Name: an antimicrobial fabric using nano-herbal encapsulation of essential oils
Uploaded: 2023-04-07T14:35:00.000+00:00
Duration: 7 min 47 s
Description: Watch this Scientific Journal Video about An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils at JoVE.com

This study was funded by &#34;Applied Research, Innovation and Entrepreneurship Services&#34; (ARIES), Centennial College, Canada.

1. Preparation of nanoparticles
<ol>
	<li>Nano-herbal encapsulation
	<ol>
		<li>Prepare 50 mL of 1% (v/v) acetic acid. 
		CAUTION: Glacial acetic acid is an irritant, which can cause severe skin burns and eye damage. Wear a full-length lab coat, nitrile gloves, and goggles, and work under a fume hood.</li>
		<li>Prepare chitosan solution (1.2% w/v) by dissolving 0.6 g of chitosan flakes (medium molecular weight) in 50 mL of 1% acetic acid (prepared above). Agitate overnight (O/N) at room temperature (R/T) to get a homogeneous emulsion.</li>
		<li>Add 0.5 g of Tween 80 and stir (1,000 rpm) at 60 &#176;C for 2 h to get a homogeneous solution. Bring the solution to R/T before adding bioactive compounds (carvacrol or thymol).</li>
		<li>Add 0.75 g of carvacrol (or thymol) dropwise or gradually while stirring (1,000 rpm) the mixture for 20 min at R/T. The weight ratio of chitosan to bioactive compound is 1:1.25.</li>
		<li>Add 50 mL (0.5% w/v) of TPP dropwise to the mixture while stirring at R/T. Continue stirring for 30 min to get a homogeneous emulsion.</li>
		<li>Prepare a negative control by following the same procedure but without the addition of bioactive compounds.</li>
	</ol>
	</li>
	<li>Purification
	<ol>
		<li>Centrifuge the emulsion at 10,000 &#215; g for 30 min at 4 &#176;C and collect the formed NPs (pellet) after decanting the supernatant. Reserve the supernatant to test the encapsulation efficacy.</li>
		<li>Wash the particles (from step 1.2.1) using aqueous Tween 80 (1% v/v) with double the volume of pellets formed to remove the unbound or free bioactive compounds. Make sure to disturb the pellet by vortexing until a homogeneous solution is formed in each wash step.</li>
		<li>Wash the particles (from step 1.2.2) with deionized water twice to get rid of impurities.</li>
		<li>Reconstitute the NPs by resuspending the pellet (from step 1.2.3) in 30 mL of deionized water. Store the NPs at 4 &#176;C for up to 6 months. 
		&#8203;NOTE: The experiment can be paused at this stage.</li>
	</ol>
	</li>
	<li>Characterization
	<ol>
		<li>Dilute the NPs in deionized water until the ultraviolet-visible (UV-Vis) readings between 250 to 400 nm fall within the range (absorbance &#62;1).</li>
		<li>Record the UV-Vis absorption spectra of the NPs over the wavelengths ranging from 250 to 400 nm using a UV-Vis spectrophotometer.</li>
		<li>Store an aliquot (1 mL) of NPs at R/T for a range of durations (e.g., 1 to 6 months) and test the antimicrobial effect by the cylinder plate method along with a fresh sample.</li>
	</ol>
	</li>
	<li>Coating on fabric
	<ol>
		<li>Apply NPs on a woven cotton fabric using a pad-dry-cure method7,21.
		<ol>
			<li>Immerse the fabric swatches in the solution containing NPs mixed with a fabric binder for 3 min until soaked. Then, pass the swatches through a two-roller laboratory padder to remove excess liquid.</li>
			<li>Oven-dry the swatches at 100 &#176;C for 30 min and cure at 130 &#176;C for 10 min.</li>
			<li>Finally, wash the swatches in an ultrasound bath for 15 min to remove unbound NPs. 
			NOTE: Alternatively, follow the simplified method described below.</li>
		</ol>
		</li>
		<li>Cut the cotton fabric (or lab coat swatches) into 10 cm x 10 cm squares.</li>
		<li>Immerse the cut pieces in 2 mL of the synthesized NP (10%) in a plastic container (12 cm x 12 cm) for 2 min at R/T. Remove excess liquid by air drying.</li>
		<li>Heat press at 100 &#176;C for 3 min to bind the NPs.</li>
		<li>Remove unbound NPs by rinsing in aqueous Tween 80 (2% w/v) and dry in the oven at 100 &#176;C for 30 min. 
		NOTE: This antimicrobial fabric can be stored for 2 years at R/T.</li>
	</ol>
	</li>
	<li>Wash durability
	<ol>
		<li>Cut the fabric into 50 mm x 50 mm square swatches.</li>
		<li>Place the swatches in 50 mL of warm tap water (40 &#176;C) in a glass/plastic container and add two drops of regular detergent (non-antimicrobial).</li>
		<li>Rinse on a rocking platform shaker or magnetic stirrer for 30 min.</li>
		<li>Air dry or incubate at 60 &#176;C for 2 h and test for antimicrobial efficacy.</li>
	</ol>
	</li>
</ol>
2. Cylinder plate assay for screening of nanoparticles
<ol>
	<li>Prepare trypticase soy agar (TSA), trypticase soy broth (TSB), antibiotic base agar (ABA), and antibiotic seed agar (ASA), according to manufacturers&#39; instructions, and sterilize the media by autoclaving at 121 &#176;C at 15 psi for 15 min.</li>
	<li>Subculture the microbes (from stock) of interest, against which the efficacy tests are performed, on freshly prepared TSA plates (or any appropriate media) by the three-way streak plate method and incubate to produce purity plates. (Recommended species: S. aureus, E. coli, P. aeruginosa, and C. albicans.)</li>
	<li>Inoculate the cultures from fresh purity plates in TSB (10 mL tubes) at 35 &#176;C O/N with moderate shaking (100-200 rpm).</li>
	<li>Aliquot 5.0 mL of the molten ASA in each of the test tubes. The number of tubes corresponds to the number of microorganisms tested.</li>
	<li>Pour 20 mL of presterilized molten ABA into each Petri plate (100 mm x 20 mm) aseptically to form the base layer. The number of agar plates corresponds to the number of microorganisms to be tested. Wait until the media get solidified.</li>
	<li>After solidification of the base layer, inoculate each molten ASA with an overnight culture from step 2.3 (1.0 mL, prewarmed to 35 &#176;C) and immediately transfer the contents (mixture of ASA and culture) to the surface of an ABA plate. Swirl the plate to distribute the molten agar layer evenly. Ensure that the seed layer appears smooth and free from bumps or bubbles.</li>
	<li>Place up to six stainless steel cylinders (6 mm x 6 mm x 10 mm; preautoclaved), evenly spaced on a hexagonal pattern, per agar plate using a pair of sterile tweezers (Figure 1).</li>
	<li>Load the cylinders with the synthesized NPs of different volumes (30 &#181;L, 50 &#181;L, 75 &#181;L, and 100 &#181;L) to be screened for antimicrobial effects. The negative control is the one without any bioactive compounds.</li>
	<li>Incubate common bacterial pathogens (e.g., E. coli, S. aureus, P. aeruginosa) at 35 &#176;C for 24 h and fungal species (e.g., C. albicans) at R/T for 3 days.</li>
	<li>Measure the diameter of the clear zone and compare the effectiveness of the synthesized NPs. 
	&#8203;NOTE: This test can be used to prescreen the best NPs to be coated on the fabric.</li>
</ol>
3. Parallel streak method (modified from AATCC 147)
<ol>
	<li>Material preparation
	<ol>
		<li>Cut the NP-treated fabric into 50 mm x 25 mm swatches.</li>
		<li>Prepare Mueller-Hinton agar (MHA), TSA, and TSB, according to manufacturers&#39; instructions, and sterilize the media by autoclaving at 121 &#176;C at 15 psi for 15 min.</li>
		<li>Subculture the microbes (from stock) of interest, against which the efficacy tests are performed, on freshly prepared TSA plates (or any appropriate media) by the three-way streak plate method and incubate at 35 &#176;C for 2 days for bacterial stains and at R/T for 5 days for fungal strains to produce purity plates. (Recommended species: S. aureus, E. coli, P. aeruginosa, and C. albicans.)</li>
	</ol>
	</li>
	<li>Spiking of microbial cultures
	<ol>
		<li>Inoculate the cultures from fresh purity plates in TSB (10 mL tubes) at 35 &#176;C O/N with moderate shaking (100-200 rpm).</li>
		<li>Dilute the O/N cultures to 1.5 &#215; 108 colony forming units (CFU)/mL or corresponding to the turbidity of 0.5 McFarland standard (usually a 1/10 to 1/20 dilution for most healthy cultures).</li>
		<li>Inoculate the diluted culture above using a 4 mm sterile loop as follows. Load a loopful of broth culture and transfer it to the surface of the MHA agar plate by making five parallel streaks, each 6 cm in length and 1 cm apart, according to Figure 2. Do not refill the loop.</li>
		<li>Gently press the fabric swatch using a sterile spatula across the five streaks so that the fabric is in the center and touches all five streak lines. Incubate at 35 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Qualitative evaluation of antimicrobial efficacy
	<ol>
		<li>Examine the incubated plate for the interruption of growth along the streaks beyond the edges of the fabric (clear zone indicates inhibition of growth).</li>
		<li>Calculate the average width of a zone of inhibition along the streak line (W) on either side of the fabric swatch using equation (1). 
		W = (T - D)/2&#160; &#160; (1) 
		W = width of clear zone; T = total length of the clear zone, including the swatch width; D = width of the fabric swatch (25 mm).</li>
	</ol>
	</li>
</ol>
4. Quantitative log reduction method (modified from AATCC 100)
<ol>
	<li>Experimental preparation
	<ol>
		<li>Cut the fabric into 50 mm x 50 mm square swatches.</li>
		<li>Prepare TSA, TSB, Letheen broth, and phosphate-buffered saline (PBS), according to manufacturers&#39; instructions, and sterilize the media by autoclaving.</li>
		<li>Prepare O/N cultures of microbes of interest, against which the efficacy tests are performed, by inoculating isolated colonies from purity plates in sterile TSB and incubating at 35 &#176;C for 18-24 h. (Recommended species: S. aureus, E. coli, P. aeruginosa, and C. albicans.)</li>
	</ol>
	</li>
	<li>Spiking of microbial cultures
	<ol>
		<li>Dilute the O/N cultures to 1.5 &#215; 108 CFU/mL or corresponding to the turbidity of 0.5 McFarland standard (usually a 1/10 to 1/20 dilution for most healthy cultures).</li>
		<li>Determine the appropriate volume of cultures for spiking by measuring the liquid holding capacity of the fabric as follows.
		<ol>
			<li>Add a series of volumes of the diluted broth culture (e.g., 100-500 &#181;L) onto fabric swatches (in separate Petri plates) and choose the volume such that the fabric swatch absorbs the water fully and leaves no residual/free liquid. The liquid holding capacity differs from the type and thickness of the fabric. For regular cotton lab coats, it is roughly 200 &#181;L.</li>
			<li>Spike the volume (liquid-holding capacity determined above [e.g., 200 &#181;L]) of each culture onto the swatches placed in sterile Petri plates. The number of swatches corresponds to the number of tests (e.g., fabric tested immediately and after washing [and the laundering cycles tested]), and the antimicrobial effects tested over the contact time (after a set time/day [e.g., 30 min, 2 h, Day 1-3]). 
			&#8203;NOTE: Use a micropipette with aerosol filter tips (to prevent contamination of the pipette in subsequent uses) to inoculate the cultures on the swatches with even distribution.</li>
			<li>For the negative control, use the same type of untreated fabric swatches. Perform the negative control for each corresponding test-microbial species, contact period, different fabrics, and wash cycles.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Recovery of microbes by viable plate counts
	<ol>
		<li>Allow the inoculated swatches (both treated and untreated) to air-dry inside the Petri plates (lids ajar) at R/T for the required contact period/s to be tested (e.g., 0 min, 30 min, 60 min, etc., or even days for long-term effects). Always include a &#34;0 min&#34; to represent an immediate effect and neutralizing efficacy.</li>
		<li>Transfer the swatches aseptically to separate sterile centrifuge tubes (50 mL) and screw the caps tightly.</li>
		<li>Add Letheen broth (any relevant neutralizing buffers) to make a 1/100 dilution (e.g., 19.8 mL for an inoculum of 200 &#181;L).</li>
		<li>Close the centrifuge tubes with screw caps tightly and vortex for 1 min at medium speed.</li>
		<li>Serially dilute the suspension with the sterile PBS in subsequent 1/10 dilutions, such that the colony counts of the &#34;untreated&#34; group becomes too low to count (TLTC).</li>
		<li>Plate the dilutions (0.1 mL) on appropriate media plates, which support the growth of the microorganism (e.g., TSA for bacteria or Sabouraud dextrose agar [SDA] for fungi) or any media that optimize the growth and provide good contrast to make the colony counting accurate.</li>
		<li>Incubate the bacterial plates at 35 &#176;C for 2 days and the fungal plates at R/T for 5 days.</li>
		<li>Count the viable CFUs directly using a colony counter or using imaging software (e.g., CFU AI).</li>
		<li>Calculate the microbial log reduction (R) due to antimicrobial fabric using equation (2): 
		R = <img alt="Equation 1" src="/files/ftp_upload/65187/65187eq01.jpg" style="margin: auto;" /> &#215; 100&#160; &#160; &#160;(2) 
		A = log value of the number of CFUs recovered from untreated fabric; B = log value of the number of CFUs recovered from treated fabric.</li>
	</ol>
	</li>
</ol>

Antimicrobial Efficacy of Nano-Herbal-Coated Fabrics

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The authors have no conflicts of interests.

The antimicrobial efficacy of biocides is conventionally tested by quantitative assays, such as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), in which the bacteria are immersed in an antimicrobial liquid for 24 h. However, these assays are not suitable for coated fabrics, where the liquid interface is lacking and the biocides are diffused slowly along the fabric fibers. Therefore, many standard fabric tests have been established, such as AATCC 147, ISO 20645, AATCC 100, and JIS L 19...

The protective white coat is a mandatory personal protective equipment (PPE) item in microbiology laboratories and healthcare facilities, and it protects from direct exposure to pathogens, spills, and burns. These cotton coats promote microbial growth due to many factors-the woven fabric provides attachment sites and aeration, cotton and starch used in the manufacturing process along with exfoliated epithelial cells from the user supply nutrients, and the proximity to the user gives warmth and moisture. The accumulation of microbes on textiles can also cause health problems such as allergies and nosocomial infection, unpleasant odors, and fabric deterioration1.
Unlike regular clothing, protective coats are infrequently washed or disinfected, as found in many surveys2,3. Many studies show evidence of lab coats acting as a vector of microbial transmission and the risk of nosocomial infections in the healthcare setting2,4, particularly resistant strains3 such as methicillin-resistant Staphylococcus aureus (MRSA); thus, they raise health concerns of PPE, which is meant to protect from microbial contamination. There are not enough cross-sectional studies on lab coat-associated infections in the context of Biosafety Level 2 (BSL-2) facilities or microbiology teaching labs, but many regulatory authorities restrict the use of lab coats within the containment level. However, many academic institutions in North America struggle to meet the requirements due to practical constraints, such as laundering and storing inside the facility, the incidents of wearing lab coats in public areas such as cafeterias and libraries are common. One practical solution to these issues is the application of antimicrobial agents in textile finishing.
Antimicrobial fabrics are gaining increasing popularity in sportswear, activewear, and socks, mainly intended to reduce body odor. However, the use of these fabrics is not common in PPE development, except for some silver-coated cotton masks and healthcare garments5. We report the development of an antimicrobial fabric for lab coats, which inhibits common pathogens found in BSL-2 labs and renders effective protection from the cross-contamination of common pathogens.
Currently, a variety of antimicrobial fabrics and finishings are available in the market, but most of these use heavy metal colloidal particles (e.g., silver, copper, zinc), organometallics, or synthetic chemicals such as triclosan and quaternary ammonium compounds, which are not environmentally friendly1 and may lead to health issues such as skin irritation and allergies6. Some synthetic formulations pose concerns due to non-target microbes, such as normal flora or inducing antimicrobial resistance (AMR). The US Food and Drug Administration (FDA) regulates commercial antimicrobial fabrics, which must be non-toxic to the user and free from eco-toxicity. Therefore, antimicrobial fabrics based on natural biocides that inhibit a broad spectrum of microbes are preferable. Essential oils (EOs) are used widely as antimicrobial and therapeutic agents, but their use in antimicrobial finishing is limited due to their durability6,7,8. Based on our knowledge and market research on nano-herbal finishing8, no herbal-based antimicrobial fabric is commercially available. This is because synthetic coatings are easy to manufacture and have long durability. A few nano-herbal-coated textiles reported only for research purposes include neem7, moringa9, and curry leaves9.
The present study uses two bioactive components extracted from oregano EOs, carvacrol and thymol, which are effective against a wide range of bacterial pathogens and viruses but are generally recognized as safe for humans10. However, these bioactive components are volatile, and therefore their antimicrobial potential is short-lived if applied directly to the fabric. Nano-herbal encapsulation is a process in which bioactive components or drugs are loaded inside a polymeric shell that protects the core from environmental degradation, and thus enhances the shelf life. In addition, the small size of the polymeric particles, which generally range from 10 nm to 100 nm, enhances the efficacy of the application and slows the release of the bioactive compounds onto the fabric. These bioactive compounds are used for various purposes, such as food preservation10, but not for textile coating.
Among many polymeric encapsulants, chitosan is an attractive candidate due to many of its attributes, such as nontoxicity, biodegradability, mucoadhesivity, and biocompatibility11. It is a natural polysaccharide, obtained by the deacetylation process from chitin, which is found in seashells and fungal cell walls. It is used in biochemical and food preservation applications such as drug or protein delivery11,12,13, controlled release14, and antimicrobial films10. Chitosan is not readily soluble in water but forms a colloidal suspension in acidic media. Bioactive molecules are loaded into chitosan nanoparticles (NPs) by a simple two-step ionic gelation method14,15,16. In this process, hydrophobic bioactive compounds such as carvacrol and thymol form an oil-in-water emulsion, which is aided by a surfactant, Tween 80. Subsequently, a polyanionic compound, pentasodium tripolyphosphate (TPP), is used to form the cross-linkages between the amino groups along the polycationic polymer molecules and phosphate groups of TPP molecules to stabilize the complex. This complexation process solidifies the bioactive compounds within the matrix of chitosan, which is subsequently purified and coated onto cotton swatches to produce antimicrobial fabric.
The nano-formulations must be tested first for antimicrobial effectiveness in emulsion form before being applied to the fabric. This can be conveniently evaluated by a qualitative method, such as Kirby-Bauer disk diffusion, well diffusion, and the cylinder plate assay. However, the cylinder plate assay17 provides the flexibility to load varying volumes of the formulation and compare the zone of clearance. In this method, the antimicrobial formulations are loaded in stainless steel cylinders and placed on a soft agar layer, which is inoculated with the test microorganism or pathogen. The diameter of the zone of clearance produced against the test organism is proportional to the inhibitory potential of the antimicrobial formulation, and therefore can be used as an alternative to broth dilution methods. However, the size of the clear zones is only a comparative or qualitative measure within a specific plate unless specific standards are maintained. Antimicrobial agents act against the pathogens either by inhibiting their growth (biostatic) or killing the cells (biocidal), which can be quantified by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. However, the efficacy and behavior of the bioactive chemicals are different in their formulations (liquid state) and when coated on a substrate such as a fabric18. This is because multiple factors play a role in the efficacy, such as the stability of the adherence of the antimicrobial agents to the fabric, moisture content, substrate type, and adherence of the microbes. If the intended purpose is only bacteriostatic activity, a qualitative assay such as the &#34;Parallel Streak Method&#34;19 can provide a relatively quick and easy evaluation of diffusible antimicrobial formulation. However, if the bactericidal effects are to be determined, &#34;Assessment of Antibacterial Finishes on Textile Materials&#34;20 can be employed, which provides the log reduction of the spiked pathogen.

<table><tbody><tr><td>Acetic acid</td><td>Millipore Sigma</td><td>64-19-7</td><td></td></tr><tr><td>Antibiotic base agar</td><td>BD Difco</td><td>DF0270-17-4</td><td>Also known as Antibiotic Medium 2</td></tr><tr><td>Antibiotic seed agar</td><td>BD Difco</td><td>DF0263-17-3</td><td>Also known as Antibiotic Medium 1</td></tr><tr><td>Blood Agar (Nutrient Agar with 5% Sheep Blood)</td><td>Donated by CFIA</td><td></td><td></td></tr><tr><td>Bromcresol Purple Lactose Agar</td><td>Donated by CFIA</td><td></td><td></td></tr><tr><td>&lt;em&gt;Candida albicans&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATTC 10231</td><td></td></tr><tr><td>Carvacrol</td><td>Millipore Sigma</td><td>282197 (CAS# 499-75-2)</td><td></td></tr><tr><td>Centrifuge&amp;nbsp; Allergra X-22R Centrifuge</td><td>Beckman Coulter</td><td>Model # X-22R</td><td>Refrigerated. Wait at least 20 min or until the temperature reach the set low value (e.g., 4 &amp;deg;C) as the refrigeration takes time.</td></tr><tr><td>Chitosan Medium Molecular Weight (CS)</td><td>Millipore Sigma</td><td>448877 (CAS # 9012-76-4)</td><td></td></tr><tr><td>Clamshell Heat Press</td><td>Intiva</td><td>IM1200</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli (E. coli)&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATTC 23725</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>1205M34</td><td></td></tr><tr><td>Letheen Broth</td><td>BD Difco</td><td>DF0681-17-7</td><td>Used to neutralize antimicrobial effects. Product from different manufacturers may require to add Polysorbate 80, which is already added in Difco product.</td></tr><tr><td>Milli Q water</td><td>Millipore Sigma</td><td>ZR0Q16WW</td><td>Deionized water</td></tr><tr><td>Mueller-Hinton Agar</td><td>BD Difco</td><td>DF0252-17-6</td><td></td></tr><tr><td>Pentasodium tripolyphosphate (TPP)</td><td>Millipore Sigma</td><td>238503 (CAS# 7758-29-4)</td><td></td></tr><tr><td>Phospahte Buffered Saline (PBS)</td><td>Thermo Scientific</td><td>AM9624</td><td></td></tr><tr><td>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATTC 9027</td><td></td></tr><tr><td>Sabouraud Dextrose Agar</td><td>BD Difco</td><td>DF0109-17-1</td><td></td></tr><tr><td>Shaking incubator/ Thermo shaker</td><td>VWR</td><td>Model# SHKA2000</td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATTC 6538</td><td></td></tr><tr><td>Thymol</td><td>Millipore Sigma</td><td>T0501 (CAS# 89-83-8)</td><td></td></tr><tr><td>Trypticase Soy Agar</td><td>BD Difco</td><td>236950</td><td></td></tr><tr><td>Trypticase Soy Broth</td><td>BD Difco</td><td>215235</td><td></td></tr><tr><td>Tween 80</td><td>Millipore Sigma</td><td>STS0204 (CAS # 9005-65-6)</td><td></td></tr><tr><td>UV-Vis Spectrophometer</td><td>Thermo Scientific</td><td>GENESYS 30 (840-277000)</td><td></td></tr></tbody></table>

an antimicrobial fabric using nano-herbal encapsulation of essential oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

Initial screening of the synthesized NPs 
Following the two-step oil-in-water emulsion technique16, the bioactive compounds (carvacrol and thymol) were successfully encapsulated in chitosan. This was confirmed by UV-Vis spectrophotometry for the peak absorption of the respective bioactive compounds compared to controls, which were the chitosan NPs without any bioactive compounds. The constituted NPs were homogeneous and stable over 12 months at 4 &#176;C. The initial screenin...

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Author Spotlight: An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils  

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...

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Research

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Biology

7.8K Views.  Centennial College. Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

Video: Author Spotlight: An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils  

Title Cell Encapsulation by Droplets

My name is Hang Moon. I'm working with the professor of condoms. He and me work with the XYJ stage to make a 3D structure of cell.Hi, I am PE and Lynn and I'm working with the cell printing project with cell encapsulation and media and gels. This is solenoid be for ejection of a cell and cell mixture of aros over cell media. The so valve is activated by two signal line.The signal line is came from a dry board here. Signal came from here. That line through the that line, that one is the function first generator.So first is generated by our programmed way. So if you want, you want to change the purse, you can change the program with the that switches line or you can control that process generator to computer. The cellular valve is open and closed by the electric signal, but the, the cell is supplied through that line.The cell and the media or agro gel is supplied through the syringe syringes. The syringes inside of the water, inside of syringe is the press light by the air. The pressurized air is supplied from a tank like here the line.So the little line and the ized air is fly through the air filter. The air filter is discriminate the dust and other kind of the dirt. That is pressure generator we call it, is generating the signal used for open the solar air bell or closed the solar air valve computer is the connected through P-I-B-U-S-V connector.So we can use the USB port by directly our computer, the laptop computer. So that one is connected to that side here. And then we can control through the control laptop here.So if we want to synchronize of the movement of the stage and opening the bell is can be controlled by the same controller as a laptop. So we can control the timing when it open, when it close, and when it moves and when it stops. And we can synchronize the motion and opening bell.That stage is the motorized stage. It is automatically automatically moved by through the death signal line and motor. So motorization is 0.1 micrometers.So it can ideally it can draw the line up to 100 millimeter line. So jet axis is moving, going up and down like that way. So it control the cap between the substrate and the solar belt.So it can control the height of ejection. That substrate is the connected to the XY stages here, X here and Y there. So it's moving around a plan XY plan.So it draws some feature demo right stages is three Xs, X, Y, and G.So the jet con jet axis or XY axis is controlled by signal here. So signal line is connected to the motion controller here. That motion controller doesn't have any manual switch at all because it cannot be controlled manually.It's controlled through the laptop computer. So laptop computer is connected. The backside of the controller, we have three xs for a printing cell XY jet.So it make three different kind of signal independently. And then motion controller is communicate through the rf 2, 3, 2 connectors is directly connected to the USV. And then USV is connected to laptop computer.So laptop controlled borrower. We use the AutoCAD program, we make a gen A path like that way bam. And then it change to the machine language like that.So machine language is transferred to the like that the simple character and numbers like that. There is each position related to the X, Y, Z stages here I choose the file, contain that kind of program and then open downloading program like that way. And then I choose from file like does exact the same, the text file.Just the text file is downloaded, choose text file and automatically download from laptop to com motion controller executive and then it going the BAM character as I mentioned before, BAM. In this flask are N IH 3G three mouse fiberblast cells. And I'm going to first aspirate the old media.This process is to passage the cells and the cells are adhered to the bottom of the flask. So after aspirating the media, I'm going to put in six RYS in which is an enzyme.Okay? So after the trypsin has been put into the flask, we need to wait three to five minutes for the cells to become detached from the bottom of the flask.Now we're ready to put in the media and passage the cells into a centrifuge tube. It's a one-to-one dilution. So I'm putting in six mil of this media right here and I'm gonna wash out the saws a little bit to make sure all of them are passaged.And now the cells are ready to be centrifuge in this tube. Now we're going to centrifuge these cells at a thousand RPM three to five minutes. Now that the cells have been spun down into a pellet, I'm going to aspirate the supernatant.Now I'm going to wash out the cells with PBS. And this is to, this is for staining the cells. The stains that we are using are esterase substrates.Okay, now we we're going to centrifuge again and spin down the cells into a palate. And I'm going to aspirate the PBS and I'm going to resin the cells. I'm in media to be counted.I'm mixing the cells so that when we take the a sample of it, the concentration will be even throughout the tube. Now I'm going to take a hundred microliters of the cells to measure the cell density. Now I'm putting in a hundred microliters of type and blue, which is a stain.We're going to wait three to five minutes in order for the stain to permeate dead cell membranes. When we count the cells, the blue ones we don't count. The cells are ready to be counted with the hemo cytometer, I'm going to transfer 50 microliters of the cell mixture to each side of the hemo cytometer.Okay, I'm going to cover it with a glass slide side. There's two sides to the hemo cytometer. I'm gonna count the first side and after counting both sides, I'm going to take the average and then multiply by two because we had a one-to-one dilution with the type in blue.So basically our cell density will be 135 times 10 to the four cells per mil. For our experiment, we will be using a cell solution with a concentration of half a million cells per mil. And so since we have 13.5 million cells, I will be resus suspending the cells in 27 mil of media.We are using two different types of stains. This is a live dead assay from molecular probes. Our first stain is called calcium am.Our second stain is called AUM homodimer. It stains dead cells. We use blue excitation to get green fluorescence and green excitation to get red fluorescence For these two stains, when we're imaging our dice solution will be prepared in PBS.So first I'm going to transfer five mil of PBS into a tube phosphate buffered saline. That's a saline solution. Now I'm going to add half a microliter of calcium am per mil.Now I'm adding 2.5 microliters of calcium am to the tube and two microliters of athe homodimer per mil of PBS. Here is the btex machine. It makes the cell and media well distributed through the whole volume.Now we load the cell 200 microliter onto the seven here. And then we put on the cap, this is a tight tightly sealed through like that way here I open the valve and then it's the pressure light air comes in and then I adjust the belt like that. And then it change the pressure.So I adjust. The pressure is five PSI. The inside of tubing pressure is five PSI.From here to here, it connected through the this inside the hole. So just we open the bell here and then the cell injected with the five PSI pressure and then starting open. And then Sona valve starting operate.And then we roll the substrate. It substrate is just a simple slide glass. And then we load the proper portion and I put some sketch tape to fix the substrate like that, right?And simply I just fix it. So we are setting whole thing the cell and ary valve and the substrate during the moving the ary working, start working. So we are set up whole thing and then open ary valve start.And then we move the stage like that, right? And then we make some pattern onto the substrate pattern onto the substrate and bam character. I'm going to immerse these droplets that we printed in this dye solution that we made earlier.And now the droplets will be incubated for 10 minutes and then imaged. First I'm going to adjust the image through the eyepiece. Our first image is just a bright field image.I'm gonna turn off the light and change the filter so that we have blue excitation, which will show green fluorescence. And the third image we'll be taking is the dead cells, which is the Thm Homodimer stain. And with green light, we will get red fluorescence.

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

Deposition of Porous Sorbents on Fabric Supports

A microwave deposition technique for silanes, previously described for production of oleophobic fabrics, is adapted to provide a fabric support material that can be subsequently treated by dip coating. Dip coating with a sol preparation provides a supported porous layer on the fabric. In this case, the porous layer is a porphyrin functionalized sorbent system based on a powdered material that has been demonstrated previously for the capture and conversion of phosgene. A representative coating is applied to cotton fabric at a loading level of 10 mg/g. This coating has minimal impact on water vapor transport through the fabric (93% of the support fabric rate) while significantly reducing transport of 2-chloroethyl ethyl sulfide (CEES) through the material (7% of support fabric rate). The described approaches are suitable for use with other fabrics providing amine and hydroxyl groups for modification and can be used in combination with other sol preparations to produce varying functionality.

This report details a microwave-initiated approach for deposition of porphyrin functionalized porous organosilicate sorbents on a cotton fabric and demonstrates reduction in 2-chloroethyl ethyl sulfide (CEES) transport through the fabric resulting from this treatment.

A microwave deposition technique for silanes, previously described for production of oleophobic fabrics, is adapted to provide a fabric support material ...

Chemistry

Use of Chitosan to Enhance Photoluminescence of Curcumin for Textile Authentication

Enhanced Photoluminescence of Curcuma longa Extracts via Chitosan-Mediated Energy Transfer for Textile Authentication Applications

Dyes for security markings play a pivotal role in safeguarding the integrity of products across various fields, such as textiles, pharmaceuticals, food, and manufacturing among others. However, most commercial dyes used as security markings are costly and may contain toxic and harmful substances that pose a risk to human health. Curcumin, a natural phenolic compound found in turmeric, possesses distinct photoluminescent properties alongside its vibrant yellow color, making it a potential candidate material for authentication applications. This study demonstrates a cost-effective and eco-friendly approach to develop enhanced photoluminescent emissions from curcumin dyes for textile authentication. Curcumin was extracted from C. longa using sonication-assisted-solvent extraction method. The extract was dip-coated and dyed into the textile substrates. Chitosan was introduced as a post-mordanting agent to stabilize the curcumin and as a co-sensitizer. Co-sensitization of curcumin with chitosan triggers energy transfer to enhance its luminescent intensity. The UV-visible absorption peak at 424 nm is associated with the characteristic absorption of curcumin. The photoluminescence measurements showed a broad emission peaking at 545 nm with significant enhancement attributed to the energy transfer induced by chitosan, thus showing great potential as a naturally derived photoluminescent dye for authentication applications.

Author Spotlight: Eco-friendly Photoluminescent Textile Authentication with Curcumin

Photoluminescence is one of the most effective authentication mechanisms being used today. Utilizing and enhancing naturally sourced materials with inherent photoluminescent properties and incorporating them into fabric substrates can lead to development of green, sustainable, and functional textiles for smart applications.

Dyes for security markings play a pivotal role in safeguarding the integrity of products across various fields, such as textiles, pharmaceuticals, food, ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Bioengineering

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

 Zinc Oxide Nanoparticles Synthesis and Analysis of its Antibacterial Effects

Preparation of Zinc Oxide Nanoparticles and the Evaluation of their Antibacterial Effects

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant bacterial strains in hospitals has been attributed to the extensive and varied use of antibiotics, further exacerbating the problem of antibiotic resistance. Metal nanomaterials have been widely studied as an alternative solution for eradicating antibiotic-resistant bacterial cells. Metallic nanoparticles attack bacterial cells through various mechanisms, such as the release of antibacterial ions, generation of reactive oxygen species, or physical disruption, against which bacteria cannot develop resistance. Among the actively researched antimicrobial metal nanoparticles, zinc oxide nanoparticles, which are FDA-approved, are known for their biocompatibility and antibacterial properties. In this study, we focused on successfully developing a precipitation method for synthesizing zinc oxide nanoparticles, analyzing the properties of these nanoparticles, and conducting antimicrobial tests. Zinc oxide nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet/visible spectroscopy, and X-ray diffraction (XRD). Antibacterial tests were conducted using the broth microdilution test with the multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. This study demonstrated the potential of zinc oxide nanoparticles in inhibiting the proliferation of antibiotic-resistant bacteria.

Author Spotlight: Exploring the Antibacterial Effects of Zinc Oxide Nanoparticles in Overcoming Antibiotic Resistance

In this study, zinc oxide nanoparticles were synthesized using a precipitation method. The antibacterial effect of the synthesized particles was tested against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa bacterial strains.

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant ...

Antibacterial Graphene Oxide/Copper Nanocomposites for Resistant Bacteria

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to antibiotics. Developing&#160;antimicrobial agents that can replace existing antibiotics is essential because antibiotic-resistant bacteria have resistance mechanisms for all current antibiotics and can promote nosocomial infections. To address this challenge, in this study, we propose graphene oxide/copper (GO/Cu) nanocomposites as antibacterial materials that can replace the existing antibiotics. GO/Cu nanocomposites are characterized by transmission electron microscopy and scanning electron microscopy. They show that copper (Cu) nanoparticles are well-grown on the graphene oxide sheets. Additionally, a microdilution broth method is used to confirm the efficacy of the antimicrobial substance against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa), which are frequently implicated in nosocomial infections. Specifically, 99.8% of MRSA and 84.7% of P. aeruginosa are eliminated by 500 &#181;g/mL of GO/Cu nanocomposites. Metal nanocomposites can eradicate antibiotic-resistant bacteria by releasing ions, forming reactive oxygen species, and physically damaging the bacteria. This study demonstrates the potential of antibacterial GO/Cu nanocomposites in eradicating antibiotic-resistant bacteria.

Author Spotlight: Metallic Nanocomposites to Eliminate Antibiotic-Resistant Bacteria

Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to ...

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