- Email: [email protected]
Efficient method for functionalization of carbon nanotubes by lysine and improved antimicrobial activity and water-dispersion
Materials Letters 72 (2012) 153–156
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Efficient method for functionalization of carbon nanotubes by lysine and improved antimicrobial activity and water-dispersion Ahmad Amiri a, Hadi Zare Zardini b, Mehdi Shanbedi a,⁎, Morteza Maghrebi a, Majid Baniadam a, Behnaz Tolueinia b a b
Department of Chemical Engineering, Engineering Faculty, Ferdowsi University of Mashhad, Mashhad, Iran Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
a r t i c l e
i n f o
Article history: Received 7 September 2011 Accepted 27 December 2011 Available online 3 January 2012 Keywords: Antimicrobial Carbon nanotubes FTIR Lysine Functionalization
a b s t r a c t Here, multi-walled carbon nanotubes (CNT) were first functionalized with lysine under microwave irradiation. The water-dispersed CNT were obtained by diazonium-assisted functionalization. Formation of lysine on CNTs surface was confirmed by FTIR, TGA, Raman and TEM techniques. Then, by minimal inhibitory concentration (MIC), antimicrobial activity of functionalized and pristine CNT was studied on three Gramnegative bacteria as well as three Gram-positive bacteria. The MIC results show that functionalized CNT with lysine were more effective than pristine CNT against all studied bacteria. This simple and efficient mechanism could be used to CNT functionalization with molecules containing primary amine groups. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Most of mortality throughout the world occurs due to infectious diseases and the performed studies in this regard are abundant [1]. Nano-structure such as silver nanoparticles (Ag-NP) and carbon nanotubes (CNT) have held a prominent place in these researches. It may be due to their unique properties and various applications such as high antimicrobial activity [2,3]. Among nanostructure substances, CNTs have presented many applications due to chemical stability and unique structure. Between various applications, their antimicrobial activity has been demonstrated on different bacterial strains [3–5]. Direct contact with pristine CNT aggregates damages the cell membrane and leads to cell death [1]. But the antimicrobial activity of CNTs is limited by chemical–physical properties such as the size of two terminals [6]. To improve the antimicrobial activity of CNTs, functionalization with various groups such as metallic nanoparticles has been reported [7]. Unfortunately, the low stability of functional groups inevitably resulted in antimicrobial efficacy loss. Covalent functionalization with cationic chemical groups could improve the antimicrobial activity of CNTs. Grafted functional groups containing cationic charge are often employed, which exert activity through attack on the bacterial membrane [6,8]. In this study, CNT were functionalized by lysine (CNT-Lysine) in a rapid microwave-assisted method. Considering dispersivity of
⁎ Corresponding author. Tel.: + 98 511 8816840. E-mail address: [email protected] (M. Shanbedi). 0167-577X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2011.12.114
functionalized CNT in aqueous solutions, it is possible to study antimicrobial activity precisely. Fourier Transform Infrared Spectroscopy (FTIR), Thermo Gravimetric Analysis (TGA), Raman and Transmission Electron Microscope (TEM) were used to analyze surface functionality and morphology. Antimicrobial activity of CNT-Lysine was studied on three Gram-positive and Gram-negative bacteria by minimal inhibitory concentration (MIC).
2. Experimental 2.1. Functionalization and characterization instrument The mechanism for the diazonium reaction has been discussed previously [9–12]. First, lysine (400 mg) was mixed with dimethylacetamide (DMA) (20 ml) for 1 h at 60 °C. Then, pristine CNT (140 mg) and NaNO2 (188 mg) were sonicated with lysine in DMA. Concentrated H2SO4 (0.124 ml) was added and the mixture heated for 1 h at 60 °C [9]. To increase functionalization degree, the mixture was poured into a teflon reaction vessel (volume of 100 ml) and was placed under microwave reaction (Milestone-MicroSYNTH programmable) [12]. The abovementioned CNT suspension was heated in microwave up to 120 °C with output power of 700 W for 15 min. The resulted suspension was filtered and to remove unreacted lysine and NaNO2, the filter cake was thoroughly washed with DMA and methanol by five times, after that with abundant deionized water, and then dried in an oven for 48 h at 60 °C. Also, the pH of the washings was tested to ensure that no acid remained in the sample.
154
A. Amiri et al. / Materials Letters 72 (2012) 153–156
The FTIR spectra of powder-pressed samples were placed on KBr pellets and recorded on a Shimadzu 4300 spectrometer. The weight loss of powdered samples was measured at a heating rate of 10 °C/ min in air with TGA analyzer (TGA-50 Shimadzu). To prepare TEM samples, CNT were sonicated in ethanol solution for 10 min, and after that were dispersed on a holey carbon-coated copper grid. 2.2. Determination of MIC Here, the MIC value was defined as a concentration of CNT-Lysine/ CNT in which the absorbance of bacteria is half of the control [13]. Staphylococcus aureus (S. aureus), Streptococcus agalactiae (S. agalactiae), Streptococcus dysgalactiae (S. dysgalactiae), Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia) and Salmonella typhimurium (S. typhimurium) were used for antimicrobial assays. To determine antimicrobial activity, the stock serial dilution of 0.01875 to 0.6 mg·ml − 1 of CNT-Lysine was prepared. Since pristine CNT doesn't form stable suspension in water, it was first sonicated for 1 h just before the tests. However, 20 μl of the each sample was added to 10 5 CFU·ml − 1 of bacteria and poured into a plate. The micro plate was incubated at 37 °C for 18 h. Next, the absorbance of wells at 630 nm was read using an ELISA reader (ELX800TM) and the results were compared with those of control. Experiments were performed with various bacteria in triplicates. 3. Results and discussion In this study, the functionalization procedure is based on diazonium reaction. It is likely that the diazonium salt receives an electron from the CNT, followed by liberating N2 and producing a semistable diazonium ion, which could react with the CNT sidewall [9–11]. Also, when CNT were exposed to microwave irradiation, strong absorptions are observed and severe heating was produced. The extended π-system of CNT authorizes conductivity to enable localized heating and therefore induced superheating point. The super heating point could potentially react with semi-stable diazonium ion [11,12]. Accordingly, FTIR, TGA, Raman and TEM were employed to analyze surface functionally groups. 3.1. Fourier transform infrared spectroscopy
3571
3.2. Thermogravimetric analysis TGA curve of the pristine CNT and CNT-Lysine is shown in Fig. 2. The curve of pristine CNT shows a small mass loss in a temperature range of 0–500 °C. On the other hand, CNT-Lysine curve shows two sudden mass losses. The first one (around 100 °C) is assigned to water, while the second one (in the range of 200–230 °C) is related to the decomposition of lysine. The decomposition temperature of lysine is around 215 °C, but it was shifted to a lower temperature. This phenomenon could occur by ejection of NH2 due to formation of the diazonium ion. 3.3. Transmission electron microscope and Raman Fig. 3 presents TEM images of the pristine CNT (Fig. 3a) and the CNT-Lysine (Fig. 3b and c). The pristine CNT show smooth surface (Fig. 3a) while increased roughness of the functionalized CNT surface is evident in TEM images of CNT-Lysine. In result of CNT functionalization, part of sp 2 hybridized carbons has been changed to sp 3 hybridization and it could damage graphitic CNT [11]. As another proof, the results of Raman spectroscopy are shown in Fig. 3d. The spectra of the samples exhibit D and G bands, at ~ 1274 and 1513 cm − 1 respectively. The intensity ratio of the D&G band (ID/IG) is utilized as an evidence of the disruption of aromatic πelectrons on the surface of CNT [11]. In functionalization researches,
1634 1554 1498 1423 1331
2304
3486 3198 2868
Fig. 1 shows the FTIR spectra of the CNT-Lysine and pristine CNT. The common peaks at 2304 cm − 1 could be related to the stretching
vibration of CNT backbone. Obviously, the FTIR spectra of CNTLysine sample explain clear cues of functionalities in contrast to the pristine CNT. However, in FTIR spectra of treated samples, the peak at 1554 cm − 1 corresponds with N\H bending vibration. Also, the medium peaks at 3571 cm − 1 and 3198 cm − 1 could be obtained from N\H stretching vibration, and the splitting is indicative of a primary amine [10]. The quite sharp peak at 1634 cm − 1 is attributable to C_O stretching vibration of carboxyl groups. Also, the appeared peak at 1331 cm − 1 is in agreement with stretching vibration of C\O. The peak at 3486 cm− 1 is assigned to O\H stretching vibration. The O\H peak can be generated due to adsorbed water by CNT-Lysine or hydroxyl groups of lysine. The peaks at 2868 cm− 1 and 1423 cm− 1 are related to C\H and C\N stretching vibration. A new peak at 1498 cm− 1 is attributed to the vibration of C\C group, which can be generated due to the interaction between amino groups in lysine and CNT and/or chain of lysine. This spectrum verifies the recognition of the lysine grafted onto the CNT.
100
(a)
90 80 70
(b) TGA %
60 50 40
CNT-Lysine
30
Pristine CNT
20 10 0 0
100
200
300
400
Temperature Fig. 1. FTIR spectra of (a) the pristine CNT, (b) the CNT-Lysine.
500
600
700
(oC)
Fig. 2. TGA analysis of the pristine CNT and functionalized CNT with lysine.
A. Amiri et al. / Materials Letters 72 (2012) 153–156
155
(b)
(a)
(d)
(c)
MWCNT-Lysine
Pristine MWCNT
500
1000
1500
2000
2500
3000
Fig. 3. (a) TEM image of the pristine CNT, (b,c) TEM image of CNT-Lysine and (d) Raman analysis of pristine and functionalized CNT.
3.4. Antimicrobial assay The MIC values of CNT-Lysine and pristine CNT on Gram-negative and Gram-positive bacteria are indicated in Fig. 4. One can see the functionalized CNT by lysine exhibited the strongest antimicrobial activity compared to the pristine CNT. MIC results shows that the antimicrobial activity of CNT-Lysine against Gram-negative bacteria E. coli, S. typhimurium and K. pneumonia respectively enhances by 3.23, 2.27 and 2.55 times compared to pristine CNT. Also, increases of 2.1, 1.84 and 1.80 times against Gram-positive bacteria S. agalactiae, S. aureus and S. dysgalactiae were observed respectively. The abovementioned data suggest that CNT-Lysine is more effective against Gram-negative bacteria. This is most likely attributable
to the different structures of cell walls of these bacteria [15,16]. The similar results were repeated in the CNT/epilson-polylysine nanocomposite by Zhou et al. [6]. 14
Minimum Inhibitory Conc.(µg / ml)
the higher (ID/IG) means the higher degree of covalent functionalization. As could be seen the ID/IG of CNT-Lysine is bigger than that of the pristine CNT. Meanwhile, stable concentration of CNT-Lysine in water was attained nearly 3.1 mg·ml − 1 in deionized water. A similar study was reported by Hu et al. [14]. Therefore, all characterization tests indicate that lysine groups are attached on the surface of CNT. Also, our results confirmed that output power of 700 W and 15 min of irradiation time was appropriate to perform reaction.
CNT-Lysine 12 Pristine CNT 10 8 6 4 2 0
Fig. 4. The MIC values of pristine CNT and CNT-Lysine against some Gram-negative and Gram-positive bacteria.
156
A. Amiri et al. / Materials Letters 72 (2012) 153–156
Since the ability of CNT to kill bacteria depends on direct interaction with the bacterial membrane, increased positive charge of CNT could improve attachment with negatively charged membranes of bacteria. The positive charge could increase permeability across the membrane and decrease metabolic activity and finally kill the bacteria [7,8]. Also, antimicrobial activity of CNT-Lysine is higher than some of treated CNT that investigated in another reports. 4. Conclusion An efficient and rapid technique to functionalize CNT by lysine under microwave irradiation was reported. A diazonium reaction occurred between lysine molecules and CNT, resulting in the attachment of lysine to the nanotube surface. Covalent functionalization procedure could significantly increase aqueous solubility, which is a critical criterion for various applications. Functionalization of CNT was confirmed by FTIR, TGA, Raman and TEM. MIC results confirmed strongest antimicrobial activity of CNTLysine compared to the pristine CNT. Also, improved antimicrobial activity is particularly obvious for the gram-negative bacteria. The more effective antimicrobial of CNT-Lysine was attributed to electrostatic adsorption of bacteria membrane, due to positive charges of the lysine groups on CNT. Acknowledgment The authors would like to thank Iran Nanotechnology Initiative Council of Iran for financial support. References [1] Aslan S, Loebick CZ, Kang S, Elimelech M, Pfefferle LD, Van Tassel PR. Antimicrobial biomaterials based on carbon nanotubes dispersed in poly(lactic-co-glycolic acid). Nanoscale 2010;2:1789–94.
[2] Espinosa-Cristóbal LF, Martínez-Castañón GA, Martínez-Martínez RE, LoyolaRodríguez JP, Patiño-Marín N, Reyes-Macías JF, et al. Antibacterial effect of silver nanoparticles against Streptococcus mutans. Mater Lett 2009;63:2603–6. [3] Liu S, Ng AK, Xu R, Wei J, Tan CM, Yang Y, et al. Antibacterial action of dispersed single-walled carbon nanotubes on Escherichia coli and Bacillus subtilis investigated by atomic force microscopy. Nanoscale 2010;2:2744–50. [4] Kang S, Pinault M, Pfefferle LD, Elimelech M. Single-walled carbon nanotubes exhibit strong antimicrobial activity. Langmuir 2007;23:8670–3. [5] Kang S, Herzberg M, Rodrigues DF, Elimelech M. Antibacterial effects of carbon nanotubes: size does matter! Langmuir 2008;24:6409–13. [6] Zhou J, Qi X. Multi-walled carbon nanotubes/epilson-polylysine nanocomposite with enhanced antibacterial activity. Lett Appl Microbiol 2011;52:76–83. [7] Mohan R, Shanmugharaj AM, Sung Hun R. An efficient growth of silver and copper nanoparticles on multiwalled carbon nanotube with enhanced antimicrobial activity. J Biomed Mater Res 2011;96B:119–26. [8] Ernst WA, Thoma-Uszynski S, Teitelbaum R, Ko C, Hanson DA, Clayberger C, et al. Granulysin, a T cell product, kills bacteria by altering membrane permeability. J Immunoassay 2000;165:7102–8. [9] Chidawanyika W, Nyokong T. Characterization of amine-functionalized singlewalled carbon nanotube-low symmetry phthalocyanine conjugates. Carbon 2010;48:2831–8. [10] Ellison MD, Gasda PJ. Functionalization of single-walled carbon nanotubes with 1,4-Benzenediamine using a diazonium reaction. J Phys Chem C 2007;112: 738–40. [11] Amiri A, Maghrebi M, Baniadam M, Zeinali Heris S. One-pot, efficient functionalization of multi-walled carbon nanotubes with diamines by microwave method. Appl Surf Sci 2011;257:10261–6. [12] Vazquez E, Prato M. Carbon nanotubes and microwaves: interactions, responses, and applications. ACS Nano 2009;3:3819–24. [13] Jin L-L, Li Q, Song S-S, Feng K, Zhang D-B, Wang Q-Y, et al. Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii. Comp Biochem Phys B 2009;154:174–8. [14] Hu N, Dang G, Zhou H, Jing J, Chen C. Efficient direct water dispersion of multiwalled carbon nanotubes by functionalization with lysine. Mater Lett 2007;61: 5285–7. [15] Huang KC, Mukhopadhyay R, Wen B, Gitai Z, Wingreen NS. Cell shape and cellwall organization in Gram-negative bacteria. Proc Natl Acad Sci U S A 2008;105: 19282–7. [16] Prokhorenko IR, Zubova SV, Ivanov AY, Grachev SV. Interaction of gram-negative bacteria with cationic proteins: dependence on the surface characteristics of the bacterial cell. Int J Gen Med 2009;2:33–8.
Contents lists available at SciVerse ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Efficient method for functionalization of carbon nanotubes by lysine and improved antimicrobial activity and water-dispersion Ahmad Amiri a, Hadi Zare Zardini b, Mehdi Shanbedi a,⁎, Morteza Maghrebi a, Majid Baniadam a, Behnaz Tolueinia b a b
Department of Chemical Engineering, Engineering Faculty, Ferdowsi University of Mashhad, Mashhad, Iran Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran
a r t i c l e
i n f o
Article history: Received 7 September 2011 Accepted 27 December 2011 Available online 3 January 2012 Keywords: Antimicrobial Carbon nanotubes FTIR Lysine Functionalization
a b s t r a c t Here, multi-walled carbon nanotubes (CNT) were first functionalized with lysine under microwave irradiation. The water-dispersed CNT were obtained by diazonium-assisted functionalization. Formation of lysine on CNTs surface was confirmed by FTIR, TGA, Raman and TEM techniques. Then, by minimal inhibitory concentration (MIC), antimicrobial activity of functionalized and pristine CNT was studied on three Gramnegative bacteria as well as three Gram-positive bacteria. The MIC results show that functionalized CNT with lysine were more effective than pristine CNT against all studied bacteria. This simple and efficient mechanism could be used to CNT functionalization with molecules containing primary amine groups. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Most of mortality throughout the world occurs due to infectious diseases and the performed studies in this regard are abundant [1]. Nano-structure such as silver nanoparticles (Ag-NP) and carbon nanotubes (CNT) have held a prominent place in these researches. It may be due to their unique properties and various applications such as high antimicrobial activity [2,3]. Among nanostructure substances, CNTs have presented many applications due to chemical stability and unique structure. Between various applications, their antimicrobial activity has been demonstrated on different bacterial strains [3–5]. Direct contact with pristine CNT aggregates damages the cell membrane and leads to cell death [1]. But the antimicrobial activity of CNTs is limited by chemical–physical properties such as the size of two terminals [6]. To improve the antimicrobial activity of CNTs, functionalization with various groups such as metallic nanoparticles has been reported [7]. Unfortunately, the low stability of functional groups inevitably resulted in antimicrobial efficacy loss. Covalent functionalization with cationic chemical groups could improve the antimicrobial activity of CNTs. Grafted functional groups containing cationic charge are often employed, which exert activity through attack on the bacterial membrane [6,8]. In this study, CNT were functionalized by lysine (CNT-Lysine) in a rapid microwave-assisted method. Considering dispersivity of
⁎ Corresponding author. Tel.: + 98 511 8816840. E-mail address: [email protected] (M. Shanbedi). 0167-577X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2011.12.114
functionalized CNT in aqueous solutions, it is possible to study antimicrobial activity precisely. Fourier Transform Infrared Spectroscopy (FTIR), Thermo Gravimetric Analysis (TGA), Raman and Transmission Electron Microscope (TEM) were used to analyze surface functionality and morphology. Antimicrobial activity of CNT-Lysine was studied on three Gram-positive and Gram-negative bacteria by minimal inhibitory concentration (MIC).
2. Experimental 2.1. Functionalization and characterization instrument The mechanism for the diazonium reaction has been discussed previously [9–12]. First, lysine (400 mg) was mixed with dimethylacetamide (DMA) (20 ml) for 1 h at 60 °C. Then, pristine CNT (140 mg) and NaNO2 (188 mg) were sonicated with lysine in DMA. Concentrated H2SO4 (0.124 ml) was added and the mixture heated for 1 h at 60 °C [9]. To increase functionalization degree, the mixture was poured into a teflon reaction vessel (volume of 100 ml) and was placed under microwave reaction (Milestone-MicroSYNTH programmable) [12]. The abovementioned CNT suspension was heated in microwave up to 120 °C with output power of 700 W for 15 min. The resulted suspension was filtered and to remove unreacted lysine and NaNO2, the filter cake was thoroughly washed with DMA and methanol by five times, after that with abundant deionized water, and then dried in an oven for 48 h at 60 °C. Also, the pH of the washings was tested to ensure that no acid remained in the sample.
154
A. Amiri et al. / Materials Letters 72 (2012) 153–156
The FTIR spectra of powder-pressed samples were placed on KBr pellets and recorded on a Shimadzu 4300 spectrometer. The weight loss of powdered samples was measured at a heating rate of 10 °C/ min in air with TGA analyzer (TGA-50 Shimadzu). To prepare TEM samples, CNT were sonicated in ethanol solution for 10 min, and after that were dispersed on a holey carbon-coated copper grid. 2.2. Determination of MIC Here, the MIC value was defined as a concentration of CNT-Lysine/ CNT in which the absorbance of bacteria is half of the control [13]. Staphylococcus aureus (S. aureus), Streptococcus agalactiae (S. agalactiae), Streptococcus dysgalactiae (S. dysgalactiae), Escherichia coli (E. coli), Klebsiella pneumonia (K. pneumonia) and Salmonella typhimurium (S. typhimurium) were used for antimicrobial assays. To determine antimicrobial activity, the stock serial dilution of 0.01875 to 0.6 mg·ml − 1 of CNT-Lysine was prepared. Since pristine CNT doesn't form stable suspension in water, it was first sonicated for 1 h just before the tests. However, 20 μl of the each sample was added to 10 5 CFU·ml − 1 of bacteria and poured into a plate. The micro plate was incubated at 37 °C for 18 h. Next, the absorbance of wells at 630 nm was read using an ELISA reader (ELX800TM) and the results were compared with those of control. Experiments were performed with various bacteria in triplicates. 3. Results and discussion In this study, the functionalization procedure is based on diazonium reaction. It is likely that the diazonium salt receives an electron from the CNT, followed by liberating N2 and producing a semistable diazonium ion, which could react with the CNT sidewall [9–11]. Also, when CNT were exposed to microwave irradiation, strong absorptions are observed and severe heating was produced. The extended π-system of CNT authorizes conductivity to enable localized heating and therefore induced superheating point. The super heating point could potentially react with semi-stable diazonium ion [11,12]. Accordingly, FTIR, TGA, Raman and TEM were employed to analyze surface functionally groups. 3.1. Fourier transform infrared spectroscopy
3571
3.2. Thermogravimetric analysis TGA curve of the pristine CNT and CNT-Lysine is shown in Fig. 2. The curve of pristine CNT shows a small mass loss in a temperature range of 0–500 °C. On the other hand, CNT-Lysine curve shows two sudden mass losses. The first one (around 100 °C) is assigned to water, while the second one (in the range of 200–230 °C) is related to the decomposition of lysine. The decomposition temperature of lysine is around 215 °C, but it was shifted to a lower temperature. This phenomenon could occur by ejection of NH2 due to formation of the diazonium ion. 3.3. Transmission electron microscope and Raman Fig. 3 presents TEM images of the pristine CNT (Fig. 3a) and the CNT-Lysine (Fig. 3b and c). The pristine CNT show smooth surface (Fig. 3a) while increased roughness of the functionalized CNT surface is evident in TEM images of CNT-Lysine. In result of CNT functionalization, part of sp 2 hybridized carbons has been changed to sp 3 hybridization and it could damage graphitic CNT [11]. As another proof, the results of Raman spectroscopy are shown in Fig. 3d. The spectra of the samples exhibit D and G bands, at ~ 1274 and 1513 cm − 1 respectively. The intensity ratio of the D&G band (ID/IG) is utilized as an evidence of the disruption of aromatic πelectrons on the surface of CNT [11]. In functionalization researches,
1634 1554 1498 1423 1331
2304
3486 3198 2868
Fig. 1 shows the FTIR spectra of the CNT-Lysine and pristine CNT. The common peaks at 2304 cm − 1 could be related to the stretching
vibration of CNT backbone. Obviously, the FTIR spectra of CNTLysine sample explain clear cues of functionalities in contrast to the pristine CNT. However, in FTIR spectra of treated samples, the peak at 1554 cm − 1 corresponds with N\H bending vibration. Also, the medium peaks at 3571 cm − 1 and 3198 cm − 1 could be obtained from N\H stretching vibration, and the splitting is indicative of a primary amine [10]. The quite sharp peak at 1634 cm − 1 is attributable to C_O stretching vibration of carboxyl groups. Also, the appeared peak at 1331 cm − 1 is in agreement with stretching vibration of C\O. The peak at 3486 cm− 1 is assigned to O\H stretching vibration. The O\H peak can be generated due to adsorbed water by CNT-Lysine or hydroxyl groups of lysine. The peaks at 2868 cm− 1 and 1423 cm− 1 are related to C\H and C\N stretching vibration. A new peak at 1498 cm− 1 is attributed to the vibration of C\C group, which can be generated due to the interaction between amino groups in lysine and CNT and/or chain of lysine. This spectrum verifies the recognition of the lysine grafted onto the CNT.
100
(a)
90 80 70
(b) TGA %
60 50 40
CNT-Lysine
30
Pristine CNT
20 10 0 0
100
200
300
400
Temperature Fig. 1. FTIR spectra of (a) the pristine CNT, (b) the CNT-Lysine.
500
600
700
(oC)
Fig. 2. TGA analysis of the pristine CNT and functionalized CNT with lysine.
A. Amiri et al. / Materials Letters 72 (2012) 153–156
155
(b)
(a)
(d)
(c)
MWCNT-Lysine
Pristine MWCNT
500
1000
1500
2000
2500
3000
Fig. 3. (a) TEM image of the pristine CNT, (b,c) TEM image of CNT-Lysine and (d) Raman analysis of pristine and functionalized CNT.
3.4. Antimicrobial assay The MIC values of CNT-Lysine and pristine CNT on Gram-negative and Gram-positive bacteria are indicated in Fig. 4. One can see the functionalized CNT by lysine exhibited the strongest antimicrobial activity compared to the pristine CNT. MIC results shows that the antimicrobial activity of CNT-Lysine against Gram-negative bacteria E. coli, S. typhimurium and K. pneumonia respectively enhances by 3.23, 2.27 and 2.55 times compared to pristine CNT. Also, increases of 2.1, 1.84 and 1.80 times against Gram-positive bacteria S. agalactiae, S. aureus and S. dysgalactiae were observed respectively. The abovementioned data suggest that CNT-Lysine is more effective against Gram-negative bacteria. This is most likely attributable
to the different structures of cell walls of these bacteria [15,16]. The similar results were repeated in the CNT/epilson-polylysine nanocomposite by Zhou et al. [6]. 14
Minimum Inhibitory Conc.(µg / ml)
the higher (ID/IG) means the higher degree of covalent functionalization. As could be seen the ID/IG of CNT-Lysine is bigger than that of the pristine CNT. Meanwhile, stable concentration of CNT-Lysine in water was attained nearly 3.1 mg·ml − 1 in deionized water. A similar study was reported by Hu et al. [14]. Therefore, all characterization tests indicate that lysine groups are attached on the surface of CNT. Also, our results confirmed that output power of 700 W and 15 min of irradiation time was appropriate to perform reaction.
CNT-Lysine 12 Pristine CNT 10 8 6 4 2 0
Fig. 4. The MIC values of pristine CNT and CNT-Lysine against some Gram-negative and Gram-positive bacteria.
156
A. Amiri et al. / Materials Letters 72 (2012) 153–156
Since the ability of CNT to kill bacteria depends on direct interaction with the bacterial membrane, increased positive charge of CNT could improve attachment with negatively charged membranes of bacteria. The positive charge could increase permeability across the membrane and decrease metabolic activity and finally kill the bacteria [7,8]. Also, antimicrobial activity of CNT-Lysine is higher than some of treated CNT that investigated in another reports. 4. Conclusion An efficient and rapid technique to functionalize CNT by lysine under microwave irradiation was reported. A diazonium reaction occurred between lysine molecules and CNT, resulting in the attachment of lysine to the nanotube surface. Covalent functionalization procedure could significantly increase aqueous solubility, which is a critical criterion for various applications. Functionalization of CNT was confirmed by FTIR, TGA, Raman and TEM. MIC results confirmed strongest antimicrobial activity of CNTLysine compared to the pristine CNT. Also, improved antimicrobial activity is particularly obvious for the gram-negative bacteria. The more effective antimicrobial of CNT-Lysine was attributed to electrostatic adsorption of bacteria membrane, due to positive charges of the lysine groups on CNT. Acknowledgment The authors would like to thank Iran Nanotechnology Initiative Council of Iran for financial support. References [1] Aslan S, Loebick CZ, Kang S, Elimelech M, Pfefferle LD, Van Tassel PR. Antimicrobial biomaterials based on carbon nanotubes dispersed in poly(lactic-co-glycolic acid). Nanoscale 2010;2:1789–94.
[2] Espinosa-Cristóbal LF, Martínez-Castañón GA, Martínez-Martínez RE, LoyolaRodríguez JP, Patiño-Marín N, Reyes-Macías JF, et al. Antibacterial effect of silver nanoparticles against Streptococcus mutans. Mater Lett 2009;63:2603–6. [3] Liu S, Ng AK, Xu R, Wei J, Tan CM, Yang Y, et al. Antibacterial action of dispersed single-walled carbon nanotubes on Escherichia coli and Bacillus subtilis investigated by atomic force microscopy. Nanoscale 2010;2:2744–50. [4] Kang S, Pinault M, Pfefferle LD, Elimelech M. Single-walled carbon nanotubes exhibit strong antimicrobial activity. Langmuir 2007;23:8670–3. [5] Kang S, Herzberg M, Rodrigues DF, Elimelech M. Antibacterial effects of carbon nanotubes: size does matter! Langmuir 2008;24:6409–13. [6] Zhou J, Qi X. Multi-walled carbon nanotubes/epilson-polylysine nanocomposite with enhanced antibacterial activity. Lett Appl Microbiol 2011;52:76–83. [7] Mohan R, Shanmugharaj AM, Sung Hun R. An efficient growth of silver and copper nanoparticles on multiwalled carbon nanotube with enhanced antimicrobial activity. J Biomed Mater Res 2011;96B:119–26. [8] Ernst WA, Thoma-Uszynski S, Teitelbaum R, Ko C, Hanson DA, Clayberger C, et al. Granulysin, a T cell product, kills bacteria by altering membrane permeability. J Immunoassay 2000;165:7102–8. [9] Chidawanyika W, Nyokong T. Characterization of amine-functionalized singlewalled carbon nanotube-low symmetry phthalocyanine conjugates. Carbon 2010;48:2831–8. [10] Ellison MD, Gasda PJ. Functionalization of single-walled carbon nanotubes with 1,4-Benzenediamine using a diazonium reaction. J Phys Chem C 2007;112: 738–40. [11] Amiri A, Maghrebi M, Baniadam M, Zeinali Heris S. One-pot, efficient functionalization of multi-walled carbon nanotubes with diamines by microwave method. Appl Surf Sci 2011;257:10261–6. [12] Vazquez E, Prato M. Carbon nanotubes and microwaves: interactions, responses, and applications. ACS Nano 2009;3:3819–24. [13] Jin L-L, Li Q, Song S-S, Feng K, Zhang D-B, Wang Q-Y, et al. Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii. Comp Biochem Phys B 2009;154:174–8. [14] Hu N, Dang G, Zhou H, Jing J, Chen C. Efficient direct water dispersion of multiwalled carbon nanotubes by functionalization with lysine. Mater Lett 2007;61: 5285–7. [15] Huang KC, Mukhopadhyay R, Wen B, Gitai Z, Wingreen NS. Cell shape and cellwall organization in Gram-negative bacteria. Proc Natl Acad Sci U S A 2008;105: 19282–7. [16] Prokhorenko IR, Zubova SV, Ivanov AY, Grachev SV. Interaction of gram-negative bacteria with cationic proteins: dependence on the surface characteristics of the bacterial cell. Int J Gen Med 2009;2:33–8.