Dispersion of multiwalled carbon nanotubes in Acacia extract and it's utility as an antimicrobial agent

Abstract

This work deals with the preparation of a multiwalled carbon nanotubes (MWCNTs) dispersion in Acacia extract (AE) and checks its antimicrobial activity. UV-vis analysis, Fourier transform infra-red spectroscopy (FT-IR) and scanning electron microscopy (SEM) were used to confirm the surface adsorption of extract components on MWCNT. The dynamic light scattering (DLS) determined the zeta potential and the particle size distribution (PSD) of MWCNTs in the AE dispersion medium. To observe the MWCNT stability and dispersion in AE, Turbiscan and transmission electron microscopy (TEM) were used respectively. The MWCNTs were found dispersed till the end of day 30, which suggests that AE is a good dispersing medium due to its surfactant properties. Finally, the dispersion was tested for its applicability as an antimicrobial agent using E. coli as a model microorganism. Colony forming units (CFUs) and dehydrogenase activity were assessed to check the inhibitory effect of dispersion. To test cell damage, fluorescent-based assay was conducted. The reduction in the viable bacterial count in the test sample proved the ability of dispersion as an antimicrobial agent. We are the first to report that AE may provide an alternative to the chemical surfactant for MWCNTs dispersion, and the AE–MWCNTs dispersion can be utilized as an antimicrobial agent.

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Introduction

The unique physicochemical properties of fabricated nanoparticles (NPs) are increasing their popularity among several commercial applications. Since they have highly active surfaces, they strongly tend to adhere and form aggregates. Therefore, methods are needed to prevent the aggregation of NPs in dispersion that will be significant for their applicability in some purposeful work. For any commercial application or toxicological study, it is desirable that nanomaterials are dispersed adequately and remain stable for a specific period of time.

One of the best methods to stabilize the NPs in the dispersion is to modify the NP surface. There are several polycarboxylic acids and their salts available for dispersing NPs.¹ For dispersing hydrophobic NPs such as carbon nanotubes (CNTs) in aqueous media, the copolymer with the hydrophilic group and hydrophobic group is frequently used. The hydrophobic segment binds with the hydrophobic particle surface while the hydrophilic segments remain in contact with aqueous media and improves compatibility, resulting in good dispersion.^2,3 A varied group of chemical surfactants are available for dispersing the NPs but they themselves have a number of harmful effects on the environment. Therefore, the use of biologically originated surfactants or stabilizers should be promoted. The most important aspect of biological surfactant will be it's easy biodegradability in the environment without any harmful effect.

Some of the studies have been done with biologically originated dispersants and CNTs. People have worked with natural organic materials (NOM) like humic acid and fulvic acid, and found suitable for CNT dispersion.^4,5 Hyung and Kim (2008) and Kennedy et al. (2009) found that for dispersion of CNT, humic acid was more appropriate than fulvic acid. The lower aromaticity and the molecular mass rendered fulvic acid less capable for dispersing CNTs.^4,5 Some studies also suggested that for dispersing the same amount of CNTs larger concentration of surfactant was needed as compared to the NOM.^6,7 Various biomolecules such as proteins, amino acids, DNA, cyclodextrins, amylose, starch, and gum arabic have been evaluated as dispersants for CNTs.⁸ Such dispersion relies on the non-covalent interaction between CNTs and dispersant molecules, and requires a direct contact between the two. This resultant surface modification prevents the aggregation of CNTs and renders the dispersion stable via steric or electrostatic repulsion or by incorporation of both. In one study, Alpatova et al. (2010) used various natural (gum arabic, amylose, Suwannee River natural organic matter) and synthetic (polyvinyl pyrrolidone, Triton X-100) dispersing agents for single-walled carbon nanotubes (SWCNTs). They found that the toxicity of the dispersant strongly influence the toxicity of SWCNT suspensions. Moreover, the applicability of non-toxic dispersants for preparing biocompatible and stable aqueous suspensions of CNTs was also established.⁸

Acacia concinna is a plant of medicinal importance and found in the tropical rainforests of southern Asia. The extract of Acacia concinna pods is used for cleaning hair, to encourage hair growth, as an expectorant, emetic and purgative, antioxidants.^9–11 Saponins are the major constituents of Acacia concinna fruits. The structures of various saponins exist in the fruit have been recently recognized, and it was found that they have surfactant properties similar to dodecylbenzene sulfonates.¹² The saponin molecule usually consists of two segments, an aglycon part called sapogenin which is hydrophobic and a hydrophilic sugar part.^13,14 The aqueous extract of Acacia pods shows acidic pH (2.1) because of the acacic acid. Acacic acid is a trihydroxy-monocarboxylic acid with molecular formula C₃₀H₄₈O₅ corresponding to pentacyclic triterpenes.^12,15 Acacia concinna is also known to have antimicrobial activity.^16,17 Natarajan and Natarajan (2009) studied the anti-dermatophytic activity of Acacia extract (AE) against several fungal species. They suggested that the fungicidal activity was due to the saponins since saponins have been reported to possess the cell wall toxicity.¹⁶ In another work, Todkar et al. (2010) found that AE had a potential to inhibit growth and metabolic activities of various bacterial and fungal species.¹⁷

Because of the dual profit from the AE, i.e. utility as a surfactant as well as antimicrobial property, we used the aqueous extract of Acacia pod for the dispersion of MWCNTs and also checked its applicability as an antimicrobial agent. As per our knowledge, none of the earlier studies have used AE for dispersing MWCNT neither checked the dispersion's toxicity on microflora. This study provides the very novel report regarding the applicability of AE as a dispersant for MWCNTs, and also the antimicrobial activity of the resultant dispersion. The UV-vis spectral analysis, Fourier transform infra-red spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) were used to examine the surface adsorption of Acacia compounds on MWCNT. The dynamic light scattering (DLS) was utilized for the determination of zeta potential, Z-average and particle size distribution (PSD) of MWCNT in the AE dispersion medium. To examine the MWCNT stability and status of dispersion, Turbiscan and transmission electron microscope (TEM) were used respectively. Finally, the AE–MWCNT dispersion was tested for its antimicrobial property by taking E. coli as a model microorganism. Three approaches were used for the purpose, counting colony forming units (CFUs), checking dehydrogenase activity for effect on cellular metabolism, and fluorescent-based assay for cell damage.

Experimental

Collection of MWCNT and preparation of stock suspension

Commercially available multiwalled carbon nanotube (MWCNT) was purchased from Reinste Nano Ventures Pvt. Ltd. INDIA. Purity 95%, number of walls 3–15, length 1–10 μm and outer diameter 5–20 nm and inner diameter 2–6 nm, as mentioned by the supplier. The metal content was analyzed by using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX). Morphology was checked with field-emission gun scanning electron microscopy (FEG-SEM). The MWCNT stock suspension was prepared (10 g L⁻¹) in deionized water using ultrasound water-bath (60 min, 45 kHz, 80% power) in sweep mode and held in reserve for later uses.

Preparation and analysis of Acacia extract

The dried Acacia pods were purchased from a local market, de-seeded, and weighed (10 g) then boiled in 1 L deionized (DI) water for 20 min. The crude extract was allowed to cool down to room temperature, filtered and stored under refrigeration. The pH and chemical oxygen demand (COD) were determined using standard methods.

Analysis for surface adsorption of extract

UV-visible spectra. The MWCNTs (1 g L⁻¹) were dispersed in the AE (50 mL) so that the final concentration of MWCNT becomes 100 mg L⁻¹. The dispersions were sonicated for 60 min (power 80%, 45 kHz) in a sweep mode, and then placed in the shaker for 24 h. Samples were gathered at regular intervals and centrifuged at 12 000 rpm for 15 min to settle down the MWCNTs. Absorbance was measured in UV-vis spectrophotometer (DR 6000, HACH, USA) for the supernatant in the range 200 to 800 nm. The decrease of absorbance value with respect to time was determined.

FT-IR spectra. The extracts were also analyzed by Fourier-transform infrared (FT-IR) spectrophotometer (SHIMADZU, 8400S with DRS) to get the idea about various chemical groups adsorbed on MWCNTs. The FT-IR spectrum of the AE was carried out in a liquid state. The FT-IR spectrum of pure MWCNT was taken after drying overnight at 60 °C to remove any moisture. For FT-IR spectrums of the Acacia treated MWCNTs, the Acacia–MWCNTs dispersion (100 mg L⁻¹) was centrifuged (12 000 rpm, 15 min) and settled MWCNTs were collected, washed with DI water, and dried overnight at 60 °C to remove any moisture and analyzed.¹⁸

Scanning electron microscopy. Field Emission Gun-Scanning Electron Microscopes (FEG-SEM), model: JSM-7600F with resolution 1.0 nm (15 kV), 1.5 nm (1 kV) and magnification: ×25 to 1 000 000 was used to image the surface modification of MWCNT. The MWCNT (100 mg L⁻¹) sample was overnight agitated with AE on shaker incubator (REMI, 150 rpm, 25 °C). Further, the dispersion was centrifuged (12 000 rpm, 10 min) to pellet down the MWCNTs, washed with DI water and dried in a vacuum dryer at 60 °C. The dried MWCNT samples were analyzed on FEG-SEM.

Dynamic light scattering

The Z-average, zeta potential and particle size distribution of the dispersion were characterized using Malvern Zetasizer, Nano ZS90 after ultrasonication. The sonicated (45 kHz, 80% power, 60 min) Acacia–MWCNT dispersion (100 mg L⁻¹) was analyzed. Dispersion was also kept undisturbed for 30 days to observe the dispersion stability.

Analysis of stability and dispersion

Turbiscan. The dispersion stability of the MWCNT in AE was checked with a Turbiscan (Turbiscan Lab, France) at 25 °C for 60 min. Different concentrations of MWCNTs (1 mg L⁻¹, 10 mg L⁻¹, 100 mg L⁻¹, 1000 mg L⁻¹) were used to check the effect of concentration on stability. Soon after the sonication (45 kHz, 80% power, 60 min), the dispersions were filled into the specially designed cylindrical glass vials up to a height of about 80 mm. The dispersion stability was studied by measuring the transmittance of a pulsed near infrared light (λ = 880 nm).¹⁹ The detector inspected the entire height of the sample and acquired the transmittance data in steps of 80 mm every min for 60 min.

Transmission electron microscopy. Transmission electron microscopy (PHILIPS, Model CM200, operating voltages 20–200 kV, resolution 2.4 Å) was used to image the MWCNT dispersion in DI water and in AE to visualize the difference. The dispersions were prepared with 100 mg L⁻¹ of MWCNTs in DI water and AE. Afterwards, sonicated at 45 kHz, 80% power for 60 min in a sweep mode. Samples were again sonicated for 15 min prior to observation. The MWCNT dispersion in DI water and AE dropped on a copper grid and dried before placing inside the TEM.

Dispersion antimicrobial activity

For antimicrobial analysis, Acacia–MWCNT dispersion (MWCNTs 1 g L⁻¹) in different dilution (10⁻¹, 10⁻², 10⁻³) was sprayed on the surface of cellulose acetate coated petri-dishes (size, 80 × 15 mm) except control one, and air-dried. Ten mL of E. coli culture was poured into each petri-dish. The dishes were incubated for 60 min in a shaker incubator with slight agitation (50 rpm, 37 °C). Thereafter, three different methodologies were applied to confirm the antimicrobial property of the coating.

In a first approach, E. coli samples were collected and plated on nutrient agar medium using serial dilution method. The CFUs were counted after 24 h incubation at 37 °C.

In a second approach, a colorimetric assay with triphenyl tetrazolium chloride (TTC) was used to assess cellular metabolic activities.²⁰ The E. coli test and control samples (5 mL) were added with 5 mL TTC (5 g L⁻¹) and 2 mL glucose solution (0.1 mol L⁻¹). The samples were kept in a shaker incubator for 20 min (200 rpm) and then again incubated at 37 °C for 12 h. Afterwards, 1 mL concentrated sulphuric acid was added to the vials to stop the reaction. To extract the triphenyl formazan (TF) formed in the reaction mixture, toluene (5 mL) was added in each vial and shaken at 200 rpm for 30 min and kept still for 3 min thereafter. The samples were centrifuged at 4000 rpm for 5 min. The supernatants were collected from each tube and the absorbance was determined at 492 nm using UV-vis spectrophotometer (DR 6000, HACH, USA).

The third approach was a fluorescent-based cell viability assay. It was carried out for testing the cell damage due to antimicrobial activity of Acacia–MWCNT composite. Ten microlitres of test and control samples were collected and added with 10 μL of the fluorescent dye (propidium iodide; 10 μg mL⁻¹). The aliquots were incubated for 15 min in a closed chamber then vortexed slightly to disperse the bacterial cells. Cell suspensions were taken on a clean slide and observed under the fluorescent microscope. The red fluorescent spots were counted for non-viable bacterial cells in control as well as in the test slides for comparison.

Results and discussion

Surface adsorption of extract components on MWCNTs

UV-visible spectral analysis. The UV-visible spectral analysis was done to check the adsorption of extract components on MWCNTs. The decrease in the absorbance value of dispersion suggests that the extract compounds which were contributing to the absorbance in the UV-visible range were getting adsorbed on to the MWCNTs. The high surface area of the MWCNTs provides vast adsorption sites for the extract compounds therefore the absorbance value successively decreases by passing time. The MWCNTs with surface adsorbed extract components were finally removed via centrifugation that has caused the decrease in the absorbance value of dispersion. The results of UV-visible spectral analysis are shown in Fig. 1. The region between 200 nm to 600 nm has shown considerable decrease in absorbance value that may correlate to the adsorption of compounds fall in this region like several aromatic molecules, saccharides, alkaloids, etc.¹⁰ The maximum decrease in absorbance was achieved on 6th h, afterwards desorption of dispersion components was observed that was exhibited by the increase of absorbance. This phenomenon was due to the saturation of adsorption sites on MWCNTs as well as the release of weakly bonded molecules. These released molecules further kept increasing the absorbance of the dispersion after 6th h.

Fig. 1 UV-vis absorption spectra for adsorption of Acacia extract on MWCNTs.

FT-IR analysis for chemical groups adsorbed on MWCNT surface. FT-IR analysis was performed with AE, pristine and Acacia treated MWCNTs in the range 400 to 4000 cm⁻¹. The IR spectra obtained are given in Fig. 2. The pristine MWCNT spectrum (spectrum a) shows a peak at 2359 cm⁻¹ corresponds to the medium O–H and N–H stretching vibration. The integrity of the hexagonal structure on MWCNT was confirmed in appearance of a peak at around 1523 cm⁻¹ elucidating the existence of carbon double bonding (C=C).²¹

Fig. 2 FT-IR spectra of MWCNTs, Acacia extract and MWCNTs treated with Acacia extract.

The AE spectra (spectrum b) showed some major peaks at 3392 cm⁻¹, 2357 cm⁻¹, 1586 cm⁻¹, 1066 cm⁻¹ and 668 cm⁻¹. The peak at 3392 cm⁻¹ corresponds to various amide and amine group stretching vibrations. These nitrogenous groups may be of the various alkaloids present in the extract.¹⁰ The peak at 2357 cm⁻¹ represents the amino acids. These amino acids are the precursors of heterocyclic nitrogenous alkaloids.²² The peak at 1586 cm⁻¹ represents asymmetric CO₂⁻ stretching vibration from carboxylic groups and presence of cyclic compounds such as saponins. The peak at 1066 cm⁻¹ represents a strong C–O vibration from aromatic and α-unsaturated hydroxyl groups while the peak at 668 cm⁻¹ represents hydrocarbon (C–H) vibrations.

The newly distinguished peaks among treated MWCNT (spectrum c) (3399 cm⁻¹, 3258 cm⁻¹, 2358 cm⁻¹, 1509 cm⁻¹, 1335 cm⁻¹, 1070 cm⁻¹ and 668 cm⁻¹) suggest the presence of various groups on the MWCNT surface. The peaks such as 3399 cm⁻¹ and 3258 cm⁻¹ represents N–H stretching vibrations from imino group and other amines (–NH₂), a strong stretching vibration of O–H from ringed structure. Appearance of peak at 1509 cm⁻¹ assigns polycyclic aromatic hydrocarbon C=C stretching vibration while 1335 cm⁻¹ corresponds to the C–H and O–H deformation vibration in carbohydrate groups. The appearance of a peak at 1070 cm⁻¹ in treated MWCNT showed C–O stretching vibration from primary alcohol and ester group. The newly appeared peaks in the IR spectra of treated MWCNT supports the observations made in UV-vis analysis and confirm the adsorption of extract components on MWCNTs.

SEM analysis for surface morphology. The SEM images present a visual evidence for surface modification. The morphology of MWCNTs before and after surface modification with AE is given in Fig. 3. The MWCNT surfaces were smooth before the surface modification that becomes comparatively rough due to the adsorption of extract components on their surface. The surface-modified MWCNTs show the aggregates possibly due to the monomeric or polymeric compounds adsorbed on their surfaces which can wrap the MWCNTs and entangle to one-another during drying. Moreover, no structure damage of MWCNTs was seen after modification.

Fig. 3 SEM images of MWCNTs at 100 000× magnification; (a) MWCNTs without surface modification; (b) MWCNTs surface modified with Acacia extract.

Z-Average and zeta potential of MWCNTs

The Z-average and zeta potential (ζ) were measured with DLS. The Z-average size or Z-average mean is the cumulants mean, and is the primary and the most stable parameter measured by the DLS. Zeta potential is a function of the surface charge of dispersed particles and the degree of zeta potential is the major player for stabilizing the colloidal dispersions. The dispersed particles whose zeta potential lies close to zero (isoelectric point) probably agglomerates. While high repulsion and reduced particle aggregation occurs when the zeta values remain higher positive or negative. The real zeta value needed to keep the particles dispersed remain doubtful, and affected by several factors such as the type of solvent, concentration of ions, effective pH, and functional groups on the NP surface.²³

The Z-average and mean zeta potential values of MWCNT dispersion in AE are presented in the Table 1. The Z-average remained 247.60 ± 6.25 and 259.25 ± 2.22 on day 1st and 30th respectively. The particle size distribution (PSD) curve of MWCNTs in the dispersion is given in Fig. 4(a). The PSD show that the majority of the particle diameter lie between 50 to 1000 nm on day 1, and 100 to 1000 nm diameters on day 30. Moreover, the measure of molecular mass distribution in a given dispersion, that is polydispersity index (PDI) attained for day 1 analysis was on an average 0.30 while, day 30th analyses expressed slightly lower PDI (0.29). The PSD and PDI values suggest that during standing the settling of larger particles occurred that rendered the dispersion more uniform. The zeta potential of MWCNT in AE was −7.33 ± 5.78 mV and −6.74 ± 5.98 on day 1st and 30th respectively. On standing for 30 days, the magnitude of zeta potential was found decreased. This decrease may have occurred due to some biochemical changes in extract. The decrease in zeta accompanied with the increase in Z-average of MWCNTs. The zeta potential distribution (ZPD) of MWCNTs in AE is given in Fig. 4(b). It is a well known fact that high magnitude of zeta potential provides a better stability to the dispersion. Specially, in case of pure electrostatic stabilization, the higher electro-kinetic potential will impart the higher degree of stability. While, a biological dispersion medium such as Acacia extract had possibly imparted electrosteric stabilization due to the presence of diverse groups of organic polymeric compounds. It was assumed, that the polymeric compounds attached to the MWCNTs and form layer around them, and in addition an electric potential also would retain on the MWCNTs surface. Thus, both the electrostatic repulsion and steric restriction prevented the agglomeration even in the low zeta potential value.

Table 1 Z-Average and mean zeta potential of MWCNT in Acacia extract

Dispersion medium	Z-Average (nm)		Mean zeta potential (mV)
	Day 1^a	Day 30^a	Day 1	Day 30
a Mean ± SD (n = 3).
Acacia concinna extract + MWCNT (100 mg L^−1)	247.60 ± 6.25	259.25 ± 2.22	−7.33 ± 5.78	−6.74 ± 5.98

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Fig. 4 DLS details of MWCNTs dispersed in Acacia extract; (a) size distribution report by intensity on day 1 and 30; (b) zeta potential distribution on day 1 and 30.

Stability and dispersion analysis

Turbiscan. The stability of the Acacia–MWCNTs dispersion was analyzed using a Turbiscan for 60 min just after the sonication. The scanning period was kept 60 min to check the initial stability of the dispersion. The different concentration of MWCNTs was used to check the effect of concentration on dispersion stability. The transmitted light flux in % corresponds to the density of suspended particles in the dispersion. The difference in the particle density among the upper and the bottom layer of the sample container changes the transmission signal. The rapid increase in the transmission during the given period of study displayed that samples have been unstable and have gone through a more rapid sedimentation. The mean transmission profiles of MWCNTs in AE are given in Fig. 5. The X-axis designates the time-scale taken for analysis while the Y-axis shows the percentage transmission. The lower concentrations of MWCNTs (below 100 mg L⁻¹) have given good and discrete transmission results as shown in the figure. Another conclusion that can be drawn from results is that the stability of MWCNT in dispersion mostly depends on its own concentration as found in previous studies. The low concentration increases the inter-particle distance and therefore vander Waal's force of attraction become inactive rendering the dispersion stable. While at higher concentration the elongated MWCNTs entangles with each other causing larger floc formation that assist sedimentation. This phenomenon results in the settling of MWCNTs and increase in transmission percentage. From this transmission profile it was clear that AE can efficiently disperse the MWCNTs.

Fig. 5 Mean transmission profile for different concentration of MWCNTs in Acacia extract (a = 1000 mg L⁻¹ MWCNTs; b = 100 mg L⁻¹ MWCNTs; c = 10 mg L⁻¹ MWCNTs; d = 1 mg L⁻¹ MWCNTs).

TEM analysis of dispersed MWCNTs. The level of dispersion of MWCNTs in AE was observed using TEM and the results are shown in Fig. 6(a) and (b). With the same concentration of MWCNTs, the dispersion in DI water showed much aggregation as found in earlier studies that were due to the hydrophobic nature of MWCNTs.²⁴ However, individual fibres were visible in the case of MWCNTs dispersed in AE. The de-bundling of the aggregates is attributed to the surface functionalization of the MWCNTs. The TEM results confirm the suitability of the AE for dispersing MWCNTs.

Fig. 6 Transmission electron microscopy images (a) MWCNT cluster in deionized water; (b) MWCNT dispersed in Acacia extract.

Dispersion antimicrobial activity

To find the applicability of Acacia–MWCNT dispersion as an antimicrobial agent, E. coli culture was tested for its susceptibility towards dispersion. The antimicrobial property of Acacia–MWCNT coating was preliminary checked with the agar plating method. The inhibitory effect of Acacia–MWCNT coating on E. coli culture is summarized in the Table 2. The Acacia–MWCNT coating was found to inhibit bacterial growth and activity. The percent inhibition with respect to control was higher in culture treated with the Acacia–MWCNT coating at concentration 10⁻¹, however on further dilution (10⁻² and 10⁻³) the inhibition decreased. We can say that as we increased the dispersion concentration for coating, the percent inhibition increased. The coating not only inhibited the colony formation of E. coli, it also affected the dehydrogenase activity of bacteria. The dehydrogenase is the enzyme that plays an important role in cellular oxidation–reduction reactions. Therefore, it is generally used to examine the cellular metabolic activities.

Table 2 Percent inhibition of CFUs and dehydrogenase activity among test cultures with respect to control. Control = no dispersion coating; test 1 = culture containing 10³ times diluted dispersion; test 2 = culture containing 10² times diluted dispersion; test 3 = culture containing 10¹ times diluted dispersion

Sample	Agar plating		Colorimetric assay
	CFUs^a × 10^6 per mL	% inhibition	Absorbance^a (492 nm)	% inhibition
a Values are average of three readings.
Control	670	—	1.09	—
Test 1	600	10.45	0.57	47.01
Test 2	460	31.34	0.44	59.64
Test 3	400	40.3	0.24	77.7

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Colorimetric assay for the assessment of cell viability are important tools to study the cellular regulatory metabolism. One of the routinely applied assays make use of tetrazolium salts, a water-soluble dye that produces water-insoluble purple formazan crystals on reduction by the dehydrogenase system of active cells.²⁵ These formazan crystals thus formed are then quantified in spectrophotometer after dissolving in an organic solvent. The formazan concentration is directly proportional to the metabolically active cell count of the culture.²⁵ The increasing concentration of Acacia–MWCNT dispersion affected the dehydrogenase activity of bacteria. This impairment of the biochemical metabolism led to the reduction in cell viability and death. The results were in support of observations attained in agar plating method (Table 2).

The results of viability test after incubation with Acacia–MWCNT coating is given in Fig. 7(a)–(d). Fig. 7(a) represents the result of control E. coli cells while Fig. 7(b)–(d) represents the fluorescent images of E. coli cells collected from the test samples. The fluorescent test results supported the phenomena of cell damage. Propidium iodide is a fluorescent dye that exclusively stains cells with a damaged membrane. The cells incubated with Acacia–MWCNT coating expressed a higher number of damaged cells as compared to control. Fig. 8 is the graphical representation of average number of stained non-viable cells. With increasing concentration of the dispersion the more cell damage was evident. From these results, it can be assumed that the surfactant property of AE may have dissolved the bacterial cell membrane in a same manner as the chemical surfactant does, leading to cell damage. On the other hand, the highly stiff and rigid pointed MWCNT tubes seem to have damaged the bacterial cells. During incubation, the cell membrane damage occurs caused by MWCNT piercing leading to cell death or reduced viability.²⁶ This phenomenon justifies the more number of red fluorescent spots in dispersion treated culture. One more possibility for antimicrobial activity could be the presence of metallic residues in MWCNTs. But, the metal content measured with SEM-EDX were Al (0.24 wt%) and Mg (0.28 wt%), and with such a low concentration they would have imparted only negligible toxicity. Therefore, the MWCNT and Acacia extract can be considered as the major factors responsible for antimicrobial activity of the dispersion.

Fig. 7 Images (10×) result of fluorescent assay (a) control, (b) E. coli culture incubated with Acacia–MWCNT coating with dilution 10⁻³, (c) with dilution 10⁻², (d) with dilution 10⁻¹.

Fig. 8 Graphical representation of average number of stained non-viable cells counted.

Conclusions

In this work the dispersion of MWCNTs in AE and the antimicrobial activity of dispersion were studied. AE proved highly efficient for dispersing MWCNTs and the adsorption of extract components were capable to stabilize the MWCNT dispersion for a reasonable period as evident by the results of electron microscopy, FT-IR spectral analysis and dispersion stability. The dispersion was also found satisfactory antimicrobial against E. coli. The antimicrobial activity of dispersion can be utilized for preparing microbial resistant coatings. More detailed experiments should be conducted to explore the possible roles of different mechanisms that may favour the dispersion stability of MWCNTs in biological extracts to replace/minimize the use of chemical surfactants. Additionally, the antimicrobial property should be checked for other potent pathogens so that such dispersions can be used for commercial application.

Compliance with ethical standards

We would like to affirm that the study was carried out at Chemical Engineering Department, Sardar Vallabhbhai National Institute of Technology, Surat, INDIA. Results consist of a part of our ongoing work and have not been published previously and are not under consideration for publication elsewhere. We also affirm the approval by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form. The authors declare that there is no conflict of interest. No human and/or animal was used as an experimental subject for the present work.

Acknowledgements

We thank Chemical Engineering Department, SVNIT, Surat for providing basic infrastructure for work, MHRD for providing fellowship, Sophisticated Analytical Instrument Facilities, IIT-Bombay, Shree Dhanvantary Pharmaceutical Analysis and Research Centre, Surat and Genecare Lab, Surat for analytical facilities.

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