Textile Zinc Nanocomposite Synthesis
The nanocomposite textiles were fabricated by soaking the textiles in an aqueous ionic precursor solution, which is thermally treated to form nanoparticles in the bulk and throughout the surface of the fabrics. Typically, nanocomposites are described as at least two-phased materials with one of the components being in the nanoscale. This could either be a metal or a non-metal nanoparticle phase embedded in a macroscale support such as the fabrics in this case. The technology used to make this is called Crescoating® (Figure 1) and begins with solid seed formation from the ionic solution under heating followed by their nucleation to form defined nanoparticles. The thermal treatment was done using a convection oven which leads to the evaporation of water followed by seed formation and nucleation of the particles. The size of the particles could range from 5-500 nm, depending on the process conditions and they get embedded in the textile supports. This method improves the durability and longevity of the nanocomposite textiles, unlike conventional surface-coated products, which lose their nanoparticles over time due to multiple washing cycles.
This method’s efficacy was previously demonstrated for environmental remediation using polyurethane foam, and polypropylene, polycotton and nylon cotton fabrics for their antiviral properties.[27-30] Figure 2 shows SEM images of nanocomposite polyurethane, nylon and polyester materials.
To demonstrate the versatility of the Crescoating® technology the process has been adapted to various fabrics using different metal nanoparticles as well (Figure 3).
In this study the fabric supports used were silk, synthetic polyester, nylon cotton and polyester cotton. The synthesis of zinc nanocomposite textiles was based on a previously developed method. Briefly, the fabrics were initially submerged in precursor ionic solutions of zinc salt for 30 minutes at room temperature. They were then heated in a convection oven at 100°C for 4 hours by maintaining a thin layer of the solution over them. White precipitates were formed on the zinc supports. Any unbound precipitate was washed away by following the American Association of Textile Chemists and Colorists (AATCC) LP1: Home Laundering method and dried.
Structure and characterization:
The synthesized nanocomposites were characterized using scanning electron microscopy (6700 SEM, JEOL Inc.). Figure 4 depicts zinc nanocomposites grown in polyester fabrics. Very small zinc nanoparticles in a size range of ≤100 nm can be seen on the textile support. Nanoparticle coverage on the support is not uniform nor widespread. SEM images of untreated control fabrics can be found in Supplementary Figure S1. Unbound nanoparticles collected through hand washing the nanocomposite fabrics immediately after synthesis were analyzed using x-ray crystallography (D8 Discover, Bruker Corp.). Pattern fitting was conducted with JADE for XRD software (Materials Data Inc.). Diffraction patterns from recovered particles are also depicted in Figure 4. Zinc particles were primarily comprised of two crystalline phases of zinc hyroxide (Zn(OH)2) and zinc carbonate hydroxide (Zn5(CO3)2(OH)6 hydrozincite).
The formation of these nanoparticles follows a three step process similar to the formation of iron oxide nanoparticles previously studied. Thermal synthesis of supported nanoparticles is achieved through the formation of solid phase seeds that nucleate to form nanoparticles. For zinc, the salt precursors undergo hydrolysis to form hydroxide intermediates in the form of Zn(OH)2. In the presence of carbonate contributed from the atmosphere and/or from the degradation of the acetate counterion, zinc carbonate hydroxide particles form. The hydrozincite and zinc hydroxide phases can be seen in x-ray diffraction patterns (Figure 4).
Synthetic precipitate leachate procedure:
The retention of zinc nanoparticles in the textiles after several wash cycles was tested by a third party testing company, Pace Analytical LLC. Samples of several types of nanocomposite cotton fabrics were subjected to the synthetic precipitation leachate procedure (SPLP). These fabrics were previously washed once post manufacturing to remove loose nanoparticles from their surface. The test based on the EPA 3010A preparation and EPA 6010B analytical method was then applied to study the leaching of zinc nanoparticles from the fabrics.  The results from the study showed that the highest zinc leaching of 106,000 µg/L occurs after the first post-fabrication wash for one of the cotton types. All other values are lower and are followed by significantly lower leaching after every subsequent wash cycle (See supplementary information Table S1). This shows that the nanocomposite fabrics prepared using the novel Crescoating® method are highly durable. The highest initial leachate concentration is found to be much lower than 250,000 µg/L, which is the soluble threshold limit concentration (STLC) value for California state. Based on this limit it can be concluded that the concentration of zinc leaching from the fabrics after each laundering cycle is not significant enough to pose a health or environmental hazard. The different values of leaching observed for the three different cotton types could be a result of differences in their fiber sizes and pretreatment processes such as mercerization (oxidization of fibers for metal easier metal binding), which was done for cotton types 1 and 2.
Antimicrobial Application Test
For antimicrobial testing, zinc nanoparticles were grown on polyester, silk and nylon/cotton (50:50) textile swatches obtained from Testfabrics Inc. and Rockywoods Fabrics LLC, respectively. AATCC Test Method 100-2004 was used for antibacterial and antifungal testing of functionalized fabrics. The experiment was done in triplicates. Two bacterial species Pseudomonas aeruginosa (PA, ATCC 27853) (Gram-negative) and methicillin resistant Staphylococcus aureus (MRSA) (Gram-positive) and a fungal species Candida albicans (CA) were selected for antimicrobial testing. The results have been separated into two categories: a “before washing” test conducted on swatches that have been machine washed soon after treatment and an “after washing” test with swatches that were subjected to more machine washing and drying cycles. The “after washing” tests were conducted by a third-party testing company (See report in supplementary information). The fabric used was polyester cotton blend and the tests were conducted on the bacterial species Staphylococcus aureus (SA, ATCC 6538) (Gram-positive) and Klebsiella pneumoniae (KP, ATCC 4352) (Gram-negative). This was done to check the antimicrobial efficacy of different zinc nanocomposite fabrics against various pathogens under different conditions.
Before-wash antimicrobial test
Briefly, the fabric samples were inoculated with suspensions of bacteria in nutrient broth and rinsed. These samples were divided into two sets with different elution times, a 0-hour immediate elution and an elution after a 24-hour incubation period. The eluted solution from the inoculated fabrics was then plated and incubated for 24 hours at 37oC. Bacterial growth was quantified through colony counting on plates as pictured in Figure 5. The results are reported in % reduction and calculated by the following formula (Equation 1).
Where A is the number of bacterial colonies recovered from the inoculated treated sample fabric and B is the number of bacterial colonies recovered from the inoculated untreated control fabric, both incubated over a set contact period. For all experiments, Equation 2 was used to determine test efficacy. This calculation is a qualitative check to confirm that the initial concentration of bacteria used was enough to do the antimicrobial test. This number must be higher than 1.5.
Where a is number of bacterial colonies recovered from untreated control fabric immediately after inoculation and b is the number of bacterial colonies recovered from untreated control fabric after 24-hour incubation post inoculation. For all reported experiments, these efficacy values ranged from 1.5-3.5, confirming the effective bacterial concentration.
The above procedure was followed similarly for tests against the fungal pathogen Candida albicans. Although AATCC recommended antifungal tests for textiles differ from that of bacteria, the growth cycle of Candida albicans resembles that of bacteria, which enables antifungal testing using this modified version of the AATCC Test Method 100-2004.
The 0-hour data, shown in Table 1, for each textile showed varied antibacterial properties for Pseudomonas aeruginosa (PA), methicillin resistant Staphylococcus aureus (MRSA) and Candida albicans (CA). This could be due to the quick elution of microbial inoculations from the fabrics, leaving much less time for the nanoparticles to interact with the pathogenic cells and reduce their growth consistently. Despite this, the microbial reduction percentage for almost all samples ranged from 32-91% indicating that the nanocomposite fabrics can still induce some antimicrobial properties. For nanocomposite polyester treated with MRSA, negative value was observed and indicate that there were more bacteria recovered than control. This could be possible because of variable absorption properties of each fabric or might be because of improper mixing of bacterial concentration. “NA” is used for either negative value or indicates contamination. The 24-hour elution results on the other hand showed significant microbial reduction in samples treated with zinc nanocomposite textiles compared to untreated controls (ANOVA, p<0.05). The microbial reduction percentages were far less variable and ranged from 98 to >99.999% reduction. The percentages obtained are higher than or similar to those reported in the literature for antimicrobial cotton fabrics against Gram-positive S. aureus and Gram-negative bacteria.[36,37] Though the antimicrobial mechanism of zinc nanoparticles is not clearly studied, reports from previous studies have suggested that their photocatalytic properties generate hydrogen peroxide (H2O2) through a series of reactions in the presence of H2O. These hydrogen peroxide molecules are toxic to the microbial cells as they can penetrate the cell membrane and kill them. There have been fewer studies on the antimicrobial efficacy of these textiles on fungal pathogens. However, the results obtained here indicate that these novel nanocomposite textiles can also be used for antifungal applications.
After-wash antibacterial test
Third-party wash durability analysis:
Zinc nanocomposite polyester cotton was machine washed 50 times per AATCC LP1: Home Laundering method and the antibacterial properties were assessed separately by a third-party testing through Vartest Laboratories LLC. The bacterial reduction was found to be 0% for both Klebsiella pneumoniae (KP, Gram-negative) and Staphylococcus aureus (SA, Gram-positive) indicating that the nanocomposite textile did not exhibit antimicrobial properties during the 0-hour (immediate elution) process after 50 wash cycles (See Table 2 and supplementary information). However, the same material exhibited antimicrobial properties even after 50 wash cycles with more than 99.999% bacterial reduction for both Klebsiella pneumoniae and Staphylococcus aureus after 24-hour incubation (See Table 2 and supplementary information). While there have been some studies in the past on the application of zinc oxide nanoparticles for the fabrication of antimicrobial textiles, most of these show much lower microbial reduction percentages than the desired 99.999% range for samples washed over 20 cycles.[39,40] The zinc nanocomposite textiles prepared using the novel Crescoating® technique on the other hand could retain their antimicrobial properties even after being reused over 50 times repeatedly. Preliminary tests on samples washed for 100 cycles (see next section) also show promising antibacterial activity. Therefore, these fabrics can be successfully applied to manufacture antimicrobial textiles in the healthcare industry.
Laboratory wash durability analysis:
Zinc nanocomposite cotton fabric was washed 100 times by following the AATCC LP1: Home Laundering method. These samples were then tested against the Gram-positive Staphylococcus aureus bacteria according to AATCC test method 100. The results from the 24-hour elution samples show >99.999% reduction indicating that the nanocomposite fabrics can retain their antimicrobial behavior even after several washes. This means that the novel nanocomposite fabrics have better longevity and durability when compared to other antimicrobial textiles previously studied.[39,40]
Safety and skin irritation test
To determine whether the antimicrobial nanocomposite fabrics were safe to use as clothing, the Human Repeat Insult Patch Test (HRIPT) was conducted by Evalulabs LLC. The tests were conducted after obtaining informed consent from 50 human subjects and was carried out under the supervision of a licensed dermatologist. The protocol was approved by the Ethics Committee and the Evalulab LLC Independent Ethics Committee (IEC) prior to the tests to protect the rights of the human participants. The results indicated that the fabrics were non-irritating and hypoallergenic to human skin. The full report of this test is available in the supplementary information.
All methods and experiments in this report were performed in accordance with the University of Minnesota guidelines and other relevant regulations.