Download Microwave-Assisted Synthesis of Nanocrystalline MgO and Its Use

FULL PAPER
Microwave-Assisted Synthesis of Nanocrystalline MgO and Its Use
as a Bacteriocide
By Shirly Makhluf, Rachel Dror, Yeshayahu Nitzan, Yaniv Abramovich, Raz Jelinek, and
Aharon Gedanken*
Nanocrystalline particles of MgO were synthesized using microwave radiation in an ethylene glycol solution. The antibacterial
activities of the MgO nanoparticles were tested by treating Escherichia coli (Gram negative) and Staphylococcus aureus (Gram
positive) cultures with 1 mg mL±1 of the nanoparticles. We have examined the importance of the size effect, pH, and the form
of the active MgO species as a bactericidal agent. A clear size dependence of the nanoparticles is observed where the amount
of eradicated bacteria was strongly dependent on the particle size.
1. Introduction
Magnesium oxide is obtained mainly by the thermal decomposition of magnesium hydroxide or carbonate,[1,2] and, recently, by the sol±gel process.[3,4] The oxide morphology and
the particle size were found to depend on the preparation conditions (pH, gelling agent, calcination rate, and temperature).
The interest in nanoparticles stems from their unique properties, which differ considerably from those of bulk materials. Research in the area of nanometer-sized metal oxides in general,
and MgO in particular, was already under way in the early
years of nanotechnology.
One of the pioneers in this area is Klabunde,[5] who in 1991
reported on an improved aerogel procedure for the production
of Mg(OH)2 and MgO. The properties of the nanometer-sized
products were also studied by his group. Nanoparticles of MgO
were produced from droplets of an aqueous salt solution in a
flame spray pyrolysis reactor.[6] MgO nanoparticles are produced from nitrate, as well as from acetate salt precursor solutions, when a propane±oxygen diffusion flame is used to decompose aqueous aerosol droplets.[6] Sonochemical synthesis,
followed by supercritical drying, was used to prepare MgO
nanoparticles from Mg(OCH3)2 and Mg(OC2H5)2.[7] MgO
nanoparticles with a narrow size distribution have been successfully prepared using a chemical precipitation route in
±
[*]
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Prof. A. Gedanken, S. Makhluf
Department of Chemistry and Kanbar Laboratory for Nanomaterials at
the Bar-Ilan University Center for Advanced Materials & Nanotechnology
Bar-Ilan University
Ramat-Gan, 52900 (Israel)
E-mail: gedanken@mail.biu.ac.il
R. Dror, Prof. Y. Nitzan
Faculty of Life Sciences, Bar-Ilan University
Ramat-Gan, 52900 (Israel)
Y. Abramovich, Prof. R. Jelinek
Department of Chemistry, Ben-Gurion University
Beer Sheva, 84105 (Israel)
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
which a polymer (polyvinyl alcohol (PVA) or polyethylene glycol (PEG)) is used for the surface protection of the particle.[8]
A novel method, which combines laser vaporization of metals
with controlled condensation in a diffusion cloud chamber,
is used to synthesize nanoscale MgO nanoparticles of homogeneous size and well-defined composition.[9] Nanocrystalline
Mg(OH)2 has been synthesized by a hydrothermal reaction
using different magnesium precursors and solvents as the reactants. Subsequent thermal decomposition at 450 C gave nanometer-sized MgO.[10]
Magnesium oxide (MgO, periclase) is an exceptionally important material, with uses in catalysis,[11,12] toxic-waste remediation,[13] or as an additive in refractory, paint, and superconducting products,[14±16] as well as for fundamental and application
studies. Ultrafine metal oxide particles have found use as bactericides, adsorbents, and, specifically, catalysts.[17,18] MgO in
particular has shown great promise as a destructive adsorbant
for toxic chemical agents.[19,20]
Of the many methods employed today for the fabrication of
nanomaterials, the use of microwave (MW) radiation is the one
which perhaps requires the smallest investment in equipment,
since it can be carried out in a domestic MW oven. MWs have
found a number of applications in chemistry since 1986.[21,22]
The effect of heating is created by the interaction of the permanent dipole moment of the molecule with high-frequency
(2.45 GHz) electromagnetic radiation. The ability of any material to absorb microwave energy is expressed by its dielectric
loss factor combined with the dielectric constant.[23,24] The dielectric loss factor depends on the material, driving frequency,
and temperature.
In the last ten years, inorganic reactions conducted in a microwave oven were found to yield nanometer-sized products.[25]
Polyol solvents such as ethylene glycol (EG) are extremely
suitable for microwave reactions because of their relatively
high dipole moment and loss factor.[22]
In the current paper we report on a new method for the
preparation of nanoparticles of MgO. The synthesis was carried
out in a domestic microwave oven where Mg(CH3COO)2, dissolved in ethylene glycol, was decomposed, yielding amorphous nanoparticles of Mg(OH)2. The particles were crystal-
DOI: 10.1002/adfm.200500029
Adv. Funct. Mater. 2005, 15, 1708±1715
2. Results and Discussion
Although the typical length of the MW reaction was 1 h, we
could observe an ample amount of product even after 5 min.
The total conversion yield after 1 h was 95 %, calculated on
the basis of the amount of magnesium in the product and compared to its weight in the precursor. The concentration of the
Mg2+ ions in the mother liquid was determined at the end of
the reaction by an ethylenediaminetetraacetic acid (EDTA) titration. It was found to be reduced to 5.0 ” 10±5 M. The first
step in the identification of the reaction products was conducted by X-ray diffraction (XRD) measurements. In Figure 1
we present the diffraction patterns obtained for the as-prepared material (Fig. 1a), and the annealed product (Fig. 1d).
The diffraction peak at 2h = 58 matches the most intense peak
in the diffractions of Mg(OH)2 (Powder Diffraction File (PDF)
76-0667) The amorphous nature of the as-prepared material is
evident from Figure 1a, although we cannot determine whether
it is a ªtrueº amorphous or X-ray amorphous state. To determine the composition of the amorphous product, the as-prepared material was heated to 100, 200, and 300 C, and the
XRD was measured. The diffraction peaks of the sample annealed at 100 C (Fig. 1b) can be assigned to Mg(OH)2, for example the peak at 2h = 58.7, as well as to MgO (2h = 43 and
Adv. Funct. Mater. 2005, 15, 1708±1715
1600
1500
1400
1300
1200
1100
Intensity (a.u.)
lized by annealing at 600 C, and their antibacterial effect was
examined. Magnesium oxide prepared through an aerogel procedure (AP-MgO) yields square and polyhedral shaped nanoparticles with diameters that vary slightly around 4 nm, arranged in an extensive porous structure with considerable pore
volume. These nanoparticles were used as biocides in a comprehensive study by Klabunde and co-workers.[26,27] It has been
shown that AP-MgO/Cl2 formulations are quite active as biocides, more so than free Cl2, AP-MgO itself, or commercially
available microcrystalline MgO. The powders can be dispersed
by simply sprinkling on contaminated areas or by spraying,
such as by the ªdry fire extinguisherº method. According to
the tests, both Escherichia coli (E. coli) and Bacillus megaterium were completely killed by any of the applied nanoparticulate formulations in 20 min or less. Only limited information
(Table 2, Klabunde and co-workers[27]) is provided for the
AP-MgO nanoparticle itself.
In the current study we have demonstrated the effect of particle size, pH, and form of the active MgO species as a bactericidal agent. This bactericidal activity was tested on the Grampositive Staphylococcus aureus (Staph. aureus) bacteria, as well
as on the Gram-negative E. coli bacteria. All the bactericidal
experiments were conducted using nanocrystalline MgO. Our
results show a clear bacteriocidal activity of small MgO nanoparticles. The smaller particles are more active in killing the
bacteria, and a gradual decrease in the bacteriocidal activity
with the increase in particle size is observed. According to our
proposed mechanism, the MgO is responsible for the formation
of an active oxygen species, which is active on the cell membrane as well as inside the bacterial cell. Finally, this paper is
another demonstration of the importance of nanoparticles.[28]
FULL PAPER
S. Makhluf et al./Nanocrystalline MgO and Its Use as a Bacteriocide
1000
900
800
700
600
500
400
d
c
b
a
300
200
100
0
20
30
40
50
60
70
80
2 Theta
Figure 1. X-ray diffraction patterns of a) as-prepared amorphous Mg(OH)2,
b) heated to 100 C, c) heated to 300 C, d) heated to 600 C ® MgO.
62.2). The sample heated to 200 C shows only diffraction
peaks assigned to crystalline MgO. In brief, the annealed product clearly shows diffraction peaks that match those reported
for face-centered cubic (fcc) MgO in the PDF files (PDF
11-0293). The peaks become more intense at 300 and 600 C.
The particle size was evaluated from the peak width at half
height using the Scherrer formula. The calculated size was
11 ± 1 nm. We could not detect any diffraction lines associated
with impurities. Nanoparticles tend to aggregate and form
large agglomerates due to interparticle interaction resulting
from van der Waals', electrostatic, or magnetic forces. Since no
surfactant was added to the reaction solution such aggregation
occurs in the current case. Dynamic light scattering (DLS)
measurements of the MgO dispersed in both EG or water after
annealing revealed that the size of the agglomerate is 350 nm.
According to our interpretation the soft aggregate, upon reaching the cell membrane and due to interaction with it, is gradually dispersed in the solution and the individual nanoparticles
penetrate the cell membrane. This is demonstrated by the bacteriocidal effect, which is size dependent.
Thus, this paper is another demonstration of the nanoscale
effect, which is observed despite the aggregated nature of the
particles. Another example of this behavior is magnetic nanoparticles, which are highly aggregated, yet whose physical properties (coercivity for example) are those of a small nanoparticle. We believe that inside the bacterial cell the nanoparticles
reform the agglomerate.
To further characterize the as-prepared material and confirm
its amorphous nature, its differential scanning calorimetry
(DSC) profile was measured. It showed two exothermic peaks
at 180 and 430 C that disappeared in the second heating cycle.
The exothermic peak at 180 C is assigned to an amorphous±
crystalline transition of the Mg(OH)2 and MgO. The XRD results support the assignment of the first exothermic peak at
180 C to an amorphous±crystalline transition. The second
peak at 430 is assigned to the decomposition of Mg(OH)2
to MgO + H2O(l). The standard enthalpy of formation (DHf0)
at 298 K of these three molecules are ±924.5, ±601.7, and
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S. Makhluf et al./Nanocrystalline MgO and Its Use as a Bacteriocide
±285.8 kJ mol±1, respectively. The calculated standard enthalpy
of reaction (DH0) at 298 K is 37 kJ mol±1. Thus, the reaction is
almost thermoneutral and may become exothermic at 430 C.
This assignment is supported by the thermogravimetric analysis
(TGA) results (Fig. 2), which exhibit a sharp weight loss at
about 460 C. This weight loss of 37 % corresponds well to the
calculated weight loss for this reaction, 30 %.
No bactericidal effect is observed for the as-prepared material. C,H elemental analysis of the as-prepared product revealed
23.5 wt.-% C and 4.78 wt.-% H. It is clear that the large
amount of carbon and hydrogen is due to EG molecules residing on the surface. They are removed upon heating to 600 C.
Indeed, the amount of impurities found after the annealing
process was 3.7 wt.-% C and 1.3 wt.-% H.
The nanoscale nature of the annealed compound is illustrated in the surface area results. The Brunauer±Emmett±Teller
(BET) measurements yielded a value of 118 m2 g±1 for the material annealed at 600 C. Nanocrystalline MgO was prepared
by a modified aerogel procedure, yielding a surface area of
400±500 m2 g±1.[29] Unlike our product, which showed a smooth
surface, the aerogel product was highly porous. A further demonstration of the particle sizes is shown in the transmission
electron microscopy (TEM) images below.
Figure 3 shows the TEM images of the as-prepared (Fig. 3a)
and the annealed samples (Fig. 3b). The picture of the as-prepared material shows isolated particles with an average diameter of 6 ± 2 nm, as well as a network of connected particles.
Larger particles of 11 ± 1 nm are observed for the annealed
sample. The number of isolated particles is reduced, and sintering caused the particles to form a more intricate network spanning a diameter of 300 nm.
High-resolution TEM (HRTEM) reveals the fringes of the
MgO crystal (Fig. 4). The measured distance between the
fringes is 2.1 Š, which is in good agreement with the (111) interplanar distance.
To obtain different sizes of MgO nanoparticles, we altered
the concentration of the Mg(Ac)2 solution. The particle sizes
Figure 2. TGA curve for the as-prepared sample.
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0.1 µm
0.2 µm
Figure 3. TEM images of the a) as-prepared MgO nanoparticles and
b) MgO nanoparticles after crystallization.
Figure 4. HRTEM images of the MgO nanoparticles after crystallization.
Scale bar: 2 nm.
as a function of the concentration of the Mg(Ac)2 solution are
calculated from the TEM images using the Scion Image program, and are presented in Table 1.
2.1. Proposed Mechanism
To clarify the mechanism of the current reaction, the following control reactions were performed. Magnesium chloride was
dissolved in EG, and the reaction was repeated under
identical conditions (including solution concentration).
No product was isolated from the reaction cell. Magnesium acetate was dissolved in water, and the reaction
was repeated under the same conditions in a MW oven.
No product was obtained. We conducted the reaction in
EG under reflux at 100 C, as well as in a MW oven at
approximately 100 C, and detected no product. The
reaction was also conducted in a reflux system at the
boiling point of EG without MW radiation. The reflux
conditions were maintained for 3 h. Amorphous MgO
nanoparticles were obtained, measured after crystallization, and found to be 23 ± 2 nm in diameter. A solution
of magnesium acetate in acetonitrile was also reacted in
a microwave oven. A mixture of magnesium carbonate
and magnesium nitrate was obtained as a solid product.
The mixture was heated to 390 C and the annealed
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Adv. Funct. Mater. 2005, 15, 1708±1715
Table 1. The particle sizes as a function of the concentration of Mg(Ac)2.
Synthesis method
Concentration of the
Mg(Ac)2 solutions in EG
Particle size [nm]
Reflux system
Microwave
Microwave
Microwave
Microwave
0.093 M
0.139 M
0.121 M
0.093 M
>0.046 M
23 ± 2
18 ± 1
15 ± 1
11 ± 1.9
8±1
obtained from reacting dehydrated Mg(Ac)2 in EG in a MW
oven under the same conditions as the reaction with hydrated
Mg(Ac)2. Identical products are identified. Thus, the importance of using MW radiation is manifested in the shorter reaction time and the smaller particles that are obtained.
FULL PAPER
S. Makhluf et al./Nanocrystalline MgO and Its Use as a Bacteriocide
2.2. Bactericidal Tests
product yielded 18 nm diameter MgO particles. Based on these
control reactions, we concluded that Mg(Ac)2 and EG are active reactants according to the following:
Mg(CH3COO)2 + 2(HO±CH2±CH2±OH) ® Mg(OH)2 +
2(HO±CH2±CH2±OOCCH3)
(1)
Mg(OH)2 ® MgO + H2O
(2)
To substantiate this proposed mechanism, we analyzed the
mother-liquid composition after reaction by 13C NMR spectroscopy ((CD3)2CO-d6, ppm, 75 MHz): HO±CH2±CH2±OOCCH3;
[C(1)] 20.825, [C(2)] 60.561, [C(3)] 66.536, [C(4)] 171.93 (Merck
Index: 171.69, 66.04, 60.63, 20.82; (CH2OH)2 [C] 64.267. Ethylene glycol monoacetate is the final product according to our
proposed mechanism. The formation of MgO nanoparticles
using microwave irradiation is assisted by the intense overheating that a solvent such as EG can undergo[21,30±32] in a microwave oven. Further conformation of the proposed mechanism is
The antibacterial activities of the MgO nanoparticles were
tested by treating E. coli and Staph. aureus cultures with
1 mg mL±1 of the nanoparticles. This concentration of MgO
nanoparticles was chosen as a result of the determination of
the minimal inhibitory concentration (MIC) of the 8 ± 1 nm
size MgO nanoparticles. The MIC of these particles on Staph.
aureus and E. coli strains was found in both cases to be
0.625 mg mL±1. The test was carried out in nutrient broth as the
traditional MIC test for bacteria.[33] In further experiments,
when testing the bactericidal effects of the MgO nanoparticles,
the concentration of 1 mg mL±1, which is above the MIC, was
used. The tests were performed while the bacteria were grown
in their broth medium. The sizes of the nanoparticles were, in
increasing order: 8 ± 1, 11 ± 1, 15 ± 1, 18 ± 1, and 23 ± 2 nm. The
results of the treatments are summarized in Table 2. The best
bactericidal activity can be gained by treating both bacterial
strains with MgO nanoparticles (1 mg mL±1) of 8 ± 1 nm size.
After 1 h of treatment, less than 20 % survivors can be found
in both cultures. Treatment for 4 h results in less than 0.1 %
survivors from E. coli and only 5 % from Staph. aureus. Treatment for 1 h with nanoparticles of 11 ± 1 nm resulted in Staph.
aureus being inactivated by 10 %, and after 4 h of treatment by
Table 2. Viability of Staph. aureus and E. coli cultures after treatment with MgO nanoparticles (1 mg mL±1) of various sizes in the growth medium (C.F.U.:
colony-forming units; N/N0: survival fraction).
Nanoparticle
size [nm]
Time of
treatment [h]
E. coli
Staph. aureus
C.F.U./ml
N/N0
% Reduction in
viability
C.F.U./ml
N/N0
% Reduction
in viability
8 ± 1 nm
0
1
4
8.1”108
1.4”108
4.1”107
1.0
1.7”10±1
5.1”10±2
0
83
95
5.3”107
1.0”107
5.0”102
1.0
1.9”10±1
9.4”10-4
0
82
< 99.9
11 ± 1 nm
0
1
4
6.0”108
5.5”108
2.1”108
1.0
9.0”10±1
3.5”10±1
0
10
65
6.8”108
8.0”107
4.2”107
1.0
1.3”10±1
7.0”10±2
0
87
93
15 ± 1nm
0
1
4
4.5”108
4.0”108
2.0”108
1.0
8.8”10±1
4.4”10±1
0
12
56
5.0”108
4.0”108
1.2”108
1.0
8.0”10±1
2.4”10±1
0
20
76
18 ± 1nm
0
1
4
2.0”108
1.0”108
9.5”107
1.0
5.0”10±1
4.7”10±1
0
50
53
1.9”108
1.0”108
8.0”107
1.0
5.2”10±1
4.2”10±2
0
48
58
23 ± 2 nm
0
1
4
1.2”108
8”107
7.2”107
1.0
6.6”10±1
6”10±1
0
33
40
2”108
1.6”108
1.3”108
1.0
8”10±1
6.5”10±1
0
20
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Adv. Funct. Mater. 2005, 15, 1708±1715
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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S. Makhluf et al./Nanocrystalline MgO and Its Use as a Bacteriocide
not affect their viable number, even after 2 or 4 h of treatment.
In addition to the above tests, reagents and pH conditions were
tested for the purpose of excluding the possibility that the precursors for the MgO nanoparticles are involved in the killing
process. From Table 3 it can be seen that commercial MgO or
Mg(CH3COO)2 alone do not affect the viability of either
tested culture in saline for at least 4 h of treatment.
Since the pH of the nanoparticles was found to be 10.6 in
both saline and broth media, it was thought that the high pH
was the major factor causing the killing effect of the nanoparticles. Concern over the effect of high basic pH on the viability
of bacteria was also indicated in previous work.[33] It seems
from the experimental results shown in Table 3 that culturing
both bacteria at pH 10.6 in saline, with no nanoparticles present, does not change the number of viable cells, at least for
up to 4 h of incubation. Although relatively large particles of
18 ± 1 nm or even 23 ± 2 nm are also capable of eradicating bacteria, we assume that it is primarily the action of the smaller
sized particles that are present in the mixture and are more efficient in killing bacteria.
X-ray microanalysis, combined with scanning electron microscopy, provides a unique tool for the elemental analysis of
individual bacterial-cell contents, which were fixed by quickfreezing in liquid nitrogen. Figures 5a,b demonstrate that treatment of the bacterial cells of both strains with MgO nanoparticles of 11 ± 1 nm diameter resulted in an influx of Mg. The increase of Mg is suggested to be the result of the penetration of
individual MgO nanoparticles into the cells, as proposed earlier
in this study. It should be emphasized that the outer attached
particles have been washed and removed. It should also be
mentioned that in the E. coli strain a significant loss of phosphate can be also seen, and in parallel, a loss of sulfur, indicating a leakage of the protein content of the cell. The latter may
be the reason why E. coli is more affected by the nanoparticles
than Staph. aureus. It is worthwhile to mention that E. coli bac-
65 %. E. coli is inactivated under the same conditions but to a
greater extent. 13 % survivors are found after 1 h and after 4 h
of treatment 7 % survivors are found. The yield of inactivation
is reduced by treating the cultures with 15 ± 1 or 18 ± 1 nm particles. Staph. aureus cultures are inactivated by 56 and 53 %, respectively, after 4 h of treatment. E. coli cultures are inactivated by 76 and 58 %, respectively, after the same treatment
period. Treating both cultures with the 23 ± 2 nm MgO nanoparticles resulted in a relatively small rate of inactivation.
Treatment of both Staph. aureus or E. coli with 1 mg mL±1 and
3 mg mL±1 of commercial MgO or Mg(CH3COO)2, the precursors for the nanoparticles, resulted in no antibacterial effect.
The growth of these cultures was similar to that of the untreated control (results not shown).
The activity of the larger particles after 4 h was also examined. It was found that the 18 ± 1 nm particles, after 24 h of activity, reduced the viability of Staph. aureus by 65 %. For E. coli,
the reduction of viability after 24 h was by 67 %. The results for
the 23 ± 2 nm show that the reductions of viability for Staph.
aureus and E. coli were 53 % and 58 %, respectively. This indicates the hindrance given by the protein in the media, so that it
takes longer for the larger particles to reach the cell and kill it.
Another set of bactericidal tests was performed, but this time
in saline (0.85 % NaCl) after washing the cultures from their
growth medium. In these tests we treated the washed bacteria
with the 8 ± 1 nm particles and the larger 18 ± 1 nm particles, as
well as the 23 ± 2 nm particles. Cultures of both bacteria were
eradicated completely after 2 h of treatment (Table 3). From
the results shown in Table 3 it can be seen that even the larger
23 ± 2 nm particles have inactivated both cultures by more than
99 %. This result may indicate that the MgO nanoparticles can
eradicate bacteria more efficiently when the medium does not
contain proteins. The latter may inhibit the adherence of the
nanoparticles to the bacterial cell. The sole incubation time of
the cultures in the saline solution without the nanoparticles did
Table 3. Viability of Staph. aureus and E. coli after treatment of MgO nanoparticles (np) in saline.
Duration of treatment
Treatment
2h
C.F.U./ml
N/N0
4h
% Reduction in
viability
C.F.U./ml
N/N0
% Reduction
in viability
Staph. aureus
No treatment
MgO-np 11±1nm
MgO-np 18±1nm
MgO-np 23±2nm
Mg(CH3COO)2
MgO (comm)
Saline pH 10.6
9.0”107
0
0
8”105
9.1”107
9.2”107
8.8”107
1.00
~10-8
~10-8
8.8”10±3
1.01
1.02
9.8”10-1
No treatment
MgO-np 11±1nm
MgO-np 18±1nm
MgO-np 23±2nm
Mg(CH3COO)2
MgO (comm)
Saline pH 10.6
6.4”107
0
0
7.5”105
6.3”107
6.5”107
6.5”107
1.00
~ 6.5”10±7
~ 6.5”10±7
8.3”10-3
9.8”10-1
1.02
1.02
±
100
100
99.12
0
0
2
9.0”107
0
0
8.2”105
8.9”107
9.0”107
8.5”107
1.00
~10±8
~10±8
9.1”10±3
9.9”10±1
1.00
9.4”10±1
±
100
100
99.1
1
0
6
6.4”107
0
0
7.8”105
6.2”107
6.4”107
6.6”107
1.00
~ 6.5”10±7
~ 6.5”10±7
7.5”10-3
9.7”10-1
1.00
1.03
±
100
100
99.25
3
0
0
E. coli
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
±
100
100
99.67
2
0
0
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Adv. Funct. Mater. 2005, 15, 1708±1715
Counts
Mg
2500
a
2000
1500
1000
O
500
Na
C
Al
Ca
K
P S Cl
0
1
Counts
2000
2
3
4
5
Energy (keV)
Mg
1500
b
1000
500
0
O
C
Na
1
Al
Ca
K
P S Cl
2
3
4
5
Energy (keV)
Figure 5. The X-ray elemental spectra of a) Staph. aureus and b) E. coli
treated with MgO nanoparticles (1 mg mL±1) of 11 ± 1 nm size for 4 h.
The elemental concentrations of the treated samples (thin lines) are compared to the untreated samples (thick lines).
teria can produce superoxide dismutase that may act on the
O2± produced by the MgO, resulting in formation of H2O2 that
penetrates the E. coli cell and oxidizes its active systems.[34]
Ultrastructural changes in bacterial cells of both bacteria are
shown by TEM (Fig. 6). Small, electron-dense black dots were
observed in the cytoplasm of the MgO-nanoparticle-treated
bacteria (Figs. 6b,d, panels). In the E. coli case, a low-density
area in the middle of the cell was observed (Fig. 6d). This supported the results of the X-ray microanalysis spectra on the existence of the MgO nanoparticles in the cells. These particles
within the cells are suggested to have been re-formed from individual MgO nanoparticles that penetrated the bacterial cell
wall and cell membrane. Two possible mechanisms are proposed for the killing of the bacteria. The first proposed mechanism of the bactericidal action of the MgO nanoparticles is that
presented in a former study.[35] The produced peroxides may
act as the oxidizing agent that causes the death of bacteria. The
fact that Staph. aureus is less sensitive to the antibacterial effect
of the MgO nanoparticles may be due to production of catalase, which neutralizes H2O2 to H2O and O2. The reaction of
catalase may be either endo- or exogenic. The oxidative activity of the peroxides is conducted inside the bacterial cell. The
second mechanism is based on the high surface-to-volume ratio
of the small nanoparticles, which would result in the formation
of more active oxygen species per unit weight. The activity of
the oxygen species is mostly executed outside the cell in destroying the cell membranes. Recently, we have ultrasonically
coated 200 nm silica spheres with 4±6 nm particles of MgO. We
examined the bacteriocidal effects of the coated particles and
found that after 4 h of incubation in saline with E. coli, 96 % of
the bacteria were killed. We believe that some fragments of the
of MgO particles break away from the 4 nm particle, assisted
by the solvent, and penetrate the membrane cell. It is also clear
that part of the MgO activity is via contact with the bacterial
membrane and the production of the active oxygen species
close to the membrane, which causes the destruction of the
membrane. The reduced bactericidal effect, as compared with
the 8 ± 1 nm free MgO particles, is due to less particles penetrating the membrane. Thus, both mechanisms may contribute
to the bactericidal effect of the MgO nanoparticles.
Further support for the importance of the two mechanisms is
obtained from the following experiment. Figure 7 depicts dose±
response curves recorded through application of a colorimetric
phospholipid/polydiacetylene (PDA) biomimetic membrane
assay,[36±38] which constitutes bilayer vesicles of phospholipids
(dimyristoylphsophatydilcholine, DMPC in the experiments
described herein), and PDA that undergo dramatic visible
blue±red transformations following the introduction of membrane-active compounds. The important property of the lipid/
PDA vesicle assay, in the context of this work, is its capability to
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S. Makhluf et al./Nanocrystalline MgO and Its Use as a Bacteriocide
a
b
Figure 6. The effect of MgO nanoparticles (1 mg mL±1, size 8 ± 1 nm for
4 h) on the ultrastructure Staph. aureus (a,b) and E. coli cells (c,d). TEM of
Staph. aureus untreated cells (a), and treated cells (b). E. coli untreated
cells (c) and treated cells (d). Magnification was 60K for all the cultures.
a±c) Scale bars: 0.5 lm.
Adv. Funct. Mater. 2005, 15, 1708±1715
Figure 7. The increase of % CR (CR: colorimetric response) values that
were correlated with the concentrations of MgO nanoparticles; a) 16 nm
and b) 12 nm, in the aqueous suspension.
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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S. Makhluf et al./Nanocrystalline MgO and Its Use as a Bacteriocide
report through visible blue±red transitions upon substances that
interact with membrane bilayers,[36,37] as well as evaluate the
extent of bilayer penetration.[36,38]
The curves in Figure 7 demonstrate an increase of percentage colorimetric response (% CR) values that were correlated
with the concentrations of MgO nanoparticles in the aqueous
suspension. % CR is a numerical value calculated from the
UV-vis spectra of the vesicle suspensions and is indicative of
the extent of blue±red transition: a higher % CR corresponds
to a more red appearance of the suspensions. Figure 7 shows
that the % CR increased when both MgO nanoparticle sizes
were added to the vesicles, indicating that both particles exhibited interactions with the lipid bilayers. Furthermore, Figure 7
shows that the dose±response curve of the 16 nm MgO nanoparticles was steeper than the curve obtained for the 12 nm
nanoparticles (i.e., the same particle concentration induced
more pronounced blue±red transition in case of the 16 nm particles). This result suggests that the 16 nm nanoparticles interact more strongly with the bilayer outer surface, compared to
the smaller 12 nm vesicles. On the other hand, the latter probably penetrate the bilayer better than the 16 nm particles.
3. Conclusion
The present paper has demonstrated that small crystalline
MgO nanoparticles have an efficient bactericidal activity. The
antibacterial activities of MgO-nanoparticles were tested by
treating E. coli (Gram negative) and Staph. aureus (Gram positive) cultures with 1 mg mL±1 of the nanoparticles. The results
revealed a clear size effect where the amount of eradicated
bacteria was strongly dependent on the particle size. The size
of the particle is related to the ease in which small individual
particles penetrate the membrane of the bacteria. The MgO in
this study was prepared using a MW radiation procedure,
which shortens the preparation time. However, there is no
other advantage in using these nanoparticles over other nanometer-sized MgO synthesized by other techniques; nanocrystalline MgO kills bacteria depending only on the particle size and
not on the method of preparation. It is worth mentioning, however, that no bactericidal effect was detected when amorphous
MgO nanoparticles were applied to the same targets. This effect is under further investigation.
4. Experimental
Synthesis: Mg(CH3COO)2´4H2O of the highest commercially available purity (99 %) and ethylene glycol were purchased from Aldrich
Co. A solution of 1 g of magnesium acetate tetrahydrate in 50 mL of
ethylene glycol was placed in a 100 mL glass flask. The solution was
purged for 45 min with nitrogen prior to turning on the microwave reactor. The reaction was carried out for 60 min under nitrogen and reflux conditions in a microwave oven. The microwave-assisted reaction
was carried out for a Spectra-900 W microwave oven, with a 2.45 GHz
working frequency. The oven was modified to include a refluxing system. In all the experiments, the microwave oven was cycled as follows:
on for 21 s, off for 9 s, with the total power always at 900 W. This cycling mode was chosen in order to reduce the risk of superheating the
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2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
solvent. All reactions were conducted under a nitrogen flow. In the
post-reaction treatment, the product was washed and centrifuged
(20 C and 9000 rpm) once with the mother liquid, and a few times with
ethanol in an inert atmosphere glove box. The product was then dried
in vacuum. Crystallization of the as-prepared product was carried out
by heating a small amount of the sample in a boat crucible at a temperature of 600 C in the open air, for 4 h.
Characterization: The X-ray diffraction patterns of the products were
recorded with a Bruker AXS D8 Advance Powder X-ray Diffractometer (using Cu Ka k = 1.5418 Š radiation). Peak fitting and lattice parameter refinement were computed using the Topas and Metric programs (Bruker Analytical X-Ray Systems). Energy dispersive X-ray
analysis was performed on a scanning electron microscope (JEOLJSM-840). The morphologies and micro- or nanostructure of the products were further characterized with a JEM-1200EX transmission electron microscope and a JEOL-2010 HRTEM using an accelerating voltage of 80 kV and 200 kV, respectively. Samples for TEM and HRTEM
analysis were prepared by ultrasonically dispersing the products into
absolute ethanol, then placing a drop of this suspension onto a copper
grid coated with an amorphous carbon film and then drying the grid in
air. The particles' size was calculated from the TEM grids by applying
the Scion Image program. The TGA measurement was carried out under a stream of nitrogen, at a heating rate of 10 C min±1 using a Mettler
TGA/STDA 851. The surface area was measured using a Micromeritics
(Gemini 2375) analyzer. The nitrogen adsorption and desorption
isotherms were obtained at 77 K after heating the sample at 120 C
for 1 h. The surface area was calculated from the linear part of the
BET plot.
Bactericidal Activity Tests: Two strains were tested in this study. One
was Staph. aureus, a Gram-positive bacteria (strain 195), coagulase
and DNAse-positive, which belongs to the phage type 0[88/89/95]. The
strain was resistant to methicillin, cephalothin, gentamicin, tobramycin,
and erythromycin antibiotics. The second strain was E. coli, a Gramnegative bacteria of serotype O111B4, which was resistant to ampicilin,
chloramphenicol, tetracycline, and sulfamethoxazol trimethoprim. Both
strains were recovered from clinical material submitted to the Bacteriology Laboratory of the Meir Hospital, Kfar-Saba, Israel [39].
Bacterial Growth and the Bactericidal Test Procedure: Overnight cultures of Staph. aureus and E. coli were grown on nutrient agar (Difco,
Detroit, MI). These cultures were transferred into Luria broth (LB;
Difco) at pH 6.5, to a final volume of 25 mL, at an initial optical density (OD) of 0.1 at 660 nm and allowed to grow at 37 C with aeration.
Nanoparticles of magnesium oxide (MgO, 1 mg mL±1) were added at
the beginning of the logarithmic phase, when the cultures reached an
optical density of 0.3 OD at 660 nm. The bacterial cells were allowed
to grow with the MgO nanoparticles, at 37 C with aeration, and samples (100 lL) were taken every hour. The samples were diluted tenfold
in saline and then transferred onto brain-heart agar plates (Difco). The
plates were allowed to grow for 24 h at 37 C and counted for viable
bacteria by counting the colony-forming units in each plate. The bacterial cell concentration at the beginning of the bactericidal test (0 time)
was also determined.
To ensure that any decrease in bacterial number was likely to be due
to exposure to nanoparticle treatment, two controls were included in
the experiment, one with the absence of bacteria and nanoparticles
(negative control) and the second with bacteria, but without the presence of nanoparticles (positive control). Both controls were individually placed in LB and their viabilities were monitored.
Bactericidal Test Procedure in Saline: The bacterial cells of the tested
strains were each grown overnight, as above, on a nutrient agar. These
cultures were centrifuged, and washed twice with saline NaCl 0.145 M at
the appropriate pH. Each pellet was re-suspended as follows: one sample was re-suspended in 1 ml of saline at pH 6.5, and the second sample
was re-suspended in 1 ml of MgO nanoparticles in saline at a 1 mg mL±1
concentration. The pH of this solution was measured to be 10.6. The
third sample was re-suspended in 1 ml of saline at pH 10.6. Bacterial
cells were incubated for up to 4 h. Samples of 100 lL each were taken at
the indicated times and diluted tenfold in saline (0.145 M). Overnight
cultures of E. Coli and Staph. aureus were grown on brain-heart agar
plates (Difco) and the viable bacteria were counted.
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Adv. Funct. Mater. 2005, 15, 1708±1715
Viable Count of Bacteria: For both tests, viable bacteria were monitored by counting the number of colony-forming units from the appropriate dilution on nutrient agar plates [40]. The survival fraction N/N0
was determined by calculating the colony-forming units (C.F.U.) per
mL of the culture. The term N0 denotes the number of colony-forming
units at the beginning of treatment before adding the nanoparticles
(time 0), and N is the number of colony-forming units after the treatment for the indicated time of each sample.
Colorimetric Measurements: Particles were diluted in buffer (50 mM
Tris-HCl tris(hydroxymethyl)aminomethane HCl, 17 mM EDTA,
pH 7) to 10 mg mL±1 concentrations prior to use. After sonication for
10 min the stock solution was diluted in the same buffer to obtain the
final concentration of 5 mg mL±1. Particle quantities were calculated
according to particle weight and total weight of particles in the vesicle±
particle suspension.
Dimyristoylphosphatidylcholine (DMPC) was purchased from Sigma (St. Louis, MO). The diacetylene monomer 10,12-tricosadiynoic
acid was purchased from GFS Chemicals (Powell, OH). Vesicles containing DMPC and polydiacetylene (PDA) [2:3 molar ratio] were prepared as follows: The phospholipids and monomer constituents were
dissolved in chloroform/ethanol solution (1:1), dried together in vacuo
followed by addition of deionized water and probe sonication at 70 C.
Following sonication, the vesicle solution was allowed to stand for few
minutes at room temperature and kept at 4 C overnight, followed by
irradiation at 254 nm for 10±20 s to induce polymerization of the PDA.
The particles were added to the vesicles (0.5 mM total lipid concentration, 25 mM Tris-base buffer at pH 7) and total solution volumes
were then adjusted to 1 mL with deionized water. Recording of UV-vis
spectra was carried out at room temperature on a Jasco spectrophotometer (Jasco, Japan) approximately 30 s after addition of compounds
to allow color stabilization.
A quantitative value for the degree of blue-to-red color transition is
given by the percentage colorimetric response (CR), which is defined as:
% CR = [(PB0 ± PBI)/PB0] ” 100
(3)
where PB = Ablue/(Ablue + Ared), A is the absorbance at either the
ªblueº component in the UV-vis absorbance spectrum (~ 640 nm) or
the ªredº component (~ 500 nm). PB0 is the initial red/blue ratio of the
control sample and PBI is the value obtained for the vesicle solution
after addition of the MgO nanoparticles.
X-Ray Microanalysis (XRMA): XRMA, combined with scanning electron microscopy, was used for the elemental analysis of individual bacterial cells [41,42] whose content was fixed by deep-freezing immediately
after 4 h of treatment with the nanoparticles. Untreated cultures served
as controls. Bacterial cultures were washed twice with 0.1 M of ammonium acetate and re-suspended in 20 lL of ammonium acetate. Each suspension (20 lL) was attached to an aluminum grid, air-dried at room temperature for at least 24 h, and then coated with a layer of carbon. XRMA
was performed using an X-ray system of an eXL link type attached to a
JEOL 840 scanning electron microscope. Each spectrum corresponds to
an average determination of approximately one million cells.
Transmission Electron Microscopy: Samples of Staph. aureus and
E. coli cultures were centrifuged immediately after 4 h of treatment
with the nanoparticles. Untreated cultures served as controls. They
were fixed in 25 % gluteraldehyde/paraformaldehyde at room temperature for 1 h, washed with a veronal/acetate buffer, and then fixed in
1 % osmium tetraoxide and uranyl acetate. Cells were prepared using
an LKB Ultratome III and examined using a JEOL 1200Ex transmission electron microscope.
±
[1]
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Received: January 20, 2005
Final version: April 20, 2005
Published online: September 1, 2005
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