Design, synthesis, characterization and bioevaluation of organic [618467]

Draft Articol
Design, synthesis, characterization and bioevaluation of organic
functionalized nano sili ca
Irina Zarafu1, Ali Abed Jebur Al Taweel1, Mariana Carmen Chifiriuc2, Marcela Popa2, Daniela
Culiță3,
Petre Ioniță1,3

1University of Bucharest, Faculty of Chemistry, Panduri 90 -92, Bucharest, Romania
2University of Bucharest, F aculty of Biology and The Research In stitute of the University of
Bucharest, Bucharest, Romania
3Institute of Physical Chemistry, Spl. Independentei 202, Bucharest, Romania
email: [anonimizat]

Abstract….
Keywords: synthesis, structural analysis, biol ogical , evaluation nanoparticles, nanosilica
A very well known process for derivatization of the silica gel for different types of
chromatography is represented by the i ntroduc tion of the amino groups on the surface of silic a.
In this work we have used plain silica gel as starting material for flash chromatography . This is
easily modified by reaction with 3 -aminopropyltrimethoxysilane (APTMOS), which reacts with
the hydroxyl groups from silica, forming a hydrocarbon layer on the surface of silica, which ends
with free amino groups (noted as aminopropyl -silica, APS). These amino groups are reactive
towards general reage nts used in organic chemistry. Thus, they should straightforwardly react
with an aromatic aldehyde, a carboxylic acid or an acid chloride derivative [26].
For the application of MSNs in the field of biomedicine some parameters have to be taken
into account such as: well -controlled nucleation and growth rate of the nanoparticles in order to
create nano materials with a uniform size distribution. Several strategies were used to synthesize
nanosized particles, including the use of templating agents, base or acid catalyzed processes and
co-solvents.
In the synthetic process, there are several steps which lead to the mesoporous particle
formation: nucleation, growth and aggregation as shown in Figure 6 , where . A and B) In an
emulsion the parallel arrangement of surfactant micelles occurs. C) After the polycondensation
of alkoxysilanes the formation of m esoporous silica nanoparticles is observed. D) To achieve Formatted: LeftDeleted: for biomedical
applications
Deleted:
Deleted: ,
Deleted: ¶
Introducing amino groups on
the surface of silica is a
Deleted: , used
Deleted: .
Deleted: at the beginning as
starting material plain silica gel for
flash chromatography. ¶

Comment [WU1]: as scoate
acest parag raf
Comment [WU2]: explicarea
abrevierii

mesoporosity the surfactant template is removed. Moreover, the surface is chemically modified
to control physical properties. E) Guest molecules are loaded in the mesoporous structure. Next,
the sur face modification is completed in order to finalize the nanovalve system for drug release
[27].

Fig. 6. General scheme of surface modified mesoporous silica nanoparticles .
Th aim of this stu dy was to prepare silica nanoparticles (SiNPs) and eight organic
compounds which then interacted to f inally obtain eight nano silica functionalized with organic
materials designed for biomedical applications.
1. Silica Nanoparticles Synthesis
1.1. Synthesis of Aminopropyl – Silica Nanoparticles (APS -NP) by Sol -Gel method.
The sol -gel method is the most common method used for the synthesis of silica
nanoparticles , as the reagents are not inexpensive and harmful. The obtained nano-silica are very
fine powder s consisting of spherical particles or microspheres with a mean diameter of 150 nm
with high specific surface area (15 to 25 m2/g) [16].
Materials
Table (1). The types, chemical formula, purity, and supplier of the raw materials used in the
synthesis of Aminopropyl -Modified Silica Nanoparticles (APS -NP)
Material Chemical formula Purity Supplier
Isopropanol C3H8O (> 90 %) Sigma Aldrich
TMOS (CH 3O)4Si ( ≥99 %) Sigma Aldrich
APTMOS H2N(CH 2)3Si(OCH 3)3 ( ≥96 %) Sigma Aldrich Deleted: is
Deleted: study includes the
method of preparation of
Deleted: ,
Deleted: the synthesis of
Deleted: in parallel , then interact
(SiNPs) with each of the organic
compounds that have been
prepared under special
circumstances
Deleted: , and f
Deleted:
Deleted: we get
Deleted: organic functionalized
nano silica , to be using it in
Deleted: Silica Nanoparticles
Deleted: are
Deleted: general
Deleted: of
Deleted: . The sol -gel technique
Deleted: is
Deleted: the silica gels
manufactured are non -poisonous
matters
Deleted: ¶
N
Deleted: produced by this
method is
Deleted: a
Deleted: that
Deleted: in table (1).

Dichloroethane ClCH 2CH 2Cl ( ≥99.8 %) Sigma Aldrich
Ammonia NH 3 ( ≥99.9 %) Sigma Aldrich

PROCEDURE:
To a solution of 80 ml isopropanol and 20 ml water there have been added 2 ml
concentrated ammonia, and, under vigorous stirring, 1 ml from a mixture made by mixing 0.8 ml
tetramethoxysilane (TMOS) and 0.2 ml 3 -aminopropyltrimthoxysilane( APTMOS) ; the addition
continued hourly, by adding 0.1 ml of the previous mixture after every 20 minutes to a solution
that has been prepared at t he beginning of preparation under vigorous stirring, after this the
solution remain ing under vigorous stirring for another three hours . The final solution was left
overnight and then refrigerat ed at 4○C till use [6-26]. The solution was washed by
dichloroethane (DCE) and divided and centrifug ed; the precipitate was taken in 10 ml of (DCE)
and a bit of methanol and then centrifuge d again and filtered to obtain the nano -silica particles
which were dried and weighed , the net weight being 0.41 g [26]. This process was repeated again
to obtain a sufficient amount of SiNPs (0.866 g) required to interact with the eight organic
compounds .

Fig.7. Aminopropyl -Modified Silica Nanoparticles (APS -NP)
2. Preparation of 2- (phenyloxy methylene) -benzoyle chloride.
To a solution made by dissolving 0.22 g of 2 -(phenoxymethyl) -benzoic acid (PMBA) in
9ml of (DCE) it has been added 0.2g of thionyl chloride SOCl 2, by reflux ing it for 6 hours,
followed by vigorous stirring for 7 days. Then , the composite evaporation allowed obtaining the
2-(Phenoxymethyl) benzoyl chloride (PMBC) pure compound, which was separat ed by
centrifug ation and then drying [28] (Scheme 1) .
DCE , SOCl 2

PMBA reflux , stirring for 7 days PMBC (1)
Scheme 1. Formatted: SubscriptFormatted: CenteredDeleted: made from
Deleted: has
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been finished,
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Deleted: compound
Deleted: A
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Deleted: ¶
Deleted: we did
Deleted: process of a composite
Deleted: pure compound
Deleted: .
Deleted: The last step is the
Deleted: ion
Deleted: e
Deleted: compound
Deleted: Where the interaction … [3]
… [7]… [2]… [1]
… [5]
… [6]… [4]
… [8]

For the organic compounds 2,3 and 4 were used as ready, i.e.: 1-Adamantanecarbonyl
chloride (A CC) (2), 2,4 -dinitro -1-fluoro -benzene (DFB) (3) , 2-Chloro -2,4-dinitro -benzoyl
chloride (CDBC) (4):

3. Preparation of the other four organic compounds
The 2-(4-florophenoxymethyl) -benzoyl chloride (5) compound was prepared from 2-(4-
florophenoxymethyl) -benzoic acid (0.246 g), dry 1,2 -dichlormethane (9 ml) and thionyl chloride
(0.4 mmol) , which were placed in a round -bottomed flask equipped with a condenser and drying
tube. The mixture was refluxed for 2 hours, and stirred for 4 days at room temperature . Then the
thionyl chloride excess was separated by evaporation and the solvent was removed under
reduced pressure.
Then, the same synthesis method , as for 2-(4-florophenoxymethyl) -benzoyl chloride , was used to
prepare 2-(4-chlorophenoxymethyl) -benzoyl chloride (6), 2-(4-methylphenoxymethyl) -benzoyl
chloride (7), 2- (4-methoxy phenoxymethyl) -benzoyl chloride (8), by using a 2 -(4-
chlorophenoxymethyl) -benz oic acid (0.262 g) with (6), 2 -(4-methylphenoxymethyl) -benzoic
acid (0.242 g) with ( 7), 2 -(4-methoxy phe noxymethyl) -benzoic acid( 0.258) with(8),
respectively , as in the following reactions:

2-(4-florophenoxymethyl) -benzoic acid 2-(4-florophenoxymethyl) -benzoyl chloride Formatted: Font: Not Italic
Formatted: HighlightDeleted: As for the
Deleted: 4
Deleted: are
Deleted:
Deleted: have been prepared in
advance, where we used directly,
which are as follows
Deleted:
Deleted: other
Deleted: by
Deleted: ing
Deleted: the excess thionyl
chloride
Deleted: were
Deleted:
Deleted: which already
mentioned,we will prepare,

2-(4-chlorophenoxymethyl) -benzoic acid 2 -(4-chlorophenoxymethyl) -benzoyl chloride

2- (4-methylphenoxymethyl) – benzoic acid 2 – (4-methylphenoxymethyl) -benzoyl chloride

2- (4-methylphenoxymethyl) – benzoic acid 2 – (4-methoxy phenoxymethyl) -benzoyl chlorid

3. Coupling of Silica Nanoparticles (APS -NP) with the organic compounds.
1.Silica Nanoparticles (APS -NP) reaction with 2 -(phenyloxy methylene) -benzoic chloride ( 1)
In a volume of 25 ml of dichloromethane (DCM) there dissolved 200 mg of Aminopropyl silica
nanoparticles (APS -NP) ( with 90 mg of 2 -(phenyloxy methylene )-benzoic chloride (PMBC) at
room temperature , stirred for two days, and then separated by centrifugation ; the organic
substance functionalized nano silica has been washed by (DCM) twice and then it was dried
[26].

+
PMBC (1)
Aminopropyl -silica (APS -NP)(APS -NP) -1 Deleted: Interact
Deleted: interact
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Deleted: the substance

2. Silica Nanoparticles (APS -NP) interact ion with 1-Adaman tanecarbonyl chloride (ACC)
(2)
In a volume of 20 ml of dichloromethane (DCM) there were dissolved 100 mg of
Aminopropyl silica nanoparticles (APS -NP) with 50 mg of 1 -Adamantanecarbonyl chloride
(ACC) at room temperature with the addition 1 ml of the solution base and tri -ethyl amine ,
stirred for two days, and then separated by centrifugation ; the organic substance functionalized
nano silica has been washed by (DCM) twice and then the dried [8].
+

ACC (2) (APS -NP) (APS -NP)-2

3. Silica nanoparticles (APS -NP) reaction with with 2,4-dinitro -1-Fluoro – benzene (3)
In a volume of 10 ml of (DCM) there were dissolved 50 mg of Aminopropyl silica
nanoparticles (APS -NP) with 66 mg of 2,4 -dinitro -1-Floro – benzene (DFB) at room temperature,
then 1 ml of the solution baseband triethylamine was added and stirr ed for two days, and then
were separated by centrifugation; the organic substan ce functionalized nano silica has been
washed by (D CM) twice and then dried.
+

(DFB) (3) (APS -NP) (APS -NP)-3
4. Silica Nanoparticles (APS -NP) reaction with 2 -chloro -2,4 dinitro -benzoylchloride (4)
In a volume of 12 ml of dichloroethane (DCM) there were dissolved 50 mg of
Amin opropyl silica nanoparticles (APS -NP) with 40 mg of 2 -chloro -2,4-dinitro -benzoyl
chloride (CDBC) at room temperature, then 1 ml of the solution baseband tri -ethyl amine was
added under stirring for two days and then by centrifugation ; the organic substance
functionalized nano silica has been washed by (DCM) twice and then dried. Deleted: reaction
Deleted: By
Deleted:
Deleted: of
Deleted: b
Deleted: and
Deleted: ing
Deleted: were
Deleted: process device
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Deleted: organic
Deleted: which was obtained
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Deleted: the added
Deleted: and
Deleted: were separated
Deleted: process device,
Deleted: organic
Deleted: which was obtained
Deleted: the substance

+

(CDBC) (4) (APS -NP) (APS -NP)-4
5.Silica Nanoparti cles (APS -NP) reaction with 2-(4-florophenoxymethyl) -benzoyl ch loride
(5)
In a volume of 20 ml of dichloromethane (DCM) and 1 ml of triethylamine (Et 3N) there were
dissolved 100 mg of nano silica particles with 200 mg in 2-(4-florophenoxymethyl) -benzoyl
chloride (1) at room temperature and stirr ed for two days, then separated by centrifugation and
after this the compound has been washed by (DCE) twice and then dry ed [8-26].
Then, in the same way as the nanosilica particles (APS -NP) interact ed with 2- (4-
florophenoxymethyl) -benzoyl chloride (5) , the nanoparticle s interact ed with the last three
compounds , respectively 2- (4-chlorophenoxymethyl) -benzoyl chloride (6), 2- (4-
methylphenoxymethyl) -benzoyl chloride (7), 2- (4-methoxy phenoxymethyl) -benzoyl chloride
(8) , Thus, four nanoscale organic compounds were obtained in the following reactions:

+

(APS -NP) (APS -NP) -5
+

(APS -NP) (APS -NP) -6 Deleted: interact
Deleted: By
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compound
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interaction
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Deleted: will
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Deleted: , as

+

(APS -NP) (APS -NP)-7
+

(APS -NP) (APS -NP)-8
Fig.19. Chemical procedures to obtain th e derivatizednanosilica partic les.

4. Characterization
1.Infrared spectroscopy (IR)
Samples were analyzed by a device with the following specifications:
– Thermo Electron Corporation Nicolet 4700 FT -IR Spectrome ter
– Smart Collector Accesory for diffuse reflectance
Performance Specifications
1-Spectral Range (Standard) 350 -17800 cm
2-Spectral Ra nge (Option, CsI Optics) 200 –6400cm -1
3-Spectral Range (Option , Extended -Range Optics) 375 – 11000cm -1
4-Spectral Range (Option, Multi -Range Optics) 15 –27000 cm -1 (Option)
5-Optical Resolution, 0.4 cm -1 6-Peak -To-Peak Noise (1 minute scan)
7-Wavenumber Precision 0.01 cm -1 8- Number of Scan Velocities 13
Infrared (IR) spectroscopy is the absorption measurement of different IR frequencies by a
sample positioned in the path of an IR beam. The main goal of the IR spectroscopic analysis is to
determine the chemical functional groups in the sample. Thus an IR s pectrum of a compound is
considered as the fingerprint for its identification [29]. In this study, we have obtained the IR – Deleted: an

spectra of the eight nano silica functioned with substances organic derived from Aminopropy l
silica nanoparticles (APS -NP) ( in figs. 21,22 and 23 ).
2. Dynamic Light Scattering (DLS)
Samples were analyzed by a device with the following specifications:
Beckman Coulter DeslaTM Nano common –Delsa™NanoC Zeta Potential and Submicron
Particle Size Analyzer.

Attributes
1-Molecular Weight: 103 – 3 × 107 Daltons 2- Size Range for Zeta Potential: 0.6 nm – 30 µm.
3-Electrophoretic Mobility: ± 1.2 × 10 -3 cm2/V.s 4-Zeta Potential: ± 200 MV.
5-Temperature Range 15 to 90 °C 6-Power Requirements 0 -300V
Scattering Angle (s)
1-Size: 15ș, 165ș, Zeta Potential: 15ș, 30ș 2-Sample Concentration: 10 ppm to 40%
3-Conductivity Range: Up to 200 ms /cm
Particle s ize can be determined by measuring the random changes in the intensity of light
scattered from a suspension or solution. This technique is commonly known as dynamic light
scattering (DLS), but is also called photon correlation spectroscopy (PCS) and quasi -elastic
lights DLS is most commonly used to analyze nanoparticles [30]. The basic principle is simple:
The sample is illuminated by a laser beam and the fluctuations of the scattered light are detected
at a known scattering angle θ by a fast photon detect or.
For the purposes of particle measurement, the shift in light frequency is related to the size
of the particles causing the shift. Due to their higher average velocity, smaller particles cause a
greater shift in the light frequency than larger particle s. It is this difference in the frequency of
the scattered light among particles of different sizes that is used to determine the sizes of the
particles present. In this study, the dynamic light scattering (DLS) of the eight substances and the
prepared Ami nopropyl silica nanoparticles (APS -NP), as described in Chapter 2 above, were
measured. The results were as in figure (24) in the third chapter of this thesis
3.Transmission Electron Microscopy ( TEM)
The samples were dispersed in ethanol and some droplets of the suspension were
deposited on a Lacey carbon foil supported on a Cu grid. TEM was performed on a Tecnai F30
(FEI, field emission gun (FEG), SuperTwin lens (point resolution CA. 0.2 nm), operated at 300
KV).
The TEM operates on the same basic princ iples as the light microscope, but uses
electrons instead of light. Because the wavelength of electrons is much smaller than that of light, Deleted: substances,
Deleted: functioned nano silica
Deleted: and are as

the optimal resolution attainable for TEM images is many orders of magnitude better than that
from a light microscop e. Thus, TEMs can reveal the finest details of internal str ucture -in some
cases as small as individual atoms [31].
The beam of electrons from the electron gun is focused into a small, thin, coherent beam
by the use of the condenser lens. This b eam is restricted by the condenser aperture, which
excludes high angle electrons. The beam, then strikes the specimen and parts of it are transmitted
depending upon the thickness and the electron transparency of the specimen. This transmitted
portion is fo cused by the objective lens into an image on phosphor screen or cha rge coupled
device (CCD) camera [32].
The image strikes the phosphor screen and light is generated, allowing the user to see the
image. The darker areas of the image represent those areas of the sample that fewer electrons are
transmitted through while the lighter areas of the image represent those areas of the sample.
In this study, particle size was measured for only two substances,Aminopropyl silica
nanoparticles (APS -NP) and) APS -NP) -6 substance, and the results were shown in fig. (25).
5.Biological tests
After the eight material organic functionalized nano silica have been obtained, and the identity of
these materials has been confirmed , they have been evaluated for their antimicrobial properties
by quantitive assay, allowing to establish the Minimum inhibitory concentration (MIC) and
minimum biofilm eliminating concentration (MBEC) against bacterial and fungal strains , i.e.
Enterococcus Faecalis , Pseud omonas aeruginosa , Staphylococcus aureus , Escherichia coli and
Candida albicans [33].
The MIC is defined as the lowest concentration of a chemical which prevents the visible growth
of a microbial liqui d culture [34]. MIC testing was performed by dissol ving 10 mg of each of
the eight compounds ((APS -NP)-1, (APS -NP)-2, (APS -NP)-3, (APS -NP)-4, (APS -NP)-5, (APS –
NP)-6, (APS -NP)-7, (APS -NP)-8 ) in 1 ml of Dimethyl sulfoxide (DMSO), obtaining a stock
solution of 10 mg/ ml, from which binary serial dilutions ranging from ≤0. 009 mg/ml to 5 mg/ml
have been achieved . Standar d microbial suspensions of 0.5 McFarland density prepared in sterile
saline have been used. By using 96 multi -well microplates , 10 binary dilutions of the tested
compounds have been p erformed in a total volume of 100 μl of nutrient broth [34-35]. The wells
11 and 12 of each row were assigned as negative and positive controls . The antimicro bial activity
of the DMSO solvent has been also assessed. After the inoculation, the plates were incubated for
24 h at 37 OC. After 24 hours the absorptio n of the well content was measured at OD620 nm (fig.
26,27,28,29,30). Inhibition of bacterial growth is observed when the solution in the well
remained clear after incubation. MIC was defined as the lowest concentration that completely
inhibit ed the visible growth of microorganisms. A fter reading the MIC, the plates were washed
by tap water to remove non -adherent cells and then washed with alcohol to stabilize and fix the
bacteria adherent on the plastic wells wall s, then fi lled with 150 μl of alcohol for 15 minutes, then
dyed with 130μl of 1% violet crystal for 10 minutes, and then washed with tap water. The stained
biofilm formed on the plastic walls was resuspended with acetic acid 33%, then transferred to the
spectrophotometer for measuring the OD a t OD492 nm and establishing the MBEC , defined as Formatted: Font: Not BoldFormatted: Font: Not Bold
Formatted: Font: Not BoldDeleted: the …identified by the
characterization …we wi ll now …
carry out the biological tests of
these eight substances. The
effectiveness of …will be
examined of these eight substances
on five strains of bacteria, which
are as follows
Deleted: PROCEDURE: ¶
a.
Deleted: Minimum inhibitory
concentration …(…)…assay :
bacterium … (in other words, at
which it has bacteriostatic
activity). This is in difference to
the minimum bactericidal
concentration (MBC) which is the
concentration resulting in
microbial death In other words, the
concentration at which it is
bactericidal. ¶
The MIC of a chemical is
determined by preparing solutions
of the chemical in vitro at
increasing concentrations,
incubating the solutions with the
separate batches of cultured
bacteria, and measuring the results
using agar dilution or broth
microdilution. Results have been
graded into susceptible (often
called sensitive), intermediate, or
resistant to a particular antibiotic
by using a cut off point …¶
… M…T…: Minimum
inhibitory concentration (MIC)
testing …ution …1…i.e.…the
concentrati on of each compound
was …. Minimum inhibitory
concentrations (MICs) …and also
for (MBEC) Assay …¶
A extracts from colonies of five
species of bacteria ( Enterococcus
Faecalis , Pseudomonas
aeruginosa , Staphylococcus
aureus , Escherichia coli , Candida
albicans ) were diluted with
distilled water, so that each species
is approximated to …standards ….¶
McFa rland standards are used as a
reference to adjust the turbidity of
bacterial suspensions so that the
number of bacteria will be within a
given range to standardize
microbial testing. ¶
…the Plate …which contains 8
rows and 12 columns, in the first
10 wells of the first row were
placed … …is a nutritional medium
for the growth of bacteria , and
then adde d 100 μl of the first the
substance (Organic functionalized
nano silica) (APS -NP)-1 on the
same ten wells, and then we did … [9]
… [10]

the lowest concentration which prevents the biofilm development figures (31,32,33,34,35) [34 –
36].
Results and discussion
A.Infrared spectrum
In this study, we have obtained eight IR –spectra of the mater ial organic functionalized
nano silica ( Figs. 21,22,23).
The organic materials functionalized nano silica were characterized by FTIR
spectroscopy between 4000 and 390 cm-1 and represented in f igures (21,22,23 ). At the first
glance, the IR spectra of all the solid materials look very similar. The absorption bands observed
at around 1075 cm-1 indicate the presence of vibration of the Si -O-Si bond which is strong in all
samples. The intensities of these bands are decreased in the samples of silica functionalized with
methyl and phenyl groups which indicate the hydrophobic behavior of the functionalized silica.
The main features of the spectra of silica functionalized by a methyl group were the
characteristic b and for aliphatic C -H stretching vibration of a methyl group at around 3000 -2800
cm-1. At the oth er wide peak of a small appear at 2925cm-1 represents an asymmetric stretch out
of a plane for alkyl group. Small peaks around 2900 cm-1 are due to the alkyl chains [16].
The spectra of all the samples show the absorption band at about 470 cm-1 which
corresponds to the Si -O rocks vibration where the oxygen atom moves per pendicular to the Si -O-
Si plane .
The band appeared at about 800 cm-1 may be due to the Si -O is bending vibration where the
oxygen move at right angles to the Si -Si lines in the Si -O-Si plane. While in figures 21 (A, B) , 22
(D) carbonyl groups are present almost at the peaks 1650 cm-1. The intensity of some peaks is
very small, and this is explained by the low ratio between organic and inorganic material. This
low ratio explains also the similarity of all the IR spectra [10] . For the figure (22 C) a peak of the
weak appears at 1548cm-1 represents a Nitro group [29].
Furthermore, the preparation of sample (APS -NP) at the figure 1 (A) displayed peaks at
790–800 cm−1 and 440 –450 cm−1 corresponding to the O -Si-O symmetric stretching and Si -O
bending vibration modes, respectively. The main features of the spectra of silica functionalized
by an ethyl group where the characteristic band for aromatic =C -H stretching vibration aromatic
ring around 3100 cm-1.
At the other wide peak of a small appear at 2925cm-1 represents an asymmetric stret ch
out of a plane for alkyl group, s mall peaks around 2900 cm-1 are due to the alkyl chains, as in f ig
22 (D, E ) .
The absorption peaks of phenyl group are observed at which associated with the
stretching vibration of ring C -H are clearly identified at 686 cm-1. The structure of the materials
in figures (21,22,23) was identified by using FTIR spectrometry for determini ng the success of
the reaction he spectra are given in the following figures along with the characteristic wide peak
descriptions the peak secondary amine groups almost appear at 3350cm-1 by weak absorption.
Formatted: HighlightDeleted: ¶
b. Minimum biofilm eliminating
concentration (MBEC) Assay: A
biofilm is any group of
microorganisms in which cells
stick to each other and often also
to a surface. These adherent cells
become embedded within a slimy
extracellular matrix that is
composed of extracellular
polymeric substances (EPS).
Microbes form a biofilm in
response to many factors, which
may include cellular recognition of
specific or non -specific attachment
sites on a surface, nutritional cues,
or in some cases, by exposure of
planktonic cells to sub -inhib itory
concentrations of antibiotics. ¶
After the MIC test was
completed, minimum biofilm
eliminating concentration
(MBEC) was tested on the same
plates as the MIC test. After
washing the plates by tap water,
then fi ll all the wells 150mm of
alcohol fo r 15minutes, and then
unload all the wells of the plates in
the wash basin, and then the
process of dyeing by the crystal
violet by the f ullness of all wells
by adding 130 μl of 1% crystal
violet per well for 10 minutes,
after coloring, all plates were
washed with tap water. The
remaining stain was removed by
washing extensively with DI water
a. Rinse 3 times with 250 µL of
sterile DI water, pipetting water
out, then Let t he plates dry
completely, then a ll of the plates
are filled with acetic acid at a
concentration of 33%, then
transferred to the
spectrophotometer for measuring
MBEC at OD492 nm . The purpose
of the violet crystal dye is to
determine the amount of biofilm
for the purpose of measurement.
The results were as in figures
(31,32,33,34,35) [34 -36].¶
:¶
1-Enterococcus Faecalis :
Enterococcus faecalis –formerly
classified as part of the group D
Streptococcus system is a Gram –
positive, commensal bacterium
inhabiting the gastrointestinal
tracts of humans and other
mammals.Like other species in the
Deleted: as in Figs.
Deleted: ,
Deleted: organic
Deleted: the
Comment [WU3]: ??? … [11]

These results provide evidence that the prepara tion of functionalizedsilica nanoparticles was
successful.While appearing peak of the absorption medium at 3170 cm-1 represents a phenyl
group in this region .

Fig.21. (A) (B) IR spectra (cm-1) of the material (APS -NP) -1,(APS -NP) -2 Respectively.

C D

E

F

Fig.22. (C) (D) ( E) (F) IR spectra (cm-1) of the material (APS -NP) -3,(APS -NP) -4 , (APS -NP) –
5 ,(APS -NP) -6 Respectively.

G

H

Fig.23. (G) (H) IR spectra (cm-1) of the material (APS -NP) -7,(APS -NP) -8 respectively.

B. Dynamic Light Scattering (DLS)
In this study , Dynamic Light Scattering (DLS) was measured for the eight materials that
were prepared, and also for Aminopropyl silica nanoparticles (APS -NP). W here all samples were
measured using methanol.

To confirm the formation of monolayer modification, silica nanoparticles before and
after the surface modifications were precisely characterized by dynamic light scatt ering (DLS)
spectrophotometer, as shown in Figure (24) .The particle characteristics data obtained on silica
nanoparticles particles is indicated in Table (2). The presence of bigger particles (diameter >500
nm) in the samples m ay be due to high temperature, as stated in the literature, reporting that a
higher temperature can cause agglomeration to occur more readily. This analysis shows that size
of particle can be affected by the trend of the samples to form agglomerates, which may be due
to the influence of Brownian motion. It was found that dynamic light scattering is affec ted by the
organic function of oligosiloxanes in hybrid nanocomposite solutions [30-37]. The intensity of
the distribution in some samples consists of two peaks and in other samples of three peaks
depending on the nature of the sample as shown in Fig (24 ). The reason for the size of the
particle diameter, which is more than 1000 nm, is to surround the solvent molecules o f the
particles, where measure the size of the diameter is the size of the part icle with the solvent
molecule a s shown in table (2).
A
B

C
D
Deleted: was
Deleted: their diameter
Deleted: it was reported that
Deleted: the
Deleted: will
Deleted: ,
Comment [WU4]: neclar

E
F

G
H

I

Fig.24. Scale intensity distribution of (DLS) technique for materials nanoparti cals (APS -NP)
(A), (APS -NP)-1(B), (APS -NP)-2 (C), (APS -NP)-3 (D), (APS -NP)-4 (E) , (APS -NP)-5(F),
(APS -NP)-6(G) , (APS -NP)-7(H), (APS -NP)-8 (I).

Intensity Distribution
Diameter(d) (nm) Nr. Materials
Nanoparticles
Peak 1 Peak 2 Peak 3 Diameter(d)
medium
(nm)

Polydispersity
Index (p.i)

1 (APS -NP) 581.1 10,849.8 –––- 927.0 0.243
2 (APS -NP) -1 123.4 668.4 17,902.5 935.4 0.367

3 (APS -NP) -2 1,744.4 58,254.4 –––- 2668.9 0.509
4 (APS -NP) -3 1,342.7 10,996.8 –––- 1116.6 0,368
5 (APS -NP) -4 199.9 1,058.5 –––- 582.3 0.333
6 (APS -NP 5 522.2 29,245.6 –––- 1605.3 0.404
7 (APS -NP) -6 585.9 18,522.7 –––- 1121.0 0.289
8 (APS -NP) -7 684.8 18,643.7 –––- 1128.6 0.279
9 (APS -NP) -8 722.7 18,285.9 –––- 1148.3 0.279

Table (2) shows diameter (d), Polydisper sity Index (p.i) for materials nanoparticles, which
measured by DLS technique and Diameter (d) medium.
C. Transmission Electron Microscopy ( TEM)
TEM images of Silica nanoparticles (APS -NP) (A, B, C, D) and (APS -NP) -6 (E, F) after
modifying the silica nanoparticles surface (figure 25) show clusters of primary particles with a
spherical shape and a 50 –200 nm diameter. The primary particles aggregate to form a cluster –
like structure, suggesting that the calcination temperature may induce particle aggregation. The
TEM images suggest that t he calcination temperature plays an important role in the formation of
silica manoparticles materials. The result shows that the particle size has a better uniform
distribution[38]. Deleted: modified
Deleted: , as shown in
Deleted:
Deleted: (
Deleted: , the
Deleted: ed
Deleted: d
Deleted: ed

A
B
C

D
E
F

Fig.25. TEM image of particle size distribution of nanosilica particles (APS -NP) (A, B, C, D)
and (APS -NP) -2 (E, F)

D.Biological tests
The antimicrobial activity of eight the organic material functionalized nano silica (APS -NP) was
tested in vitro on both Gram-positive and Gram-negative bacteria , as well as funga l strains.
Enterococcus faecalis , formerly classified as part of the Streptococcus genus, sero group D is a
Gram -positive, commensal bacterium inhabiting the gastrointestinal tracts of humans and other
mammals. Like other species in the genus Enterococcus, E. faecalis can cause life -threatening
infections in humans, especially in the nosocomial (hospital) environment, where the naturally
high levels of antibiotic resistance contribute to its pathogenicity . Staphylococcus aureus is a
Gram-positive, round -shaped bacteriu m frequently found in the nose, respiratory tract, and on
the skin.. Although S. aureus is not always pathogenic, it is a common cause of skin infections,
including abscesses, respiratory infections such as sinusitis, and food poisoning.
Pseudomonas aeruginosa is a common Gram -negative, rod -shaped bacterium that can cause
disease in plants and animals, including humans. A species of considerable medical importance,
P. aeruginosa is a multi drug resistant pathogen recogni zed for its ubiquity , its intrinsically
advanced antibiotic resistance mechanisms, and its association with serious illnesses – hospital –
acquired infections such as ventilator -associated pneumonia and various sepsis syndromes.
Escherichia coli is a Gram-negative, facultative anaerobic, rod -shaped, coliform bacterium of the
genus Escherichia that is commonly found in the lower intestine of warm -blooded organisms.
Most E. coli strains are harmless, but some s erotypes can cause serious food poisoning in their Formatted: Font: Italic
Formatted: Font: Italic
Formatted: Font: Italic
Formatted: Font: Not Bold
Formatted: Font: Italic
Formatted: Font: Not Bold
Formatted: Font: Italic
Formatted: Font: Not Bold
Formatted: Font: ItalicDeleted: ¶
Deleted: capability
Deleted: organic
Deleted: to inactivate bacteria
were
Deleted: g
Deleted: g

hosts, and are occasionally responsible for product recalls due to food contamination. Candida
albicans is a type of yeast that is detected in the gastrointestinal tract and mouth of 40-60% of
healthy adults . It is usually a commensal organism, but it can become pathogenic in
immunocompromised individuals such as the HIV -infected patients.
The MIC of the obtained compounds determined against these five microbial strains ranged from
≤0. 009 mg/ml to 5 mg/ml. The DMSO solvent did not show any antimicrobial activity, thus not
interf ering with the assay of the tested compounds.
The functionalized s ilica nanoparticles did not exhibit any significant antimicrobial effect ,
excepting S6 which exhibited a moderate antimicrobial activity on the E. faecalis strain ( 0.625
μg/ml) and Se6 and S8 on the on the E. coli strain (1.25 μg/ml).

Fig.26 a. Formatted: Font: Not Bold
Formatted: Justified
Formatted: Font: ItalicDeleted: was
Deleted: as the lowest
concentration that totally inhibits
visible bacterial growth. Minimum
inhibitory concentrations (MICs)
Deleted:
Deleted: ing
Deleted: By observing the
results, t
Deleted:
Deleted: model showed no effect
on the disruption of bacterial
growth as shown in figures
(26,27,28,29,30 ) which prove that
the recorded effect mainly come
from the substances addition. ¶
Deleted: The adherence was
quantified spectrophotometrically
(optical density) at 620 nm for
MIC and 492 for MBEC. ¶
The results of MIC were
shown in the following five forms,
where form (26) represents a test
of the MIC effect on the bacteria
of Enterococcu s Faecalis strain,
while Fig. 27, 28, 29 and 30 are
the same MIC test for the eight the
material organic functionalized
nano silica (APS -NP) on
Pseudomonas aeruginosa ,
Staphylococcus aureus , Esherchia
coli, Candida albicans
respectively. ¶
The eight the ma terial organic
functionalized nano silica were
applied on five types of bacteria
as mentioned in table (3.2) ¶
Symbol
Deleted: A form representing
the effectiveness of MIC for eight
compounds on the Pseudomonas
aeruginosa type of bacteria …. [12]

Fig.26b.

Fig.26c . Deleted: 27. A form representing
the effectiveness of MIC for eight
compounds on Enterococcus
Faecalis type of bacteria .
Deleted: 28 A form representing
the effectiveness of MIC for eight
compounds on the Staphylococcus
aureus type of bacteria

Fig.2 6d.

Fig.26e
Fig. 26a -e Intensity of microbial planktonic growth in the presence of binary dilutions of the
tested compounds ..
2. Minimum biofilm erradicating concentration (MBEC) assay Deleted: 9. A form representing
the effectiveness of MIC for eight
compounds on the Escheri chia
colitype of bacteria
Deleted: 30. A form representing
the effectiveness of MIC for eight
compounds on the Candida
albicans type of bacteria
Deleted: eliminating

A biofilm is any group of microorganisms in which cells stick to each other and often also to a
surface. These adherent cells are embedded within a slimy extracellular matrix that is composed
of extracellular polymeric substances (EPS). Microbes form a biofilm in response to many
factors, which may include cellular recognition of specific or non -specific attachment sites on a
surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub -inhibitory
concentrations of antibiotics.
In this stu dy, we have assessed the efficien cy of the obtained compounds agains t biofilm
embedded microbial cells. The calcula ted MBEC values revealed that the eight nanoscal e
organic compounds did not significa ntly influence the biofilm growth of the tested strains ( Fig.
(27 a -e)
.

Fig.27a
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: JustifiedFormatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: JustifiedFormatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: JustifiedFormatted: JustifiedFormatted: Justified
Formatted: Justified
Formatted: Justified
Formatted: JustifiedDeleted: Gram -positive bacteria
are bacteria that give a positive
result in the Gram stain test, which
is traditionally used to quickly
classify bacteria into tw o broad
categories according to their cell
wall. Gram -positive bacteria take
up the crystal violet stain used in
the test, and then appear to be
purple -colored when seen through
a microscope. This is because the
thick peptidoglycan layer in the
bacterial c ell wall retains the stain
after it is washed away from the
rest of the sample, in the
decolorization stage of the test. ¶
Gram -negative bacteria cannot
retain the violet stain after the
decolorization step; alcohol used in
Deleted: test and after
Deleted:
Deleted:
Deleted: a
Deleted: had
Deleted: a positive effectin
Deleted: 31. A form representing … [14]
… [15]… [13]
… [16]

Fig. 27b.

Fig. 27c.
Deleted: 32. A form representing
the effectiveness of MBEC for
eight compounds on the
Pseudomonas aeruginosa type of
bacteria
Deleted: 33. A form representing
the effectiveness of MBEC for
eight compounds on the
Staphylococcus Aureus type of
bacteria

Fig. 27d.

Fig. 27e
Fig. 2 7a-e Intensity of microbial biofilms growth in the presence of binary dilutions of the
tested compounds

CONCLUSIONS Deleted: 34. A form representing
the effectiveness of MBEC for
eight compounds on Escherichia
Coli type of bacteria
Deleted: 35. A form representing
the effectiveness of MBEC for
eight compounds on the Candida
Albicans type of bacteria.

Duringthisstudy, Aminopropylsilicananoparticles(APS -NP) were prepared by the sol – gel
method, and then used them to interactwitheightorganic compounds, in order to configure eight
organic material functionalized nano silica . The hybrid compounds were synthesized following
the interaction of 2-(phenoxymethyl) -benzoic acid (PMBA) , 1 -adamantanecarbonyl chloride
(ACC), 2 -4-dinitro -1-floro -benzene (DFB), 2 -chloro -2,4 dinitro -benzoyl chloride (CDBC) , 2-
(4-floropheno xymethyl) -benzoyl chloride(5) ,2 – (4-chlorophenoxymethyl) -benzoyl chloride (6)
,2- (4-methylphenoxymethyl) -benzoyl chloride (7) ,2 – (4-methoxy phenoxymethyl) -benzoyl
chloride (8) with Aminopropyl silica nanoparticles(APS -NP) in spe cific conditions , the
following organic nanomaterials (APS -NP)-1, (APS -NP)-2, (APS -NP)-3, (APS -NP)-4 (APS –
NP)-5, (APS -NP)-6, (APS -NP)-7, (APS -NP)-8 being obtained . The obtained structures were then
characterized by psysico -chemical methods. TEM examination has revea led that the obtained
nano particles have a spherical shape and their size ranges from 50 -200 nm to few micrometers.
The averag e nanoparticle s diameters were measured, and finally the presence of the functional
groups was confirmed by the IR measurements.
– The assessment of the antimi crobial activity of the obtained nanomaterials against different
Gram-positive and Gram-negative bacterial and fu ngal strains in planktoni c and biofilm growth
state revealed that S4, S6, and S8 materials proved to be the most efficient against E. faecalis and
E. coli strains , both of them being used as microbial indicators of faecal pollution in different
meda, suggesting the potential of the obtained materials to be used as d isinfectant agents.
Formatted: Font: Italic
Formatted: Font: ItalicDeleted: –
Deleted: itwasprepared
Deleted: wereconsidered
effective most important amine
group for nanoparticalorganic
compounds,
Deleted: then
Deleted: thesenanoparticle
Deleted: s to
Deleted: the
Deleted: organic
Deleted: ,
thenstudytheirproperties and
seetheir structures by meas uring
the infrared radiation.
Deleted: ¶
-Size the material organic
functionalized nano silica was
characterizated using TEM
technology, Where was obtained
some of the particles have a
spherical shape and Its size ranges
from 50 -200 nm to few
micrometers. Then, using the DLS
technique
Deleted: is
Deleted: Then preparation and
use organic compounds following
2-(phenoxymethyl) -benzoic acid
(PMBA) , 1 -adamantanecarbonyl
chloride (ACC), 2 -4-dinitro -1-
floro -benzene (DFB), 2 -chloro -2,4
dinitro -benzoyl chloride (CDBC) ,
2- (4-floropheno xymethyl) –
benzoyl chloride (5),2 – (4-
chlorophenoxymethyl) -benzoyl
chloride (6) ,2 – (4-
methylphenoxymethyl) -benzoyl
chloride (7) ,2 – (4-methoxy
phenoxymethyl) -benzoyl chloride
(8) where interaction with
Aminopropyl silica
nanoparticles(APS -NP) under
special circumstances, wher e was
obtained Nanomaterials organic
following (APS -NP)-1, (APS -NP)-
2, (APS -NP)-3, (APS -NP)-4 (APS –
Deleted: effectiveness
Deleted: of
Deleted: Minimum inhibitory
concentration (MIC) and minimum
biofilm eliminating concentration
Deleted: ).
Deleted: -Overall, we envisage
that further development of these
nanoparticles will provi de a variety … [17]
… [18]
… [19]

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Then washed t

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into two parts, then separated by

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e, after that we take t he precipitate and add to both the tu

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where the total value of the weight for s ilica nano particles (SiNPs) was reached
(0.866 g).

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after this has been under

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process of a composite output for

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Where the interaction equation as follows:

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the

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identified by the characterization

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we wi ll now

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carry out the biological tests of these eight substances. The effectiveness of

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will be examined of these eight substances on five strains of bacteria, which are as follows

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Minimum inhibitory concentration

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(

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)

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assay :

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bacterium

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(in other words, at which it has bacteriostatic activity). This is in difference to the minimum
bactericidal concentration (MBC) which is the concentration resulting in microbial death In
other words, the concentration at which it is bactericidal.
The MIC of a chemical is determined by preparing solutions of the chemical in vitro at
increasing concentrations, incubating the solutions with the separate batches of cultured
bacteria, and measuring the results using agar dilution or broth microdilution. Results have
been graded into susceptible (often called sensitive), intermediate, or resistant to a particular
antibiotic by using a cut off point

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M

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T

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: Minimum inhibitory concentration (MIC) testing

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ution

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1

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i.e.

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the concentrati on of each compound was

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. Minimum inhibitory concentrations (MICs)

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and also for (MBEC) Assay

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A extracts from colonies of five species of bacteria ( Enterococcus Faecalis , Pseudomonas
aeruginosa , Staphylococcus aureus , Escherichia coli , Candida albicans ) were diluted with
distilled water, so that each species is approximated to

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standards

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.
McFa rland standards are used as a reference to adjust the turbidity of bacterial suspensions so
that the number of bacteria will be within a given range to standardize microbial testing.

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the Plate

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which contains 8 rows and 12 columns, in the first 10 wells of the first row were placed

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is a nutritional medium for the growth of bacteria , and then adde d 100 μl of the first the
substance (Organic functionalized nano silica) (APS -NP)-1 on the same ten wells, and then
we did the work of serial dilution, was added 200 μl of bacteria Enterococcus Faecalis for
inoculation on the same ten wells mentioned abo ve

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W

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control

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,while 12 as control

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test of each plate, where only 11 Broth was placed in the well and 12 was placed in the well
the colonies of bacteria with broth

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And so on the same way for the rest of the seven other compounds and using

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Dimethyl sulfoxide (DMSO) medium as a control, with each type of four types other bacteria
previousl y mentioned .

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all

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we transfer all the plates to the spectrophotometer for the purpose of measuring

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materials

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.The results were as in figures

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extract

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s

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the

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ll

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with salt water to rid the bacteria of the broth, or

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s of the well .

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b. Minimum biofilm eliminating concentration (MBEC) Assay: A biofilm is any group of
microorganisms in which cells stick to each other and often also to a surface. These adherent
cells become embedded within a slimy extracellular matrix that is composed of extracellular
polymeric substances (EPS). Microbes form a biofilm in response to many factors, which may
include cellular recognition of specific or non -specific attachment sites on a surface,
nutritional cues, or in some cases, by exposure of planktonic cells to sub -inhib itory
concentrations of antibiotics.
After the MIC test was completed, minimum biofilm eliminating concentration
(MBEC) was tested on the same plates as the MIC test. After washing the plates by tap water,
then fi ll all the wells 150mm of alcohol fo r 15minutes, and then unload all the wells of the
plates in the wash basin, and then the process of dyeing by the crystal violet by the f ullness of
all wells by adding 130 μl of 1% crystal violet per well for 10 minutes, after coloring, all
plates were w ashed with tap water. The remaining stain was removed by washing extensively
with DI water a. Rinse 3 times with 250 µL of sterile DI water, pipetting water out, then Let
the plates dry completely, then a ll of the plates are filled with acetic acid at a concentration of
33%, then transferred to the spectrophotometer for measuring MBEC at OD492 nm . The
purpose of the violet crystal dye is to determine the amount of biofilm for the purpose of
measurement. The results were as in figures (31,32,33,34,35) [34 -36].
:
1-Enterococcus Faecalis : Enterococcus faecalis –formerly classified as part of the group D
Streptococcus system is a Gram -positive, commensal bacterium inhabiting the gastrointestinal
tracts of humans and other mammals.Like other species in the genus Enterococcus, E. faecalis
can cause life -threatening infections in humans, especially in the nosocomial (hospital)
environment, where the naturally high levels of antibiotic resistance found in E. Faecalis
contribute to its pathogenicity.

2-Pseudomon as aeruginosa : is a common Gram -negative, rod -shaped bacterium that can
cause disease in plants and animals, including humans. A species of considerable medical
importance, P. aeruginosa is a multidrug resistant pathogen recognised for its ubiquity, its
intrinsically advanced antibiotic resistance mechanisms, and its association with serious
illnesses – hospital -acquired infections such as ventilator -associated pneumonia and various
sepsis syndromes.
3-Staphylococcus aureus : is a gram -positive, round -shape d bacterium that is a member of the
Firmicutes, and is frequently found in the nose, respiratory tract, and on the skin. It is often
positive for catalase and nitrate reduction and is a facultative anaerobe that can grow without
the need for oxygen. Althou gh S. aureus is not always pathogenic, it is a common cause of
skin infections, including abscesses, respiratory infections such as sinusitis, and food
poisoning.
4-Escher ichia coli : is a gram -negative, facultatively anaerobic, rod -shaped, coliform
bacteri um of the genus Escherichia that is commonly found in the lower intestine of warm –
blooded organisms (endotherms). Most E. Coli strains are harmless, but some stereotypes can
cause serious food poisoning in their hosts, and are occasionally responsible for product
recalls due to food contamination.
5-Candida albicans : is a type of yeast that is a common member of the human gut flora. It
does not proliferate outside the human body.It is detected in the gastrointestinal tract and
mouth in 40 -60% of healthy adu lts. It is usually a commensal organism, but can become
pathogenic in immunocompromised individuals under a variety of conditions. It is one of the
few species of the Candida genus that causes the human infection candidiasis, which results
from an overgrow th of the fungus. Candidiasis is, for example to, often observed in HIV –
infected patients.

A B C D E
Fig.20. Images of microscopic strains of bacteria are (A) Enterococcus Faecalis , (B)
Pseudomonas aeruginosa , (C) Staphylococcus aureus , (D) Escherichia coli , (E) Candida
albicans .
PROCEDURE:
a.Minimum inhibitory concentration (MIC) assay : is the lowest concentration of a chemical
which prevents the visible growth of a bacterium (in oth er words, at which it has
bacteriostatic activity). This is in difference to the minimum bactericidal concentration
(MBC) which is the concentration resulting in microbial death In other words, the
concentration at which it is bactericidal.
The MIC of a ch emical is determined by preparing solutions of the chemical in vitro at
increasing concentrations, incubating the solutions with the separate batches of cultured

bacteria, and measuring the results using agar dilution or broth microdilution. Results have
been graded into susceptible (often called sensitive), intermediate, or resistant to a particular
antibiotic by using a cut off point [34].
MIC Testing: Min imum inhibitory concentration (MIC) testing was performed by
dissolution 10 mg of each of the eight compounds ((APS -NP)-1, (APS -NP)-2, (APS -NP)-3,
(APS -NP)-4, (APS -NP)-5, (APS -NP)-6, (APS -NP)-7, (APS -NP)-8 ) in 1 ml of Dimethyl
sulfoxide (DMSO), i.e. t he concentrati on of each compound was 10 mg/ ml. Minimum
inhibitory concentrations (MICs) ranging from ≤0. 009 mg/ml to 5 mg/ml and also for
(MBEC) Assay.
A extracts from colonies of five species of bacteria ( Enterococcus Faecalis , Pseudomonas
aeruginosa , Staphylococcus aureus , Escherichia coli , Candida albicans ) were diluted with
distilled water, so that each species is approximated to McFarland standards.
McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so
that t he number of bacteria will be within a given range to standardize microbial testing.
By using the Plate 96, which contains 8 rows and 12 columns, in the first 10 wells of the
first row were placed 100 μl of nutrient broth is a nutritional medium f or the growth of
bacteria , and then added 100 μl of the first the substance (Organic functionalized nano silica)
(APS -NP)-1 on the same ten wells, and then we did the work of serial dilution, was added
200 μl of bacteria Enterococcus Faecalis for inocula tion on the same ten wells mentioned
above [34 -35].
Wells 11 and 12 were assigned as control negative,while 12 as control positive test of
each plate, where only 11 Broth was placed in the well and 12 was placed in the well the
colonies of bacte ria with broth. And so on the same way for the rest of the seven other
compounds and using Dimethyl sulfoxide (DMSO) medium as a control, with each type of
four types other bacteria previously mentioned .
After the inoculation, all the plates were incubate d for 24 h at 37 OC.
After 24 hours we transfer all the plates to the spectrophotometer for the purpose of
measuring the absorptio n of the materials at OD620 nm .The results were as in figures
(26,27,28,29,30). Inhibition of bacterial growth is observed when the solution in the well
remained clear after incubation. MIC was defined as the lowest extract concentration that
completely inhibits the growth of microorganisms. All the plates were washed with salt water
to rid the bacteria of the broth, or to remove non -adherent cells and then washed with alcohol
to stabilize and fix the bacteria on the walls of the well.
b. Minimum biofilm eliminating concentration (MBEC) Assay: A biofilm is any group of
microorganisms in which cells stick to each oth er and often also to a surface. These adherent
cells become embedded within a slimy extracellular matrix that is composed of extracellular
polymeric substances (EPS). Microbes form a biofilm in response to many factors, which may
include cellular recogniti on of specific or non -specific attachment sites on a surface,
nutritional cues, or in some cases, by exposure of planktonic cells to sub -inhibitory
concentrations of antibiotics.
After the MIC test was completed, minimum biofilm eliminating concent ration
(MBEC) was tested on the same plates as the MIC test. After washing the plates by tap water,

then fi ll all the wells 150mm of alcohol for 15 minutes, and then unload all the wells of the
plates in the wash basin, and then the process of dyeing by the crystal violet by the f ullness of
all wells by adding 130 μl of 1% crystal violet per well for 10 minutes, after coloring, all
plates were washed with tap water. The remaining stain was removed by washing extensively
with DI water a. Rinse 3 times with 25 0 µL of sterile DI water, pipetting water out, then Let
the plates dry completely, then a ll of the plates are filled with acetic acid at a concentration of
33%, then transferred to the spectrophotometer for measuring MBEC at OD492 nm . The
purpose of the violet crystal dye is to determine the amount of biofilm for the purpose of
measurement. The results were as in figures (31,32,33,34,35) [34 -36].

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The adherence was quantified spectrophotometrically (optical density) at 620 nm for MIC
and 492 for MBEC.
The results of MIC were shown in the following five forms, where form (26) represents a
test of the MIC effect on the bacteria of Enterococcu s Faecalis strain, while Fig. 27, 28, 29
and 30 are the same MIC test for the eight the material organic functionalized nano silica
(APS -NP) on Pseudomonas aeruginosa , Staphylococcus aureus , Esherchia coli , Candida
albicans respectively.
The eight the ma terial organic functionalized nano silica were applied on five types of
bacteria as mentioned in table (3.2)
Symbol S1 S2 S3 S4 S5 S6 S7 S8 S9
Substance (APS –
NP)-1 (APS –
NP)-2 (APS –
NP)-3 (APS –
NP)-4 (APS –
NP)-5 (APS –
NP)-6 (APS –
NP)-7 (APS –
NP)-8 DMSO

Table (3) Shows the eight the material organic functionalized nano silica, and DMSO
substance .
1. Minimum inhibitory concentration (MIC) assay
After comparing all the results obtained with readings of control negatives adsorption, can
be observed from figures (27,28,29) that no reasonable effect was detected for Silica
nanoparticles substances on ( Pseudomonas aeruginosa , Staphylococcus aureus , Escherichia
coli ) strains of bacteria, d ifferent ratios were recorded, but they did not reach the required
level.
Figure 26 i ndicate an effective result of (S6) sample of the Enterococcus Faecalis type of
bacteria. However the effected did not indicate at all concentrations, that the readi ng for MIC
can be detected at ( 0.625) concentration for sample (S6) of the eight substances
concentrations.
Candida albicans type of bacteria w as also shown an inhibition, i ndicate an effective result
under the effect of using substances S5 and S6, figure (30) exposes the detected efficiency of
the samples S5, S6 at MIC (0.312 and 0.009) concentrations of the samples, respectively.

As men tioned before the S5, S6 and S2 samples proved their efficiency as substance can
be used to determine eliminate the effect of the most popular types of bacteria in our life.
More studies should achieve to ensure expert them with other types of bacteria. Al so more
studies must be achieved on the un effective types of samples to indicate them with other
nutrition and types of bacteria.
By observing the results, the DMSO model showed no effect on the disruption of bacterial
growth as shown in figures (26,27,28 ,29,30 ) which prove that the recorded effect mainly
come from the substances addition.

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Gram -positive bacteria are bacteria that give a positive result in the Gram stain test, which is
traditionally used to quickly classify bacteria into tw o broad categories according to their cell
wall. Gram -positive bacteria take up the crystal violet stain used in the test, and then appear to
be purple -colored when seen through a microscope. This is because the thick peptidoglycan
layer in the bacterial c ell wall retains the stain after it is washed away from the rest of the
sample, in the decolorization stage of the test.
Gram -negative bacteria cannot retain the violet stain after the decolorization step; alcohol
used in this stage degrades the outer memb rane of gram -negative cells, making the cell wall
more porous and incapable of retaining the crystal violet stain. Their peptidoglycan layer is
much thinner and sandwiched between an inner cell membrane and a bacterial outer
membrane, causing them to take up the counterstain (safranin or fuchsine) and appear red or
pink.
Despite their thicker peptidoglycan layer, gram -positive bacteria are more receptive to
antibiotics than gram -negative, due to the absence of the outer membrane [39].
Both gram -positive and gram -negative bacteria commonly have a surface layer called an S –
layer. In gram -positive bacteria, the S -layer is attached to the peptidoglycan layer (in gram –
negative bacteria, the S -layer is attached directly to the outer membrane) . Specific for gram –
positive bacteria is the presence of teichoic acids in the cell wall.
By observing the

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test and after comparing all the results obtained with the negative control, we can say

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a positive effectin different concentrations , where the potent biofilm inhibition activity was
detected against two types only of types of bacteria it is ( Staphylococcus aureus , Candida
albicans ) as shown in Fig (33, 35)respectively , and the results were shown in the table (4)
below , while no any reasonable effect was detected for the other three types it is
(Enterococcus Faecalis , Pseudomonas aeruginosa , Candida albicans ) as shown in Fig
(31,32, 34 ) , this may be due to the non -format ion of these t ypes of biofilm, b ecause of the
negative control results the same results of the positive control .
Type of bacteria
Staphylococcus aureus Type of bacteria
Candida albicans

Symbol of sample (MBEC) Symbol of sample (MBEC)
S1 5 mg/ml S1 1.25 mg/ml
S2 2.5 mg/ml S2 1.25 mg/ml
S3 2.5 mg/ml S3 1.25 mg/ml
S4 2.5 mg/ml S4 0.625 mg/ml
S5 2.5 mg/ml S5 5 mg/ml
S6 2.5 mg/ml S6 5 mg/ml
S7 2.5 mg/ml S7 0.625 mg/ml
S8 2.5 mg/ml S8 1.25 mg/ml
DMSO 5 mg/ml DMSO 1.25 mg/ml

Table (4) showing the effect of inhibition of eight nanoparticles on biofilm for two types of
bacteria ( Staphylococcus aureus ,Candida albicans )

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31. A form representing the effectiveness of MBEC for eight compounds on the
EnrerococcusFaecalis type of bacteria.

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Then preparation and use organic compounds following 2 -(phenoxymethyl) -benzoic acid
(PMBA) , 1 -adamantanecarbonyl chloride (ACC), 2 -4-dinitro -1-floro -benzene (DFB), 2 –
chloro -2,4 dinitro -benzoyl chloride (CDBC) , 2- (4 -floropheno xymethyl) -benzoyl
chloride (5),2 – (4-chlorophenoxymethyl) -benzoyl chloride (6) ,2 – (4-methylphenoxymethyl) –
benzoyl chloride (7) ,2 – (4-methoxy phenoxymethyl) -benzoyl chloride (8) where interaction
with Aminopropyl silica nanoparticles(APS -NP) under special circumstances, wher e was
obtained Nanomaterials organic following (APS -NP)-1, (APS -NP)-2, (APS -NP)-3, (APS –
NP)-4 (APS -NP)-5, (APS -NP)-6, (APS -NP)-7, (APS -NP)-8 respectively.
–

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Minimum inhibitory concentration (MIC) and minimum biofilm eliminating concentration
(MBEC) for eight a nanoscal organic compounds (APS -NP) was examined on five types of
bacteria ( Enterococcus Faecalis , Pseudomonas aeruginosa , Staphylococcus aureus ,
Esherchia coli , Candida albicans ) in vitro on both gram -positive and gram -negative bacteria,
the adherence was quantified spectrophotometrically (optical density) at 620 nm for MIC and
492 for MBEC.
-As mentioned before the S5, S6 and S2 samples proved their efficiency as substance can be
used to determine eliminate the effect of the most popular types of bacteria in our life . More
studies should achieve to ensure expert them with other types of bacteria. Also more studies
must be achieved on the un effective types of samples to indicate them with other nutrition
and types of bacteria.
-By observing the MBEC test and after com paring all the results obtained with the negative
control, we can say that eight a nanoscal organic compounds had a positive effect on the
inhibition of biofilm two types only of types of bacteria, it is ( Staphylococcus aureus ,

Candida albicans ), while no any reasonable effect was detected for the other three types it is
(Enterococcus Faecalis , Pseudomonas aeruginosa , Candida albicans

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-Overall, we envisage that further development of these nanoparticles will provi de a variety of
advanced tools for molecular biology, genomics, proteomics and medicine. Moreover, such
nano composites display much potential applications in biomedical field, such as
photodynamic therapy, cell imaging, selective recognition and so on. Th ese strategies might
be universal methods for constructing hybrid organic – inorganic nanomaterials which can be
widely applied in biomedical field.