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One-step functionalization of gold and silver nanoparticles by ampicillin
Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Materials Letters journal homepage: www.elsevier.com/locate/matlet
One-step functionalization of gold and silver nanoparticles by ampicillin Ye Eun Hur a,1, Sojung Kim a,1, Jae-Hyun Kim b, Song-Hyun Cha b, Myung-Jin Choi b, Seonho Cho b, Youmie Park a,b,n a
College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, Republic of Korea National Creative Research Initiatives (NCRI) Center for Isogeometric Optimal Design, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea
b
art ic l e i nf o
a b s t r a c t
Article history: Received 20 December 2013 Accepted 3 May 2014
This report describes a one-step functionalization of gold and silver nanoparticles with ampicillin in which ampicillin is also used as a reducing agent to convert Au3 þ and Ag þ to gold and silver nanoparticles, respectively. This process eliminates the use of other chemical reducing agents to provide a completely green synthetic route. The nanostructure, morphology, crystallinity, and reaction yield of the nanoparticles were investigated by spectroscopic and microscopic techniques. Spherical and amorphous nanoparticles were synthesized with diameters of 10–33 nm. Remarkably, the newlyprepared NPs exert excellent antibacterial activities against Streptococcus pyogenes (minimum inhibitory concentration 0.14–1.09 μg/mL). This system enables the straightforward functionalization of gold and silver nanoparticles with ampicillin using a simple, one-step process. & 2014 Published by Elsevier B.V.
Keywords: Ampicillin Gold nanoparticles Silver nanoparticles One-step process Antibacterial activity Streptococcus pyogenes
1. Introduction The advent of multidrug resistant (MDR) bacteria has necessitated the development of novel categories of antibiotics that effectively block or subvert bacterial growth. Metallic nanoparticles are an effective platform for antibacterial nanotechnology. In particular, silver nanoparticles (AgNPs) have shown promise as bactericidal agents for MDR bacteria [1–4]. Gold nanoparticles (AuNPs) and AuNPs functionalized/mixed with antibiotics are also effective antibacterial agents and enhance the antibacterial activity of antibiotics [5–11]. Brown et al. have reported that the functionalization of AuNPs and AgNPs with ampicillin destroys MDR bacteria [12]. They prepared citrate-stabilized NPs using NaBH4 as a reducing agent and then mixed the NPs with ampicillin to generate ampicillin-functionalized NPs [12]. Kora et al. observed an enhancement of the antibacterial activity of AgNPs when they were combined with streptomycin, ampicillin, or tetracycline [13]. The combination of ampicillin with biogenic AgNPs synthesized by the fungus Trichoderma viride yielded a synergistic effect [14]. In these previous studies [12–14], the authors performed the
n Corresponding author at: College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, Republic of Korea. Tel.: þ 82 55 320 3884; fax: þ 82 55 320 3940. E-mail address: [email protected] (Y. Park). 1 These authors contributed equally to this work.
experiments in two separate steps: preparation of the NPs and functionalization of the NPs with antibiotics. In the present work, we develop a one-step synthesis of ampicillin–functionalized AuNPs and AgNPs (referred to hereafter as ampicillin–AuNPs and ampicillin–AgNPs). In this one-step synthesis, ampicillin functions as the reducing agent, thereby enabling a greener process by avoiding the use of other chemical reducing or capping agents. The ampicillin–functionalized NPs were characterized by inductively coupled plasma mass spectrometry (ICP-MS), Fourier transform infrared spectroscopy (FTIR), UV–visible spectrophotometry, high-resolution X-ray diffraction (HR-XRD), high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and field emission scanning electron microscopy (FE-SEM).
2. Experimental Materials and instruments: Ampicillin trihydrate, silver nitrate, Q4 and hydrochloroauric acid trihydrate (HAuCl4 3H2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade. AFM images were acquired with a Dimensions Icons (Bruker Nano, Santa Barbara, CA, USA) operated in tapping mode. A Shimadzu UV-1800 was used to acquire UV–visible spectra (Shimadzu Corporation, Kyoto, Japan). A JEM3010 (JEOL, Tokyo, Japan) at 300 kV was used to obtain HR-TEM
http://dx.doi.org/10.1016/j.matlet.2014.05.032 0167-577X/& 2014 Published by Elsevier B.V.
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
Y. Eun Hur et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
3. Results and discussion Functionalization and yield of AuNPs and AgNPs: Surface plasmon resonance bands emerged at 527 nm with a pink color for the ampicillin–AuNPs and at 390 nm with a yellow-green color for the ampicillin–AgNPs (Fig. 1A). The reaction yield was 96.7% and 94.1% for the ampicillin–AuNPs and ampicillin–AgNPs, respectively, as determined by ICP-MS. FT-IR spectra: As depicted in Fig. 1B, the FT-IR spectra changed greatly upon the synthesis of the NPs. The intense band for ampicillin at 1774 cm 1 disappeared completely, suggesting that the C ¼ O functional group on ampicillin contributed to the synthesis of the NPs. The stretching vibration of the primary and secondary amines was observed at 3509 cm 1 and 3446 cm 1 for the ampicillin standard. These two bands merged, broadened, and shifted to 3418 cm 1 and 3436 cm 1 during the synthesis of the ampicillin–AuNPs and ampicillin–AgNPs, respectively, indicating that the amine functional group was involved in the synthesis and/or was complexed to the surface of the NPs. HR-XRD analyses: As shown in Fig. 1C, the diffraction peaks of the ampicillin–AuNPs at 38.31, 44.31, 64.81, and 77.61 represent the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the Au crystal. The (1 1 1) plane of the ampicillin–AgNPs was also observed at 38.11, confirming the crystallinity of the AgNPs. HR-TEM images and size histogram: HR-TEM revealed spherical ampicillin–AuNPs with an average diameter of 18.717 2.90 nm among 51 discrete NPs. Large and small ampicillin–AgNPs with diameters of 33.967 4.96 nm from 20 discrete NPs and 10.82 7 3.06 nm from 36 discrete NPs, respectively, were observed. Both spherical and amorphous AgNPs were observed. AFM and FE-SEM images: As shown in Fig. 4A–C, AFM revealed spherical ampicillin–AuNPs. Spherical and amorphous ampicillin– AgNPs were clearly visualized in the AFM images (Fig. 4D–F). The AFM images correlated well with the HR-TEM images. The FE-SEM images also provided information about the shapes of the NPs (Fig. 5). Antibacterial activities: Interestingly, the antibacterial activity of NPs against Gram-positive bacteria was higher than that of Gram-negative bacteria (Table 1). In addition, ampicillin–AgNPs
0.7 527 nm
0.6 390 nm
a
0.5 Absorbance
images. A JSM-7100F SEM with an accelerating voltage of 15 kV was used to collect FE-SEM images (JEOL, Tokyo, Japan). An ELAN 6100 (Perkin-Elmer SCIEX, Waltham, MA, USA) was used for ICPMS. To obtain the supernatant containing unreacted Au3 þ or Ag þ for ICP-MS analysis, the NPs were subjected to ultracentrifugation in an Eppendorf centrifuge 5424R (Eppendorf AG, Hamberg, Germany) at 21,000 6100 (Perkin-Elmer SCIEX, Waltham, MA 6100 (Perkin-Elmer SCIEX, Waltham, MA 6100 (Perkin-Elmer SCIEX, Waltham, MA g for 30 min at 21 1C for AuNPs and 21,130 g for 45 min at 18 1C for AgNPs. A high-resolution X-ray diffractometer (Bruker D8 Discover, Bruker, Germany) with a CuKα radiation source (λ¼ 0.1541 nm) was used to obtain HR-XRD data. FT-IR spectra were recorded on a Varian 640-IR using the KBr pellet method (Agilent Technologies, Santa Clara, USA). Functionalization of AuNPs and AgNPs by ampicillin: Ampicillin– AuNPs were prepared by mixing a HAuCl4 3H2O solution (1 mM in deionized water, 150 mL) with ampicillin (1 mM in deionized water, 50 mL). To prepare ampicillin–AgNPs, a silver nitrate solution (1 mM in deionized water, 300 mL) was mixed with ampicillin (0.5 mM in deionized water, 100 mL). The final volume of each mixture was adjusted to 1 mL with deionized water. The reaction was conducted in an 80 1C oven for 15 h (ampicillin–AuNPs) or 24 h (ampicillin–AgNPs). For antibacterial activities, twenty strains of Gram-positive and Gram-negative bacteria were utilized. Minimum inhibitory concentration (MIC) values were measured according to our previous report [15].
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Fig. 1. (A) UV–visible spectra: (a) ampicillin–AuNPs before oven incubation; (b) ampicillin–AuNPs after oven incubation; (c) ampicillin–AgNPs before oven incubation; (d) ampicillin–AgNPs after oven incubation. (B) FT-IR analyses: (a) ampicillin; (b) ampicillin–AuNPs; (c) ampicillin–AgNPs. (C) HR-XRD analyses: (a) ampicillin–AuNPs; (b) ampicillin–AgNPs. The asterisk (n) indicates the presence of contaminants.
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
Y. Eun Hur et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Percentage (%)
average diameter : 18.71 ± 2.90 (nm)
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Diameter (nm) Fig. 2. HR-TEM images of ampicillin–AuNPs. The scale bar represents (A) 50 nm, (B) 10 nm, and (C) 5 nm. The size histogram is shown in (D).
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Fig. 3. HR-TEM images of ampicillin–AgNPs. The scale bar represents (A) 50 nm and (B) 5 nm. The size histograms are shown in (C) and (D).
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Y. Eun Hur et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 4. AFM images of ampicillin-AuNPs (A)–(C) and ampicillin–AgNPs (D)–(F). (A) 2-D height (500 nm 500 nm), (B) 3-D height (500 nm 500 nm), (C) 3-D phase (500 nm 500 nm), (D) 3-D height (2.5 mm 2.5 mm), (E) 3-D amplitude error (1 mm 1 mm), and (F) 3-D phase (500 nm 500 nm).
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
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Fig. 5. FE-SEM images of ampicillin–AuNPs (A) and (B) and ampicillin–AgNPs (C) and (D). The magnifications of the images are (A) 65,000 , (B) 45,000 , (C) 110,000 , and (D) 450,000 . Table 1 Antibacterial activity of ampicillin–AuNPs and ampicillin–AgNPs. MIC values were calculated based on the ampicillin concentration. Strain name Staphylococcus Staphylococcus Staphylococcus Streptococcus Streptococcus Streptococcus Pseudomonas Pseudomonas Pseudomonas Pseudomonas Escherichia Escherichia Escherichia Escherichia Escherichia Salmonella Klebsiella Klebsiella Enterobacter Enterobacter Escherichia Streptococcus a
aureus SG511 aureus 285 aureus 503 pyogenes 308A pyogenes T12A pyogenes 77A aeruginosa 9027 aeruginosa 1592E aeruginosa 1771 aeruginosa 1771M coli 078 coli TEM coli 1507 coli DC0 coli DC2 typhimurium oxytoca 1082E aerogenes 1522E cloacae P99 cloacae 1321E coli pneumoniae
CCARM no.
Ampicillin–AuNPs (μg/ml)
Ampicillin–AgNPs (μg/ml)
NOR (μg/ml)a
0201 0204 0205 0206 0207 0208 0219 0223 0224 0225 0230 0235 0236 0237 0238 0240 0248 0249 0252 0253 0010 0031
4.37 4 8.74 4 8.74 0.55 1.09 0.55 4 8.74 4 8.74 4 8.74 2.18 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4.37
1.09 8.74 8.74 0.14 0.27 0.27 48.74 48.74 48.74 2.18 48.74 48.74 48.74 48.74 4.37 4.37 48.74 48.74 48.74 4.37 48.74 1.09
r 0.25 1 1 8 1 1 1 0.5 1 0.5 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 2
Norfloxacin was used as a standard antibiotic.
was slightly more effective compared to ampicillin–AuNPs on Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Salmonella typhimurium, Enterobacter cloacae and Streptococcus
pneumonia. The lowest MIC values were observed on Streptococcus pyogenes 308A (0.14 μg/mL) which is far lower than that of a standard antibiotic, norfloxacin (8 μg/mL).
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
Y. Eun Hur et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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4. Conclusions This study reports the facile synthesis of ampicillin–functionalized AuNPs and AgNPs by a one-step process that requires no additional chemicals as reducing agents. Among the tested Grampositive and Gram-negative bacteria, the newly-prepared NPs exert the highest antibacterial activities against S. pyogenes Figs. 2 and 3. Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean government: the Ministry of Education (NRF-2012R1A1A2042224) and the Ministry of Science, ICT & Future Planning (NRF-2010–18282). The authors would also like to thank Ms. Sang Hui Jun for assistance with the preparation of this manuscript.
[2] Pelgrift RY, Friedman AJ. Adv Drug Deliv Rev 2013;65:1803–15. [3] Naqvi SZ, Kiran U, Ali MI, Jamal A, Hameed A, Ahmed S, et al. Int J Nanomed 2013;8:3187–95. [4] Rai MK, Deshmukh SD, Ingle AP, Gade AK. J Appl Microbiol 2012;112:841–52. [5] Badwaik VD, Willis CB, Pender DS, Paripelly R, Shah M, Kherde YA, et al. J Biomed Nanotechnol 2013;9:1716–23. [6] Pender DS, Vangala LM, Badwaik VD, Willis CB, Aguilar ZP, Sangoi TN, et al. Front Biosci 2013;18:993–1002. [7] Annamalai A, Christina VL, Sudha D, Kalpana M, Lakshmi PT. Colloids Surf B Biointerfaces 2013;108:60–5. [8] Burygin GL, Khlebtsov BN, Shantrokha AN, Dykman LA, Bogatyrev VA, Khlebtsov NG. Nanoscale Res Lett 2009;4:794–801. [9] Gu H, Ho PL, Tong E, Wang L, Xu B. Nano Lett 2003;3:1261–3. [10] Pissuwan D, Cortie CH, Valenzuela SM, Cortie MB. Trends Biotechnol 2010;28:207–13. [11] Nirmala Grace A, Pandian K. Colloids Surf A: Physicochem Eng Asp 2007;297:63–70. [12] Brown AN, Smith K, Samuels TA, Lu J, Obare SO, Scott ME. Appl Environ Microbiol 2012;78:2768–74. [13] Kora AJ, Rastogi L. Bioinorg Chem Appl 2013;2013:871097. [14] Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Nanomed: Nanotechnol Biol Med 2010;6:103–9. [15] Im AR, Han L, Kim ER, Kim J, Kim YS, Park Y. Phytother Res 2012;26:1249–55.
References [1] Lara HH, Garza-Treviño EN, Ixtepan-Turrent L, Singh DK. J Nanobiotechnol 2011;9:30.
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
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Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
One-step functionalization of gold and silver nanoparticles by ampicillin Ye Eun Hur a,1, Sojung Kim a,1, Jae-Hyun Kim b, Song-Hyun Cha b, Myung-Jin Choi b, Seonho Cho b, Youmie Park a,b,n a
College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, Republic of Korea National Creative Research Initiatives (NCRI) Center for Isogeometric Optimal Design, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea
b
art ic l e i nf o
a b s t r a c t
Article history: Received 20 December 2013 Accepted 3 May 2014
This report describes a one-step functionalization of gold and silver nanoparticles with ampicillin in which ampicillin is also used as a reducing agent to convert Au3 þ and Ag þ to gold and silver nanoparticles, respectively. This process eliminates the use of other chemical reducing agents to provide a completely green synthetic route. The nanostructure, morphology, crystallinity, and reaction yield of the nanoparticles were investigated by spectroscopic and microscopic techniques. Spherical and amorphous nanoparticles were synthesized with diameters of 10–33 nm. Remarkably, the newlyprepared NPs exert excellent antibacterial activities against Streptococcus pyogenes (minimum inhibitory concentration 0.14–1.09 μg/mL). This system enables the straightforward functionalization of gold and silver nanoparticles with ampicillin using a simple, one-step process. & 2014 Published by Elsevier B.V.
Keywords: Ampicillin Gold nanoparticles Silver nanoparticles One-step process Antibacterial activity Streptococcus pyogenes
1. Introduction The advent of multidrug resistant (MDR) bacteria has necessitated the development of novel categories of antibiotics that effectively block or subvert bacterial growth. Metallic nanoparticles are an effective platform for antibacterial nanotechnology. In particular, silver nanoparticles (AgNPs) have shown promise as bactericidal agents for MDR bacteria [1–4]. Gold nanoparticles (AuNPs) and AuNPs functionalized/mixed with antibiotics are also effective antibacterial agents and enhance the antibacterial activity of antibiotics [5–11]. Brown et al. have reported that the functionalization of AuNPs and AgNPs with ampicillin destroys MDR bacteria [12]. They prepared citrate-stabilized NPs using NaBH4 as a reducing agent and then mixed the NPs with ampicillin to generate ampicillin-functionalized NPs [12]. Kora et al. observed an enhancement of the antibacterial activity of AgNPs when they were combined with streptomycin, ampicillin, or tetracycline [13]. The combination of ampicillin with biogenic AgNPs synthesized by the fungus Trichoderma viride yielded a synergistic effect [14]. In these previous studies [12–14], the authors performed the
n Corresponding author at: College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, Republic of Korea. Tel.: þ 82 55 320 3884; fax: þ 82 55 320 3940. E-mail address: [email protected] (Y. Park). 1 These authors contributed equally to this work.
experiments in two separate steps: preparation of the NPs and functionalization of the NPs with antibiotics. In the present work, we develop a one-step synthesis of ampicillin–functionalized AuNPs and AgNPs (referred to hereafter as ampicillin–AuNPs and ampicillin–AgNPs). In this one-step synthesis, ampicillin functions as the reducing agent, thereby enabling a greener process by avoiding the use of other chemical reducing or capping agents. The ampicillin–functionalized NPs were characterized by inductively coupled plasma mass spectrometry (ICP-MS), Fourier transform infrared spectroscopy (FTIR), UV–visible spectrophotometry, high-resolution X-ray diffraction (HR-XRD), high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and field emission scanning electron microscopy (FE-SEM).
2. Experimental Materials and instruments: Ampicillin trihydrate, silver nitrate, Q4 and hydrochloroauric acid trihydrate (HAuCl4 3H2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade. AFM images were acquired with a Dimensions Icons (Bruker Nano, Santa Barbara, CA, USA) operated in tapping mode. A Shimadzu UV-1800 was used to acquire UV–visible spectra (Shimadzu Corporation, Kyoto, Japan). A JEM3010 (JEOL, Tokyo, Japan) at 300 kV was used to obtain HR-TEM
http://dx.doi.org/10.1016/j.matlet.2014.05.032 0167-577X/& 2014 Published by Elsevier B.V.
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
Y. Eun Hur et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
3. Results and discussion Functionalization and yield of AuNPs and AgNPs: Surface plasmon resonance bands emerged at 527 nm with a pink color for the ampicillin–AuNPs and at 390 nm with a yellow-green color for the ampicillin–AgNPs (Fig. 1A). The reaction yield was 96.7% and 94.1% for the ampicillin–AuNPs and ampicillin–AgNPs, respectively, as determined by ICP-MS. FT-IR spectra: As depicted in Fig. 1B, the FT-IR spectra changed greatly upon the synthesis of the NPs. The intense band for ampicillin at 1774 cm 1 disappeared completely, suggesting that the C ¼ O functional group on ampicillin contributed to the synthesis of the NPs. The stretching vibration of the primary and secondary amines was observed at 3509 cm 1 and 3446 cm 1 for the ampicillin standard. These two bands merged, broadened, and shifted to 3418 cm 1 and 3436 cm 1 during the synthesis of the ampicillin–AuNPs and ampicillin–AgNPs, respectively, indicating that the amine functional group was involved in the synthesis and/or was complexed to the surface of the NPs. HR-XRD analyses: As shown in Fig. 1C, the diffraction peaks of the ampicillin–AuNPs at 38.31, 44.31, 64.81, and 77.61 represent the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the Au crystal. The (1 1 1) plane of the ampicillin–AgNPs was also observed at 38.11, confirming the crystallinity of the AgNPs. HR-TEM images and size histogram: HR-TEM revealed spherical ampicillin–AuNPs with an average diameter of 18.717 2.90 nm among 51 discrete NPs. Large and small ampicillin–AgNPs with diameters of 33.967 4.96 nm from 20 discrete NPs and 10.82 7 3.06 nm from 36 discrete NPs, respectively, were observed. Both spherical and amorphous AgNPs were observed. AFM and FE-SEM images: As shown in Fig. 4A–C, AFM revealed spherical ampicillin–AuNPs. Spherical and amorphous ampicillin– AgNPs were clearly visualized in the AFM images (Fig. 4D–F). The AFM images correlated well with the HR-TEM images. The FE-SEM images also provided information about the shapes of the NPs (Fig. 5). Antibacterial activities: Interestingly, the antibacterial activity of NPs against Gram-positive bacteria was higher than that of Gram-negative bacteria (Table 1). In addition, ampicillin–AgNPs
0.7 527 nm
0.6 390 nm
a
0.5 Absorbance
images. A JSM-7100F SEM with an accelerating voltage of 15 kV was used to collect FE-SEM images (JEOL, Tokyo, Japan). An ELAN 6100 (Perkin-Elmer SCIEX, Waltham, MA, USA) was used for ICPMS. To obtain the supernatant containing unreacted Au3 þ or Ag þ for ICP-MS analysis, the NPs were subjected to ultracentrifugation in an Eppendorf centrifuge 5424R (Eppendorf AG, Hamberg, Germany) at 21,000 6100 (Perkin-Elmer SCIEX, Waltham, MA 6100 (Perkin-Elmer SCIEX, Waltham, MA 6100 (Perkin-Elmer SCIEX, Waltham, MA g for 30 min at 21 1C for AuNPs and 21,130 g for 45 min at 18 1C for AgNPs. A high-resolution X-ray diffractometer (Bruker D8 Discover, Bruker, Germany) with a CuKα radiation source (λ¼ 0.1541 nm) was used to obtain HR-XRD data. FT-IR spectra were recorded on a Varian 640-IR using the KBr pellet method (Agilent Technologies, Santa Clara, USA). Functionalization of AuNPs and AgNPs by ampicillin: Ampicillin– AuNPs were prepared by mixing a HAuCl4 3H2O solution (1 mM in deionized water, 150 mL) with ampicillin (1 mM in deionized water, 50 mL). To prepare ampicillin–AgNPs, a silver nitrate solution (1 mM in deionized water, 300 mL) was mixed with ampicillin (0.5 mM in deionized water, 100 mL). The final volume of each mixture was adjusted to 1 mL with deionized water. The reaction was conducted in an 80 1C oven for 15 h (ampicillin–AuNPs) or 24 h (ampicillin–AgNPs). For antibacterial activities, twenty strains of Gram-positive and Gram-negative bacteria were utilized. Minimum inhibitory concentration (MIC) values were measured according to our previous report [15].
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80
Fig. 1. (A) UV–visible spectra: (a) ampicillin–AuNPs before oven incubation; (b) ampicillin–AuNPs after oven incubation; (c) ampicillin–AgNPs before oven incubation; (d) ampicillin–AgNPs after oven incubation. (B) FT-IR analyses: (a) ampicillin; (b) ampicillin–AuNPs; (c) ampicillin–AgNPs. (C) HR-XRD analyses: (a) ampicillin–AuNPs; (b) ampicillin–AgNPs. The asterisk (n) indicates the presence of contaminants.
Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i
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Percentage (%)
average diameter : 18.71 ± 2.90 (nm)
30
20
10
0 ~15
15~17 17~19 19~21 21~23
23~
Diameter (nm) Fig. 2. HR-TEM images of ampicillin–AuNPs. The scale bar represents (A) 50 nm, (B) 10 nm, and (C) 5 nm. The size histogram is shown in (D).
50
25
average diameter : 10.82 ± 3.06 (nm)
Percentage (%)
average diameter : 33.96 ± 4.96 (nm)
Percentage (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
3
20 15 10 5
40 30 20 10 0
0 26~28 28~30 30~32 32~34 34~36 36~38
Diameter (nm)
38~
4~6
6~8
8~10 10~12 12~14
14~
Diameter (nm)
Fig. 3. HR-TEM images of ampicillin–AgNPs. The scale bar represents (A) 50 nm and (B) 5 nm. The size histograms are shown in (C) and (D).
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
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Fig. 4. AFM images of ampicillin-AuNPs (A)–(C) and ampicillin–AgNPs (D)–(F). (A) 2-D height (500 nm 500 nm), (B) 3-D height (500 nm 500 nm), (C) 3-D phase (500 nm 500 nm), (D) 3-D height (2.5 mm 2.5 mm), (E) 3-D amplitude error (1 mm 1 mm), and (F) 3-D phase (500 nm 500 nm).
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
5
Fig. 5. FE-SEM images of ampicillin–AuNPs (A) and (B) and ampicillin–AgNPs (C) and (D). The magnifications of the images are (A) 65,000 , (B) 45,000 , (C) 110,000 , and (D) 450,000 . Table 1 Antibacterial activity of ampicillin–AuNPs and ampicillin–AgNPs. MIC values were calculated based on the ampicillin concentration. Strain name Staphylococcus Staphylococcus Staphylococcus Streptococcus Streptococcus Streptococcus Pseudomonas Pseudomonas Pseudomonas Pseudomonas Escherichia Escherichia Escherichia Escherichia Escherichia Salmonella Klebsiella Klebsiella Enterobacter Enterobacter Escherichia Streptococcus a
aureus SG511 aureus 285 aureus 503 pyogenes 308A pyogenes T12A pyogenes 77A aeruginosa 9027 aeruginosa 1592E aeruginosa 1771 aeruginosa 1771M coli 078 coli TEM coli 1507 coli DC0 coli DC2 typhimurium oxytoca 1082E aerogenes 1522E cloacae P99 cloacae 1321E coli pneumoniae
CCARM no.
Ampicillin–AuNPs (μg/ml)
Ampicillin–AgNPs (μg/ml)
NOR (μg/ml)a
0201 0204 0205 0206 0207 0208 0219 0223 0224 0225 0230 0235 0236 0237 0238 0240 0248 0249 0252 0253 0010 0031
4.37 4 8.74 4 8.74 0.55 1.09 0.55 4 8.74 4 8.74 4 8.74 2.18 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4 8.74 4.37
1.09 8.74 8.74 0.14 0.27 0.27 48.74 48.74 48.74 2.18 48.74 48.74 48.74 48.74 4.37 4.37 48.74 48.74 48.74 4.37 48.74 1.09
r 0.25 1 1 8 1 1 1 0.5 1 0.5 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 r 0.25 2
Norfloxacin was used as a standard antibiotic.
was slightly more effective compared to ampicillin–AuNPs on Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Salmonella typhimurium, Enterobacter cloacae and Streptococcus
pneumonia. The lowest MIC values were observed on Streptococcus pyogenes 308A (0.14 μg/mL) which is far lower than that of a standard antibiotic, norfloxacin (8 μg/mL).
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1 2 3 4 5 6 7 Q5 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
4. Conclusions This study reports the facile synthesis of ampicillin–functionalized AuNPs and AgNPs by a one-step process that requires no additional chemicals as reducing agents. Among the tested Grampositive and Gram-negative bacteria, the newly-prepared NPs exert the highest antibacterial activities against S. pyogenes Figs. 2 and 3. Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean government: the Ministry of Education (NRF-2012R1A1A2042224) and the Ministry of Science, ICT & Future Planning (NRF-2010–18282). The authors would also like to thank Ms. Sang Hui Jun for assistance with the preparation of this manuscript.
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Please cite this article as: Eun Hur Y, et al. One-step functionalization of gold and silver nanoparticles by ampicillin. Mater Lett (2014), http://dx.doi.org/10.1016/j.matlet.2014.05.032i