubated overnight in 500 μL of LB medium inoculated with the
E. coli cells in log-phase at a ratio of 1:100 (
v/
v). For bactericidal efficacy, the film was incubated overnight in 500 μL of LB medium containing 1 × 10
9 cells·mL
−1 of
E. coli. The culture broth was sampled, and viable bacterial cells were counted at the end of each incubation period. Then, the film was gently washed with DW to remove remaining cells. The CCNF-DA/AgNPs film was incubated again as described above, and the process repeated for 5 days. The growth-inhibiting and bactericidal efficacies were calculated following (Equation 1) and (Equation 2).
where,
N is the mean number of surviving bacterial colonies in the sampled culture broth incubated on the CCNF-DA/AgNPs film;
M is the mean number of colonies in the sampled culture broth incubated without the CCNF-DA/AgNPs film (control); and
N0 is the initial mean number of colonies in the culture broth inoculated on the CCNF-DA/AgNPs film.