1 23

Journal of Nanoparticle ResearchAn Interdisciplinary Forum forNanoscale Science and Technology ISSN 1388-0764Volume 16Number 4 J Nanopart Res (2014) 16:1-14DOI 10.1007/s11051-014-2346-x

Fabrication and characterization ofantibacterial nanoparticles supported onhierarchical hybrid substrates

Anil K. Karumuri, AdamA. Maleszewski, Dhawal P. Oswal,Heather A. Hostetler & SharmilaM. Mukhopadhyay

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media Dordrecht. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

RESEARCH PAPER

Fabrication and characterization of antibacterialnanoparticles supported on hierarchical hybrid substrates

Anil K. Karumuri • Adam A. Maleszewski •

Dhawal P. Oswal • Heather A. Hostetler •

Sharmila M. Mukhopadhyay

Received: 3 October 2013 / Accepted: 1 March 2014

� Springer Science+Business Media Dordrecht 2014

Abstract The effectiveness of many nanomaterial-

based devices depends upon their available surface

area. Isolated nanoparticles (NPs) can offer high-

surface area, but are prone to environmental loss and

pollution. Whereas those supported on solid substrates

are limited by the specific surface area (SSA) of the

support. The SSA limitation of traditional supports can

be addressed by attaching NPs on specially designed

hierarchical structures having unusually high SSA,

thereby maximizing the nanomaterial advantage with-

out the risks of using loose nano-powders. In this

research, hierarchical structures were fabricated by

grafting carbon nanotubes (CNT) on carbon and

subsequently decorated with strongly attached silver

nanoparticles (AgNP) via controlled reduction of

silver salts in the presence of reducing and capping

agents. Microstructure characterization revealed that

along with other processing parameters, reduction

temperature can be used to control NP morphology.

For this substrate morphology, fine and uniformly

dispersed AgNP were obtained at 60 �C, whereas

significant particle coalescence and increase in parti-

cle size occurred at 80 �C. Mechanical durability of

AgNP–CNT attachments on the substrate was tested in

harsh ultrasonic conditions and found to be impres-

sive, with no detectable AgNP loss even when the

larger substrate begins to fail. The antibacterial

effectiveness of these structures was tested in multiple

testing modes against Gram-negative Escherichia coli

(E. coli, JM109). It was seen in each case that AgNP

attached on CNT-grafted hierarchical substrates

showed significantly higher reduction of E. coli com-

pared to AgNP attached directly on the starting porous

supports without CNT grafting. These results indicate

that AgNP attached to hierarchal hybrid supports can

lead to compact and powerful antibacterial devices for

chemical-free disinfection devices of the future.

Keywords Hierarchical structures � Carbon

nanotubes � Silver nanoparticles � Antibacterial

properties � Health effects

Introduction

Silver (Ag) is a known antibacterial element for many

centuries and has been extensively used for antibac-

terial applications in textiles (Jiang et al. 2005),

cosmetics (Kokura et al. 2010), surgical tools (Lee

A. K. Karumuri (&) � A. A. Maleszewski �S. M. Mukhopadhyay

Department of Mechanical and Materials Science

Engineering, Center for Nanoscale Multifunctional

Materials, Wright State University, Dayton, OH 45435,

USA

e-mail: karumuri.2@wright.edu

D. P. Oswal � H. A. Hostetler

Department of Biochemistry and Molecular Biology,

Wright State University, Dayton, OH 45435, USA

123

J Nanopart Res (2014) 16:2346

DOI 10.1007/s11051-014-2346-x

Author's personal copy

et al. 2007; Eby et al. 2009), and water treatment

applications (Davies 1997). It is also known that

antibacterial effectiveness of any Ag-based devices is

greatly influenced by the amount of Ag surface area

available for interaction with environmental microbes.

In the past few years, nanoparticles of silver (AgNP)

have gained considerable attention owing to their high

ratio of surface to volume (specific surface area or

SSA). However, standalone AgNP can cause handling

problems, material loss, and environmental safety

issues when they are deployed for treatment. Nano-

particles (NPs) in the environment can enter the

human body through multiple routes (Mukhopadhyay

2012) and it is observed that long-term accumulation

of Ag in the human body can cause health problems

(Asharani et al. 2008; Wijnhoven et al. 2009).

Hazards of AgNP contamination, both health and

environmental, can be greatly reduced by attaching

these particles to larger substrates (Jain and Pradeep

2005; Lv et al. 2009; Dankovich and Gray 2011;

Mpenyana-Monyatsi et al. 2012; Rathnayake et al.

2012). The limiting factor in these cases is the SSA,

the support can offer. Porous supports having higher

SSA than flat surfaces are often used for surface-

sensitive applications and their SSA can be increased

to some extent by controlling structural parameters

such as increasing percentage porosity and decreasing

pore dimensions (Mukhopadhyay et al. 2009). How-

ever, the SSA improvements using such measures

often pose other problems. For instance, higher

porosities result in lower mechanical strengths and

structural instability, effectively making the structures

weaker. For applications requiring fluid permeability,

very small pore sizes impart extremely high pressure

drops across the material. Such problems can be

overcome by enhancing structurally adequate porous

structures with ‘‘nano-hair’’ like coatings which

increase the SSA without compromising the structural

integrity (Mukhopadhyay et al. 2009).

In this research, we have selected carbon-based

porous structures due to their low density, chemical

stability, and high thermal and electrical conductivi-

ties. The conductivity properties make this substrate

attractive for future applications since it is already

known that antibacterial efficacy of the AgNP would

increase with application of heat and electric field

(Zhu et al. 2002). In an earlier paper (Karumuri et al.

2013), it was shown that AgNP deposited on com-

mercial carbon foam can be useful for reducing

bacteria in water. This study extends that concept to

develop significantly more powerful devices, wherein

dense arrays of carbon nanotubes (CNT) have been

grafted on porous carbon foams to create a multi-scale

hierarchical morphology. SSA of these CNT-grafted

structures is known to be several orders of magnitude

higher than their as-received counterparts, even in the

simplest of devices (Mukhopadhyay et al. 2009;

Barney et al. 2012a). This approach provides the

additional advantage that the surface area, permeabil-

ity, and other properties can be tuned in future by

controlling the coverage density, morphology, and

length of the attached CNTs. Some other applications

that have already benefited from this approach are bio

sensing (Maurer et al. 2012), cell scaffolding (Maurer

et al. 2011), thermal transport (Barney et al. 2012b),

and degradation of volatile solvents in water (Vijwani

et al. 2012). In this study, the CNT-grafted hierarchi-

cal structures serve as host sites for AgNP, allowing

accommodation of large amounts of NPs, which can

improve the bacteria degradation functionality of the

hybrid solid.

There are several established methods to create

AgNP. Physical methods include thermal decomposi-

tion (Navaladian et al. 2006), laser ablation (Pyatenko

et al. 2004; Brito-Silva et al. 2010), c-ray reduction

(Chen et al. 2007), microwave irradiation (Yin et al.

2004), and sonochemical synthesis (Manoiu 2010),

which produce stand-alone NPs in powder form.

Biogenic or green synthesis of AgNPs is extensively

reported in the literature (Fayaz et al. 2010; Awwad

et al. 2013); they nucleate at bacterial cells and/or

supernatant fluid, and also form as isolated particles

mono-dispersed in the medium.

In this study, our aim is to nucleate and grow the

AgNP on the selected hierarchical substrates which is

possible by controlled in situ chemical reduction of

silver salts in the presence of suitable reducing and

stabilizing agents (Guzman et al. 2009, Dankovich and

Gray 2011). We have opted for this approach in this

study, which is expected to provide more controllable

NP distribution and stronger support-NP bonding.

The morphology and particle size distribution of

AgNP coated CNT-grafted structures were character-

ized using the field emission scanning electron micro-

scope (FESEM). Surface chemistry and crystal

structure of AgNP-coated surfaces were characterized

by X-ray photoelectron spectroscopy (XPS) and X-ray

diffraction (XRD), respectively. Also, the antibacterial

2346 Page 2 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy

properties of these specimens were examined against

Escherichia coli (E. coli, JM109, K12 derivative).

Materials and methods

Materials

Selected supports

Two kinds of carbon support structures were used in

this study: highly oriented pyrolytic graphite (HOPG)

(HOPG ZYH grade provided by Molecular Imaging)

and reticulated vitreous carbon (RVC) foam (grade-80

pores per inch provided by Ultramet Inc). The HOPG

is a standard flat carbon substrate having well-defined

geometry, and hence suitable for in-depth character-

ization and analysis. The RVC foam is a porous carbon

structure suitable for deployment in water purification

devices. Open porosity and density of as-received

RVC foam are approximately 97 % and 0.045 g/cc,

respectively.

Methods

CNT-grafted hierarchical carbon structures

CNTs were grafted on aforementioned support struc-

tures to create hierarchical morphology. CNT grafting

is a two-step process as described in an earlier paper

(Mukhopadhyay et al. 2009). It involves deposition of

silicon oxide (SiOx) buffer layer followed by CNT

deposition via thermal CVD method using xylene and

ferrocene as carbon and catalyst sources, respectively.

The growth parameters for SiOx buffer layer and

subsequent CNT grafting were optimized in-house to

maximize through-thickness CNT across the RVC

foam.

Synthesis of AgNP on support structures

For synthesis of AgNP, Silver nitrate was used as the

metal precursor [AgNO3 (Sigma Aldrich; 99.99 %)],

dimethyl sulfoxide was the selected reducing agent

(Rodriguez-Gattorno and Diaz 2002) [DMSO (Sigma

Aldrich; 99.5 %)], and tri sodium citrate was used as

the stabilizing agent (Rodriguez-Gattorno and Diaz

2002) (Na3C6H5O7, MP bio; 99 %).

Deposition AgNP is also a two-step process:

deposition of AgNO3 on support structures followed

by in situ chemical reduction in DMSO–citrate

solution at the selected temperature. In the former

step, support structures were soaked in AgNO3

solution at concentration 0.24 M for 24 h. In the latter

step, AgNO3-coated samples were reduced in 1 mM

DMSO–citrate solution for 2 h at a set temperature. As

the AgNP nucleated from AgNO3, DMSO–citrate

solution gradually turns from colorless liquid to light

yellow to pinkish. The actual shade can often depend

on size of AgNP, but the overall color indicates

formation of AgNP. Subsequently, measurement of

AgNP size and distribution were made by microstruc-

ture analysis. Silver nanoparticles were synthesized on

four types of supports: CNT–HOPG, bare HOPG,

CNT–RVC, and bare RVC foam.

Characterization methods

Microstructure, chemical, and crystallographic char-

acterization of the AgNP on CNT-grafted carbon

surfaces were performed using FESEM, XPS, and

XRD, respectively.

Microstructure characterization

Detailed structural analysis of CNT-grafted carbon

structures before and after AgNP functionalization

was performed by FESEM (JEOL 7401F). Both

secondary (SE) and backscattered (BE) imaging

modes were used for microstructure analysis. Particle

size distributions of AgNP were measured from the

images using the Scandium imaging software.

Surface chemistry

Surface chemistry of AgNP-modified structures were

characterized using XPS system from Kratos (Axis

Ultra) using mono-chromatized AlKa (1,486.6 eV) as

X-ray source. The survey scans along with high-

resolution fine scans of all individual elements presented

were taken. CASA software was used for data analysis

and curve fitting. Peaks were shifted for charge correc-

tion taking C 1 s (284.5 eV) as a reference, a well-

established value for HOPG (Arroyo-Ramırez et al.

2009).

J Nanopart Res (2014) 16:2346 Page 3 of 14 2346

123

Author's personal copy

Crystal structure

Crystallographic orientation of synthesized AgNP on

carbon structures were studied using X-ray mini

diffractometer, MD-10, using a monochromotized

Cu Ka radiation at 25 kV and 0.4 mA.

Mechanical characterization/durability test

It has been reported earlier (Mukhopadhyay et al.

2009; Mukhopadhyay and Karumuri 2010) that CNT

grown on carbon supports using this technique are

very strongly attached to the supports, resulting in a

strong base for AgNP functionalization. In this study,

the idea is to test the mechanical durability of the

AgNP attachment on the CNT. The hybrid structures

comprising AgNP attached on CNT–HOPG were

agitated in ultrasonic bath in progressively stronger

power and time until the substrate began to fail

(42 kHz for 10 s). Microstructural analysis was per-

formed before and after the test to count total number

of NPs and their size distribution.

Antibacterial performance test

Influent water preparation

Escherichia coli, K12 derivative, was selected as a test

microbe due to its role as an indicator of fecal

contamination in water. Preparation of E. coli culture

was discussed in earlier reports (Karumuri et al. 2013),

and hence details are not provided here. The concen-

tration of prepared influent water (E. coli dispersed

pure water) was 103 CFU/ml and used for testing.

Antibacterial performance of the prepared samples

[11.5 mm (diameter) 9 4 mm (thickness)] was tested

in two modes; Rotation and Filtration.

Antibacterial performance via rotation

In case of rotation mode, the sample was taped to the

wall of a glass jar filled with 500 ml influent water.

The jar was tumbled at a speed of 60 RPM for 40 min.

50 ll aliquots of the water sample were acquired at 0,

5, 10, 20, and 40 min and streaked on agar plates.

Antibacterial performance via filtration

In case of filtration test, influent water was passed

through a cartridge fitted with the foam sample to be

tested. Schematic representation of the experimental

set up was shown in an earlier paper (Karumuri et al.

2013). 50 ll aliquots of the water sample were

acquired at the start and the end of each filtration

step, and streaked on three agar plates in order to

measure the change of E. coli activity with each pass.

The agar plates were incubated in an incubator at

37 �C for 20 h, and bacteria colonies were counted

visually. Results are reported as a fraction of E. coli

survival (colonies formed from the water sampled

after the treatment over initial E. coli load).

Disinfection zone test

Disinfection zone test was performed to study the

antibacterial mechanism of prepared structures. 50 ll

influent water with CFU concentration of 106/ml is

streaked over the agar plate, forming a thin moist

layer. The samples were placed gently over the agar in

different petri dishes (one sample per dish) and

incubated them at 37 �C for 20 h.

Results and discussion

Silver nanoparticles coated CNT

CNTs have been grafted on selected support structures

as explained in ‘‘Methods’’ section. Geometrical

models had been reported in earlier studies (Mukho-

padhyay et al. 2009) and showed that SSA of CNT-

grafted solids (hierarchical structures) will easily be

over 2 orders of magnitude higher than the initial as-

received solids. Subsequent Brunauer–Emmett–Teller

(BET) surface area measurements (Barney et al.

2012a) of these structures had confirmed this predic-

tion. It must be noted that the present CNT-grafting

method has denser packing and longer CNT compared

to samples in earlier studies, and the increase in SSA

for these substrates are expected to be even higher.

For AgNP quantification purposes, we have

selected a model substrate (HOPG) as support, which

provides a well-defined flat geometry, but identical

chemistry as the foams. CNTs were grafted on the

HOPG (CNT–HOPG) graphite followed by AgNP

2346 Page 4 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy

deposition (using technique outlined in ‘‘Methods’’

section). The size, distribution, and density of AgNP

depend on several process parameters such as molarity

of silver nitrate solution, molarity of DMSO–citrate

solution, and the reduction temperature. In this study,

the other parameters were optimized for a given

substrate, and the reduction temperature varied, with

the idea of using that as the parameter to control NPs

distribution in future.

Influence of reduction temperature

Microstructure analysis of AgNP on the selected

substrates was carried out at 2 kV accelerating voltage

and backscatter electron (BE) mode. The BE images

of CNT–HOPG, the control sample, are shown in

Fig. 1a. It can be seen in Fig. 1a that the CNT–HOPG

sample contained small but detectable amount of NPs

even prior to AgNP deposition. These are iron

nanoparticles (FeNP) which served as catalysts for

CNT growth during the CVD process. These remain in

the substrates, and are, therefore, analyzed carefully to

provide an estimate of background NP counts on

CNT-grafted substrates, as seen in Table 1.

Silver nanoparticles reduced on CNT–HOPG at 60

and 80 �C are shown in Fig. 1b, c, respectively. For

comparison, AgNP was also grown directly on the

HOPG surface (without CNT attachment). Figure 1d

shows the SEM image of AgNP on HOPG reduced at

60 �C. Particle sizes are similar to those on Fig. 1b.

However, the number density of AgNP per unit area of

substrate is expected to be very significantly lower in

the 2D distribution of Fig. 1d compared to that on

hierarchical substrate of Fig. 1b.

The 60 �C reduced samples contained a very large

number of well-dispersed AgNP with a mean diameter

Fig. 1 Silver nanoparticles grown on CNT–HOPG; as-grafted CNT (a) AgNP on CNT–HOPG at 60 �C (b) AgNP on CNT–HOPG at

80 �C (c), AgNP on HOPG (d)

Table 1 Summary of average AgNP particle size and PPL

measurements

Sample Total

images

Average

particle size

(nm)

Particle per

CNT length

(/lm)

CNT–HOPG 6 8.1 ± 5.3 9.2 ± 4

AgNP on CNT–

HOPG @ 60 �C

18 3.53 ± 2.63 106 ± 24

AgNP on CNT–HOPG

@ 80 �C

14 7.45 ± 4.19 82 ± 20

J Nanopart Res (2014) 16:2346 Page 5 of 14 2346

123

Author's personal copy

of less than 5 nm (Fig. 1b). The samples reduced at

80 �C showed significant AgNP deposition, but these

particles have coalesced, yielding larger clusters

(Fig. 1c) of agglomerated patches as large as several

micrometers. Closer inspection of the 80 �C reduced

samples indicates that they consist of component

individual particles having larger diameters. This is

discussed more in the next section.

Analysis of particle size distribution

Multiple BE images at magnification of 250,0009

(implying analysis area just over 180,000 nm2) from

all three samples were analyzed for particle size

distribution, and summarized in Table 1. The BE

micrographs were loaded in Scandium� software, and

counted both computationally and manually for cross

checking. It must be noted that it was not possible to

separate the AgNP from the background FeNP, and the

size distribution results include all visible particles.

However, it is clear that the background FeNP are very

small in number, and account for only a small fraction

of the total particle count. What is significant is that

AgNP reduced at 60 �C shows a mean diameter of

about 3–4 nm, whereas those reduced at 80 �C show a

higher mean diameter in the 7–8 nm range.

Figure 2a shows the histogram of AgNP size

distribution from the 60 and 80 �C samples. This is

divided into three regions based on the particle sizes:

3 nm, 3 B xB7 nm, and 7 nm (Fig. 2b). In case of

AgNP grown at 60 �C, percentages of particles fall in

the aforementioned regions are 56.79, 34.22, and

8.97 %, respectively. For samples grown at 80 �C, the

percentages are 32.11, 38.79, and 28.88 %. The shift

Fig. 2 Particle size

distribution of AgNP grown

at 60 and 80 �C (a),

Percentage of AgNP fall

between three size

categories (b)

2346 Page 6 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy

toward larger particle size for 80 �C samples is very

clear. It has been reported earlier that nucleation and

growth rates of metallic NPs are known to ripening

with temperature (Zhu et al. 2007). The clusters of

Fig. 1c imply that these phenomena may be starting at

as low as 80 �C for these particles, resulting in larger

NPs and noticeable aggregation.

Number of nanoparticles per CNT length (PPL)

For this analysis, a total length of CNT that falls within

the depth of focus of the instrument operating at

working distance of 1.5 mm was measured. It must be

emphasized that, unlike counting all visible particles

as done for size distribution analysis, only particles

that were clearly seen to be attached to the in-focus

nanotubes were considered for this analysis. From

such measurements, the average particle per length

(PPL) of CNT counted was 9.2 ± 4/lm for control

sample, 106 ± 24/lm for 60 �C sample, and 82 ± 20/

lm for 80 �C sample (Table 1). Overall, it is clear that

the AgNP formed at 60 �C yielded smaller NPs with

denser and more uniform distribution on CNT, and

hence selected for the further disinfection studies.

Surface chemistry of synthesized AgNP

Surface chemistry studies were performed on HOPG

surfaces before and after the reduction of precursor

salts in order to monitor chemical changes. The XPS

fine scans of Ag 3d and N 1 s is shown in Fig. 3a, b,

respectively. Due to close proximity in binding

energies (BE) of metallic Ag and its compounds

(Phenomena et al. 1991; Bukhtiyarov et al. 2003; Ma

et al. 2011; Wei et al. 2011), the position of the Ag 3d

(Fig. 3a) peak cannot be used to monitor the chemical

changes during reduction. The N 1 s peak may be the

only indicator which is seen to be greatly reduced after

AgNP formation. The N/Ag ratio decreased upon

reduction, but was not completely eliminated. This

may indicate either incomplete reduction of nitrates on

the outermost surface or adsorption of nitrogen-

containing species from the environment.

Crystal structure of synthesized AgNP

The diffraction pattern of carbon (Vijwani and

Mukhopadhyay 2012), AgNO3 (Carotenuto et al.

2000), and AgNP grown on carbon were shown in

Fig. 4a–c, respectively. However, once AgNO3 was

reduced on carbon, all peaks related to AgNO3 were

completely missing, while all the carbon-related peaks

were retained along with extra new peak. The extra

new peak at 38.3� was observed (Fig. 4c) which

corresponds to silver face-centered cubic (111) plane.

This indicates that the formed AgNP is highly

crystalline and is in agreement with reported diffrac-

tion data (Mpenyana-Monyatsi et al. 2012) for stand-

alone AgNP and AgNP grown on CNT.

Considering the low intensity of the major (111)

peak in the small amount of detectable silver, the

absence of other Ag related peaks at 44.4� and 64.6�correspond to (200) and (220), respectively (Mpenya-

na-Monyatsi et al. 2012) is not surprising. The

complete reduction of all nitrate peaks in XRD

indicates that most of the AgNO3 precursor salt is

reduced during reduction step. However, some traces

of N-containing species may be remaining on the

surface, as indicated by the residual N 2p peak in the

XPS spectrum of Fig. 3.

Fig. 3 Fine scan of Ag 3d peak (a) and N 1 s (b) before and

after AgNP formation

J Nanopart Res (2014) 16:2346 Page 7 of 14 2346

123

Author's personal copy

Mechanical durability of AgNP on CNT

This test was done on AgNP deposited CNT–HOPG at

60 �C reduction temperature. Mechanical durability of

AgNP on CNT was tested as explained in ‘‘Mechan-

ical characterization/Durability test’’ section. Ultra

sonication is expected to impart significant vibrational

stresses at the roots of the nanotubes due to large

aspect ratio of the nanotubes (length/diameter ratio

*1,000). It was, however, observed that long sonica-

tion times at a given power would first damage the

substrate, where entire regions of CNT forests

attached to graphite substrate would be pulled out,

rather than shedding of individual CNT or NPs.

Microstructure 5A provides the snapshot of the early

stages of sample failure under this type of rapidly

oscillating mechanical force.

It can be seen that patches of underlying graphite

with CNT layer are the beginning to peel off

(highlighted by box in Fig. 5a). These areas were not

included in counts. Detailed microstructures were

taken of the areas that remained covered with CNTs

(Fig. 5b). It was measured that the average number of

NPs over image area (several images covering

180,000 nm2 at 250,0009 magnification) were

counted to be around 332 ± 41 and 330 ± 35 before

and after the sonication test, respectively. This result

demonstrates that NPs were still intact on surviving

CNT, even when parts of the substrate were beginning

to peel off in some regions.

Particle size distribution upon durability test

Particle size distribution of samples before and after

ultrasonication was compared and shown as a histo-

gram (Fig. 6). It can be seen that no significant

difference was observed in particle size distribution

over the test period. Overall, we can confidently

conclude that amount of NPs and their size distribu-

tions were retained even in the harshest of test

conditions. This result emphasizes the usability of

this structure for variety of purification applications.

Antibacterial performance test

Microstructure characterization

Microstructure of as-received RVC foam and its

subsequent CNT-grafted surfaces are shown in

Fig. 7a, b, respectively. Having confirmed the forma-

tion of AgNP on CNT and its mechanical durability,

AgNP were attached on CNT–RVC with slight

modification: higher molar concentration of AgNO3

was needed for precursor retention on the reticulated

foam surface compared to flat graphite. Figure 7c

shows the microstructures of the AgNP-attached

CNT–RVC foam. The interconnected struts provides

necessary structural integrity and strength, the inter-

connected pores provide flow channels for the fluid,

while the nanotubes on the walls increase SSA,

enabling deposition of unprecedented quantity of

surface-active NPs.

Antibacterial performance test

The antibacterial performance of the prepared sample

was tested as explained in ‘‘Antibacterial performance

test’’ section. The samples compared in this experi-

ment were as-received foam without AgNP (RVC),

CNT-grafted RVC foam without AgNP (CNT–RVC),

Fig. 4 XRD pattern for carbon (a), AgNO3 (b), and AgNP

attached carbon (c)

2346 Page 8 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy

AgNP attached directly on RVC foam (AgNP–RVC),

and foams where AgNP was attached on CNT-grafted

RVC foam (AgNP–CNT–RVC). Each experiment was

repeated twice, with two independent samples and

fresh lots of bacteria-infused water, and three aliquots

used at each step. Therefore, each data point is

averaged over six aliquots. Figure 8 shows the E. coli

degradation rates in the two-test modes.

Bacteria counts were seen to be more or less

constant in RVC and CNT–RVC samples, indicating

no degradation without AgNP in the current test

conditions. It is worth noting that earlier studies (Kang

et al. 2008; Lee et al. 2008; Liu et al. 2010) have

shown that both multiwalled carbon nanotubes

(MWCNT) and FeNP exhibit some antibacterial

properties. However, the toxicity effect of MWCNT

was seen to be approximately three times lower than

that of single-walled CNT (SWCNT). Moreover, the

antibacterial properties of the FeNP are known to be

effective in inert atmosphere. Iron nanoparticles

showed limited inactivation in air or oxygen environ-

ment due to passive oxide layer on NPs. In the

hierarchical structures used in this study, the chemical

bonds of the FeNP are reported to be a combination of

Fe–O, Fe–Fe, and Fe–C (Mukhopadhyay et al. 2009).

These current results conclude that the presence of

both MWCNT and FeNP cause minimal bacterial

degradation in the current test times, and measurable

E. coli degradation can only be achieved by the

presence of AgNP.

From rotation test results (Fig. 8a), it can be seen

that 40 min of treatment resulted in 34 % reduction (or

1-E. coli survived) of E. coli for AgNP–RVC samples

and 71 % for AgNP–CNT–RVC samples. From

Fig. 5 After 10 s of ultrasonication; failure of CNT/graphite layer along the graphite–graphite interface (a), instated AgNP on CNT

after the test (b)

Fig. 6 Particle size

distribution of AgNP grown

at 60 �C before and after

10 s ultrasonication

J Nanopart Res (2014) 16:2346 Page 9 of 14 2346

123

Author's personal copy

filtration test results (Fig. 8b), three percolations

resulted in 43 % bacteria deactivation in AgNP–

RVC foam and 70 % E. coli reduction in AgNP–

CNT–RVC foam. Sample active E. coli regrown on

the agar plates are shown in Fig. 9. Only agar plates at

the start and end of the test are shown for samples

AgNP on RVC foam and CNT–RVC foams.

Both these studies indicate that when antibacterial

NPs such as AgNP are supported on CNT-grafted

substrates; their potency and purification rates can be

significantly enhanced compared to those supported

on regular substrates.

Disinfection zone test

The antibacterial mechanism of silver is not yet

completely known and debatable. There are two

proposed mechanisms discussed in the literature (Feng

et al. 2000): bactericidal effect and bacteriostatic

effect. In either mechanism, antibacterial efficiency of

AgNP comes from the Ag? ion release from the

surface atoms, which will depend on surface chemical

activity, controllable by AgNP size and density. This

can be investigated by monitoring if any disinfection

zone is created around the specimens due to Ag? ion

release. Such a test was performed as explained in

‘‘Antibacterial performance test’’ section. Over the

course of 20 h incubation times, there was no detect-

able disinfection zone (Fig. 10a) observed in AgNP–

RVC foam, whereas AgNP–CNT–RVC foam showed

a distinct zone (Fig. 10b) of about 2 mm from the edge

of the sample, where bacteria did not grow. The

creation of detectable disinfection zone outside the

sample clearly indicates release of Ag? ions, which

will depend on the surface chemical activity, particle

size, and the packing density of AgNP on the substrate.

This can depend on particle size of AgNP and the

packing density of such particles on the surface.

Figure 1b, d show that size of AgNP grown on larger

substrate is similar to that on CNT-attached substrate.

Therefore, the enhancement in disinfection zone

comes from increased number of AgNP available on

the increased surface area of CNT-grafted support.

Similar trends can also be observed with other kinds

of bacteria such as Gram-positive Staphylococcus

aureus. While both Gram-positive and Gram-negative

kinds of bacteria consists of peptidoglycan layer which

protects against antibacterial agents, S. aureus’s layer

Fig. 7 Microstructure of as-received RVC foam (a), CNT-grafted strut surface (b), and AgNP attached CNT (c)

2346 Page 10 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy

is much thicker (30–100 nm) than the E. coli (only few

nanometers). This thick peptidoglycan layer on S.

aureus’s may protect formation of pits by AgNPs more

severely than thin layer of E. coli (Lee et al. 2011). On

the other hand, Gram-negative bacteria are sometimes

much more pathogenic due to lipopolysaccharide outer

membrane and develop resistance to antibiotics (Al-

exandraki and Palacio 2010). Nonetheless, AgNP is

expected to be effective against both Gram-negative

and Gram-positive bacteria, albeit at varying degree.

These studies clearly indicate that the CNT-grafted

foam, which allows higher SSA for supporting larger

density of AgNP in a given volume, provides signif-

icantly improved antibacterial efficacy in all the test

modes investigated. This approach may, therefore, be

very useful in the development of future purification

devices that are more powerful and compact.

Summary

In this investigation, CNTs were grafted on carbon

structures to create ultra-high surface area hierarchical

structures. From earlier studies, both throughFig. 8 Antibacterial performance test results; rotation (a),

filtration (b)

Fig. 9 Active E. coli regrown on agar plates from rotation test;

AgNP deposition on as-received foam @ 0 min (a), 40 min

(b) and CNT-grafted foam @ 0 min (c), 40 min (d). Filtration

test; AgNP deposition on as-received foam @ 0 pass (e), 3rd

pass (f) and CNT-grafted foam @ 0 pass (g), 3rd pass (h)

J Nanopart Res (2014) 16:2346 Page 11 of 14 2346

123

Author's personal copy

mathematical models and BET measurements, it was

estimated that CNT grafting would increase the

surface area of any starting structure by few orders

of magnitude. Silver nanoparticles were grown on

these CNT-grafted (hierarchical) supports, and the

influence of reduction temperature on NP size and

distribution was studied. The mean diameter of AgNP

is observed to be 3.53 ± 2.63 and 7.45 ± 4.19 nm at

60 and 80 �C reduction temperature, respectively.

Diffraction spectrum of AgNP foam showed FCC

crystal structure of AgNP with no traces of peaks

related to AgNO3 crystals. Mechanical durability of

AgNP on CNT showed that the prepared structures are

indeed very durable and are potential purification

devices. The antibacterial efficiency of AgNP attached

CNT–RVC structures tested via both rotation, and

filtration modes showed improved efficiency com-

pared to AgNP directly attached on RVC foam. This

result is further complemented by disinfection zone

studies which indicate distinct bacteria-free region

around the AgNP attached to CNT-grafted foam

samples, and none around AgNP directly attached on

foams. The improved antibacterial efficiency of

AgNP-deposited hierarchical structure is attributed

to the higher concentration of AgNP that can be

packed on higher surface area substrates. While there

are many studies reporting antibacterial efficiency of

CNT and NPs (Gururaj and Neelgund 2011; Prodana

et al. 2011), most involve unsupported nanomaterials

that contaminate the environment. To the best of our

knowledge, this is the first study where AgNP and

CNT are seamlessly integrated on one monolithic

structure, and its increased surface area directly

related to its antibacterial efficacy. This approach

combines nanomaterial advantage with structural

integrity and is expected to provide powerful and

compact disinfection devices in future.

Acknowledgments Financial support from the Ohio Third

Frontier Program, the Environmental Protection Agency, and

the Wright state University Ph.D. fellowship is acknowledged.

Facilities used were funded by NSF-MRI award and Ohio Board

of Regents. The authors are grateful to Ultramet Inc. for

generous supply of reticulated vitreous carbon foams.

References

Alexandraki I, Palacio C (2010) Gram-negative versus Gram-

positive bacteremia: what is more alarmin(g)? Crit Care

14:161. doi:10.1186/cc9013

Arroyo-Ramırez L, Montano-Serrano R, Raptis RG, Cabrera

CR (2009) Nanostructural formation of Pd–Co bimetallic

complex on HOPG surfaces: XPS and AFM studies.

J Nanotechnol 2009:1–5. doi:10.1155/2009/971423

Asharani PV, Lian WuY, Gong Z, Valiyaveettil S (2008) Tox-

icity of silver nanoparticles in zebrafish models. Nano-

technology 19:255102. doi:10.1088/0957-4484/19/25/

255102

Awwad AM, Salem NM, Abdeen AO (2013) Green synthesis of

silver nanoparticles using carob leaf extract and its anti-

bacterial activity. Int J Ind Chem 4:2–7. doi:10.1186/2228-

5547-4-29

Barney IT, Lennaerts DSR, Higgins SR, Mukhopadhyay SM

(2012a) Specific surface area of hierarchical graphitic

substrates suitable for multi-functional applications. Mater

Lett 88:160–163. doi:10.1016/j.matlet.2012.08.042

Barney IT, Ganguli S, Roy AK, Mukhopadhyay SM (2012b)

Improved thermal response in encapsulated phase change

materials by nanotube attachment on encapsulating solid.

J Nanotechnol Eng Med 3(3):031005

Brito-Silva AM, Gomez LA, de Araujo CB, Galembeck A

(2010) Laser ablated silver nanoparticles with nearly the

same size in different carrier media. J Nanomater

2010:1–7. doi:10.1155/2010/142897

Fig. 10 Disinfection zone test; AgNP–CNT foam contained agar plate (a), AgNP attached CNT–RVC foam (b)

2346 Page 12 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy

Bukhtiyarov V, Havecker M, Kaichev V et al (2003) Atomic

oxygen species on silver: photoelectron spectroscopy and

X-ray absorption studies. Phys Rev B 67:235422. doi:10.

1103/PhysRevB.67.235422

Carotenuto G, Pepe GP, Nicolais L (2000) Preparation and

characterization of nano-sized Ag/PVP. Eur Phys J B

17:11–17. doi:10.1007/s100510070243

Chen P, Song L, Liu Y, Fang Y (2007) Synthesis of silver

nanoparticles by c-ray irradiation in acetic water solution

containing chitosan. Radiat Phys Chem 76:1165–1168.

doi:10.1016/j.radphyschem.2006.11.012

Dankovich TA, Gray DG (2011) Bactericidal paper impreg-

nated with silver nanoparticles for point-of-use water

treatment. Environ Sci Technol 45:1992–1998. doi:10.

1021/es103302t

Davies R (1997) The development and functions of silver in

water purification and disease control. Catal Today

36:107–114. doi:10.1016/S0920-5861(96)00203-9

Eby DM, Luckarift HR, Johnson GR (2009) Hybrid antimicro-

bial enzyme and silver nanoparticle coatings for medical

instruments. ACS Appl Mater Interfaces 1:1553–1560.

doi:10.1021/am9002155

Fayaz AM, Balaji K, Girilal M et al (2010) Biogenic synthesis of

silver nanoparticles and their synergistic effect with anti-

biotics: a study against Gram-positive and Gram-negative

bacteria. Nanomedicine 6:103–109. doi:10.1016/j.nano.

2009.04.006

Feng QL, Wu J, Chen GQ, et al (2000) A mechanistic study of the

antibacterial effect of silver ions on Escherichia coli and

Staphylococcus aureus. J Biomed Mater Res 52:662–668

Gururaj M, Neelgund AO (2011) Deposition of silver nano-

particles on dendrimer functionalized multiwalled carbon

nanotubes: synthesis, characterization and antimicrobial

activity. J Nanosci Nanotechnol 11:3621–3629

Guzman MG, Dille J, Godet S (2009) Synthesis of silver

nanoparticles by chemical reduction method and their

antibacterial activity. Int J Chem Biol Eng 2:104–111

Jain P, Pradeep T (2005) Potential of silver nanoparticle-coated

polyurethane foam as an antibacterial water filter. Bio-

technol Bioeng 90:59–63. doi:10.1002/bit.20368

Jiang SQ, Newton E, Yuen CWM, Kan CW (2005) Chemical

silver plating and its application to textile fabric design.

J Appl Polym Sci 96:919–926. doi:10.1002/app.21541

Kang S, Herzberg M, Rodrigues DF, Elimelech M (2008)

Antibacterial effects of carbon nanotubes: size does matter!

Langmuir 24:6409–6413. doi:10.1021/1a800951v

Karumuri AK, Oswal DP, Hostetler HA, Mukhopadhyay SM

(2013) Silver nanoparticles attached to porous carbon

substrates: robust materials for chemical-free water disin-

fection. Mater Lett 109:83–87. doi:10.1016/j.matlet.2013.

07.021

Kokura S, Handa O, Takagi T et al (2010) Silver nanoparticles

as a safe preservative for use in cosmetics. Nanomedicine

6:570–574. doi:10.1016/j.nano.2009.12.002

Lee HY, Park HK, Lee YM, et al (2007) A practical procedure

for producing silver nanocoated fabric and its antibacterial

evaluation for biomedical applications. Chem Commun

(28):2959–2961. doi: 10.1039/b703034g

Lee C, Kim JY, Lee WI, et al (2008) Bactericidal effect of zero-

valent iron nanoparticles on Escherichia coli. Environ Sci

Technol 42:4927–4933

Lee H, Ryu D, Choi S, Lee D (2011) Antibacterial activity of

silver-nanoparticles against Staphylococcus aureus and

Escherichia coli. Korean J. Microbiol. Biotechnol.

39:77–85

Liu S, Ng AK, Xu R, et al (2010) Antibacterial action of dis-

persed single-walled carbon nanotubes on Escherichia and

Bacillus subtilis investigated by atomic force microscopy.

Nanoscale 2:2744–2750. doi:10.1039/c0nr004411c

Lv Y, Liu H, Wang Z et al (2009) Silver nanoparticle-decorated

porous ceramic composite for water treatment. J Memb Sci

331:50–56. doi:10.1016/j.memsci.2009.01.007

Ma J, Zhang J, Xiong Z et al (2011) Preparation, characteriza-

tion and antibacterial properties of silver-modified graph-

ene oxide. J Mater Chem 21:3350. doi:10.1039/

c0jm02806a

Manoiu V (2010) Obtaining silver nanoparticles by sono-

chemical methods. U.P.B. Sci Bull Ser B 72:179–186

Maurer E, Hussain S, Mukhopadhyay SM (2011) Cell growth in

a porous microcellular structure: influence of surface

modification and nanostructures. Nanosci Nanotechnol

Lett 3:110–113. doi:10.1166/nnl.2011.1128

Maurer EI, Comfort KK, Hussain SM et al (2012) Novel plat-

form development using an assembly of carbon nanotube,

nanogold and immobilized RNA capture element towards

rapid, selective sensing of bacteria. Sensors (Basel)

12:8135–8144. doi:10.3390/s120608135

Mpenyana-Monyatsi L, Mthombeni NH, Onyango MS, Momba

MNB (2012) Cost-effective filter materials coated with

silver nanoparticles for the removal of pathogenic bacteria

in groundwater. Int J Environ Res Public Health

9:244–271. doi:10.3390/ijerph9010244

Mukhopadhyay SM (2012) Nanoscale mutifunctional materials.

Wiley, Hoboken

Mukhopadhyay SM, Karumuri AK (2010) Nanotube attachment

for prevention of interfacial delamination. J Phys D Appl

Phys 43:365301. doi:10.1088/0022-3727/43/36/365301

Mukhopadhyay SM, Karumuri A, Barney IT (2009) Hierarchi-

cal nanostructures by nanotube grafting on porous cellular

surfaces. J Phys D Appl Phys 42:195503. doi:10.1088/

0022-3727/42/19/195503

Navaladian S, Viswanathan B, Viswanath R, Varadarajan T

(2006) Thermal decomposition as route for silver nano-

particles. Nanoscale Res Lett 2:44–48. doi:10.1007/

s11671-006-9028-2

Phenomena R, Petrochemicals I, Limited C (1991) XPS CORE

LIWEL SPECTRA SOME SILVER COMPOUNDS*.

J Electron Spectros Relat Phenomena 56:273–277

Prodana M, Ionita D, Ungureanu C et al (2011) Enhancing

antibacterial effect of multiwalled carbon nanotubes using

silver nanoparticles. Microscopy 6:549–556

Pyatenko A, Shimokawa K, Yamaguchi M et al (2004) Syn-

thesis of silver nanoparticles by laser ablation in pure

water. Appl Phys A 79:803–806. doi:10.1007/s00339-004-

2841-5

Rathnayake WGIU, Ismail H, Baharin A et al (2012) Synthesis

and characterization of nano silver based natural rubber

latex foam for imparting antibacterial and anti-fungal

properties. Polym Test 31:586–592. doi:10.1016/j.

polymertesting.2012.01.010

Rodriguez-Gattorno G, Diaz D (2002) Metallic nanoparticles

from spontaneous reduction of silver (I) in DMSO.

J Nanopart Res (2014) 16:2346 Page 13 of 14 2346

123

Author's personal copy

Interaction between nitric oxide and silver nanoparticles.

J Phys Chem B 106:2482–2487

Vijwani H, Mukhopadhyay SM (2012) Palladium nanoparticles

on hierarchical carbon surfaces: a new architecture for

robust nano-catalysts. Appl Surf Sci 263:712–721

Vijwani H, Agrawal A, Mukhopadhyay SM (2012) Dechlori-

nation of environmental contaminants using a hybrid

nanocatalyst: palladium nanoparticles supported on hier-

archical carbon nanostructures. J Nanotechnol 2012:1–9.

doi:10.1155/2012/478381

Wei W, Mao X, Ortiz LA, Sadoway DR (2011) Oriented silver

oxide nanostructures synthesized through a template-free

electrochemical route. J Mater Chem 21:432. doi:10.1039/

c0jm02214d

Wijnhoven SWP, Peijnenburg WJGM, Herberts CA et al (2009)

Nano-silver: a review of available data and knowledge

gaps in human and environmental risk assessment. Nano-

toxicology 3:109–138. doi:10.1080/17435390902725914

Yin H, Yamamoto T, Wada Y, Yanagida S (2004) Large-scale

and size-controlled synthesis of silver nanoparticles under

microwave irradiation. Mater Chem Phys 83:66–70.

doi:10.1016/j.matchemphys.2003.09.006

Zhu HW, Xu CL, Wu DH et al (2002) Direct synthesis of long

single-walled carbon nanotube strands. Science 296:

884–886. doi:10.1126/science.1066996

Zhu L, Lu G, Mao S et al (2007) Ripening of silver nanoparticles

on carbon nanotubes. NANO 02:149–156. doi:10.1142/

S1793292007000507

2346 Page 14 of 14 J Nanopart Res (2014) 16:2346

123

Author's personal copy