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Magnetic Nanoparticles in Medical Diagnostic Applications: Synthesis,

Characterization and Proteins Conjugation

Article in Current Nanoscience · December 2015

DOI: 10.2174/1573413712666151210230002

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 Elsevier Editorial System(tm) for Materials Science and Engineering C
Manuscript Draft

Manuscript Number:

Title: Magnetic nanoparticles in Medical Diagnostic applications. Synthesis, characterization,

functionalization and proteins conjugation

Article Type: Research Paper

Keywords: Superparamagnetic, magnetite, nanoparticles, citric acid, silica coating, functionalization,

conjugation, proteins, Bovine Serum Albumin, human natural immunoglobulin

Corresponding Author: Dr. Lori Nalbandian,

Corresponding Author's Institution: CERTH

First Author: Evdokia Patrikiadou, Diploma Chemical Engineer

Order of Authors: Evdokia Patrikiadou, Diploma Chemical Engineer; Anna Patrikidou, Dr; Eleana
Hatzidaki , Dr; Christos N Papandreou , Professor; Vassilis Zaspalis , Professor; Lori Nalbandian

Abstract: Superparamagnetic Fe3O4 nanoparticles are synthesized by the coprecipitation reaction of

ferrous and ferric salts with NaOH or NH4OH. The nanoparticles are physicochemically characterized
by X-ray diffraction, N2 adsorption, High Resolution TEM and FTIR Spectroscopy. The mean diameter
of the synthesized nanoparticles is 8-12 nm, while BET surface areas between 100-250 m2/gr are
obtained by varying the addition rate of the base alkaline solution. Nanoparticles surface is coated with
a thin silica (SiO2) layer in order to become non-toxic and biocompatible. A nearly uniform coating
with thickness 1-2 nm, is obtained using TetraEthylOrthoSilicate (TEOS). The surface of the
nanoparticles is modified by conversion of the hydroxyl groups of the silica coating into amino-groups.
The most appropriate source of amino groups proved to be 3-aminopropyltriethoxysilane (APTES).
Silanization reaction was conducted either in non-aqueous Methanol - Glycerol environment at
elevated temperature, 70°C, or at room temperature in water. Both methods proved to be equally good
for NPs functionalization. In order to enable covalent coupling with the protein molecules, the surface
amine groups of the NPs are converted, either to aldehyde groups by Glutaraldehyde, or to carboxyl
groups using glutaric anhydride. Bovine Serum Albumin (BSA) and Vena human natural
immunoglobulin (Ig) are used in order to study the protein conjugation capacity of the synthesized
nanoparticles. NPs with aldehyde surface groups display better conjugation capacity than NPs
functionalized with carboxyl groups. The conjugation experiments are conducted at room temperature,
under continuous shaking, for 2-4 h. The amount of protein attached to the nanoparticles is
determined by UV-Vis spectroscopy of the supernatant at 280nm. Conjugation of synthesized
nanoparticles to protein BSA is confirmed by FTIR spectroscopy. The conjugation capacity of the
optimized nanoparticles is higher for Ig antibody than for BSA protein, under similar reaction
conditions.
Cover Letter

CENTRE FOR RESEARCH AND TECHNOLOGY - HELLAS

CHEMICAL PROCESS AND ENERGY RESOURCES INSTITUTE
INSTITUTEENGINEERING RESEARCH INSTITUTE
6th km Harilaou – Thermi Rd• PO BOX 60361 • 570 01 Thermi, Thessaloniki
Tel.: 0030 2310 498.112 • Fax: 0030 2310 498.130
Web: http://www.cperi.certh.gr • E-mail: cperi@cperi.certh.gr

Thessaloniki, 5/1/2015

Prof. R. Narayan
Editor-in-Chief, Materials Science and Engineering C: Materials for Biological Applications

Dear editor

Please find enclosed our manuscript titled “Magnetic nanoparticles in Medical Diagnostic
applications. Synthesis, characterization, functionalization and proteins conjugation” submitted
for publication in the Materials Science and Engineering C: Materials for Biological Applications.

Thank you for your attention.

Yours sincerely,

Lori Nalbandian

Dr Chemical Engineer - Principal Researcher

Chemical Process and Energy Resources Institute (CPERI) - Center for Research and Technology
Hellas (CERTH) 6th klm Harilaou - Thermi Road, P.O.Box 60361
57001 - Themi Thessaloniki GREECE
Tel.: 30 2310 498142, Fax: 30 2310 498131, email: nalbanti@cperi.certh.gr http://www.certh.gr
*Suggest Reviewers

Suggested Reviewers

1. H. Shokrollahi
Electroceramics Group, Department of Materials Science and Engineering, Shiraz
University of Technology, Shiraz, Iran
Tel.: +98 7117353509; fax: +98 7117354520.
E-mail address: Shokrollahi@sutech.ac.ir

2. Manuel Arruebo
Aragon Nanoscience Institute (INA), University of Zaragoza, 50009 Zaragoza, Pedro
Cerbuna 12, Spain
E-mail address: arruebom@unizar.es

3. Maria Cristina Mascolo

Laboratory of Materials, Department of Civil and Mechanical Engineering, University of
Cassino and Southern Latium, G. Di Biasio 43 Street, 03043 Cassino (FR), Italy
Tel.: +39-0776-299-4346; Fax: +39-0776-299-3711
E-Mail address: mcmascolo@unicas.it

4. M. Racuciu
Lucian Blaga University, Faculty of Sciences, Victoriei Blvd., No.10, Sibiu, 550012,
Romania
E-Mail address: mracuciu@yahoo.com
Highlights (for review)

HIGHLIGHTS

 Superparamagnetic Fe3O4 NPs with diameter 8-12 nm, magnetization 60emu/g

are synthesized
 Addition of citric acid increases the dispersion stability of the nanoparticles
 NPs with a silica coating 1-2 nm have the highest conjugation capacity towards
proteins
 The NPs are functionalized with Amine groups using 3-
aminopropyltriethoxysilane
 Surface aldehyde groups have higher conjugation capacity than carboxylic
groups
 The conjugation capacity the NPs is greater towards Ig human antibody than
towards BSA
*Manuscript
Click here to view linked References

Magnetic nanoparticles in Medical Diagnostic applications.

Synthesis, characterization, functionalization and proteins conjugation

E. Patrikiadou1, A. Patrikidou2, E. Hatzidaki3, C. N. Papandreou3 V. Zaspalis1,4, L. Nalbandian1, *

1: Chemical Process & Energy Resources Institute, Center for Research and Technology - Hellas
(CPERI / CERTH), P.O. Box 60361, 57001, Thermi-Thessaloniki, Greece
2: Department of Cancer Medicine, Gustave Roussy, Cancer Campus, Grand Paris, University of
Paris-Sud, cedex 94805, Villejuif,, France
3: Department of Medical Oncology, University Hospital of Larisa and
Department of Medicine, School of Health Sciences, University of Thessaly, GREECE
4: Department of Chemical Engineering, Aristotle University, Thessaloniki, Greece

Highlights
 Superparamagnetic Fe3O4 NPs with diameter 8-12 nm, magnetization 60emu/g are
synthesized
 Addition of citric acid increases the dispersion stability of the nanoparticles
 NPs with a silica coating 1-2 nm have the highest conjugation capacity towards proteins
 The NPs are functionalized with Amine groups using 3-aminopropyltriethoxysilane
 Surface aldehyde groups have higher conjugation capacity than carboxylic groups
 The conjugation capacity the NPs is greater towards Ig human antibody than towards BSA

Abstract
Superparamagnetic Fe3O4 nanoparticles are synthesized by the coprecipitation reaction of ferrous
and ferric salts with NaOH or NH4OH. The nanoparticles are physicochemically characterized by X-
ray diffraction, N2 adsorption, High Resolution TEM and FTIR Spectroscopy. The mean diameter of
the synthesized nanoparticles is 8-12 nm, while BET surface areas between 100-250 m2/gr are
obtained by varying the addition rate of the base alkaline solution. Nanoparticles surface is coated
with a thin silica (SiO2) layer in order to become non-toxic and biocompatible. A nearly uniform
coating with thickness 1-2 nm, is obtained using TetraEthylOrthoSilicate (TEOS). The surface of the
nanoparticles is modified by conversion of the hydroxyl groups of the silica coating into amino-
groups. The most appropriate source of amino groups proved to be 3-aminopropyltriethoxysilane
(APTES). Silanization reaction was conducted either in non-aqueous Methanol – Glycerol
environment at elevated temperature, 70°C, or at room temperature in water. Both methods
proved to be equally good for NPs functionalization. In order to enable covalent coupling with the
protein molecules, the surface amine groups of the NPs are converted, either to aldehyde groups
by Glutaraldehyde, or to carboxyl groups using glutaric anhydride. Bovine Serum Albumin (BSA)
and Vena human natural immunoglobulin (Ig) are used in order to study the protein conjugation
capacity of the synthesized nanoparticles. NPs with aldehyde surface groups display better
conjugation capacity than NPs functionalized with carboxyl groups. The conjugation experiments
are conducted at room temperature, under continuous shaking, for 2-4 h. The amount of protein
attached to the nanoparticles is determined by UV–Vis spectroscopy of the supernatant at 280nm.

**
Corresponding author: Lori Nalbandian, Tel.: +30 2310 498142, Fax: +30 2310 498131, E-mail:
nalbanti@cperi.certh.gr
1
Conjugation of synthesized nanoparticles to protein BSA is confirmed by FTIR spectroscopy. The
conjugation capacity of the optimized nanoparticles is higher for Ig antibody than for BSA protein,
under similar reaction conditions.

Keywords: Superparamagnetic, magnetite, nanoparticles, citric acid, silica coating,

functionalization, conjugation, proteins, Bovine Serum Albumin, human natural immunoglobulin

1. INTRODUCTION
In recent years, the magnetic properties of nanometer sized iron oxide powders such as α-Fe2O3,
γ-Fe2O3, and Fe3O4 have been intensively studied because magnetic nanoparticles (NPs) can be
widely used in biomedical applications [1-6]. NPs have been used as magnetic drug delivery
systems [7,8], as heating mediators for cancer thermotherapy [9,10] and as contrast agents for
magnetic resonance imaging (MRI) [11-14]. Superparamagnetic iron oxide nanoparticles can be
used as contrast agents in order to improve the diagnostic value of MRI because they enhance the
tissue contrast of an area of interest by increasing the relaxation rate of water protons in their
proximity, therefore showing positive T1 contrast effect.
Although commercial preparations are already available, an intense research effort is under way
aimed at attaining the sensitivity needed for early tumor detection. Various methodologies have
been developed to synthesize magnetic nanoparticles that possess some means of distinguishing
cancerous tissue from healthy tissue in order to facilitate their preferential accumulation in one
type of tissue and provide distinct image enhancement.
Magnetic nanoparticles can be tailored to target specific organs, cells, or even molecular markers
of different diseases in vivo, with suitable surface chemistry modification. In this case magnetic
nanoparticles are capable of binding to specific target ligands such as peptides, antibodies and
proteins, through chemical bonds [12, 14-18].
Biomedical applications require that iron oxide nanoparticles should be discrete with small
particle sizes, narrow size distribution for unified physical and chemical properties and
superparamagnetic [4,6]. Superparamagnetism implies that there is not coercivity, and that the
magnetization is close to zero in the absence of an external field.
For the synthesis of Fe3O4 superparamagnetic nanoparticles coprecipitation is a fast and low-cost
method that has been widely applied, thus far [19-24]. In the coprecipitation method, first
described by Massart in 1981 [19], a 2:1 mixture of ferric and ferrous salts is precipitated through
the addition of an acid or base. Coprecipitation can provide NPs with narrow size distribution and
uniform shape and morphology, however it is very sensitive to small variations of the reaction
parameters since limited control of the synthesis process often leads to nanoparticles with broad
variation in their characteristics. Concentration, molar ratio of the reactants and pH as well as the
presence of dispersing agents have been reported as the most important factors during
coprecipitation [20- 24].
After synthesis, a coating on the nanoparticle surface is necessary to prevent aggregation, render
the NPs water-soluble, non-toxic and biocompatible, and provide functionalization for the
conjugation of biomolecules. Coating of the surface of NPs can be performed either with natural
and synthetic polymers, such as dextran, albumin and polyethylene glycol or with an inorganic
silica layer [20,22,25-27]. A silica shell protects the magnetic cores by preventing the direct contact
with additional agents linked to the silica surface thus avoiding unwanted interactions.
Furthermore, the hydroxyl groups present on the surface of the silica layer increase the reactivity
of the nanoparticles because organic molecules can be attached to their surface via covalent or
hydrogen bonding.

2
Stöber’s method [28] is the basis of most commonly used methods [26,29,30] for the silica coating
on the iron oxide nanoparticles. Use of the above methods is a rather easy way to form a silica
layer by mixing of nanoparticles, aqueous solution and TetraEthylOrthoSilicate (TEOS) in alcohol.
To control the thickness of the silica layer, optimization of the reaction parameters, such as the
alcohol type, volume ratio of alcohol to water, the feeding amount of catalyst as well as the
volume of TEOS, is required [26,30].
Nanoparticles with functional groups such as amines are of great interest as they can conjugate
with biological molecules. The hydroxyl groups on the surface of the silica layer can be converted
to amino groups by different methods [31-34]. This surface modification is commonly carried out
with alkoxysilanes with non-hydrolysable amino groups via silanization reactions. Alkoxysilanes
can react via a range of procedures including organic phase, aqueous phase, and chemical vapour
deposition of the silane [29,35-37]. Silanization in solution is the most common approach. Typical
silane concentrations lie between 0.1 and 2% (w/v) and the presence of a small amount of water
is necessary for the reaction to occur. During the surface activation reaction, the alkoxy groups of
the alkoxysilanes are hydrolysed to form silanol-containing species. Reaction of these silanes
involves four steps. Hydrolysis is followed by condensation to oligomers. The oligomers then form
hydrogen bonds with -OH groups of the substrate. Finally during drying or curing, a covalent
linkage is formed with the substrate with concomitant loss of water.
The progress of these reactions and thus the characteristics of the final surface layer depend on
the type and concentrations of the organosilane, as well as on reaction variables such as solvent
type, temperature, and time. The huge diversity of alkoxysilanes allows for different types of
nanoparticle surface functionalization.
There are two main ways to immobilize proteins on supports, physical adsorption and covalent
immobilization [14]. Comparatively, covalent immobilization can eliminate or significantly reduce
leakage of proteins through increased bond strength and increase covalent immobilization
stability. In order to covalently bind the prepared nanoparticles with affinity ligands such as
proteins and antibodies, their surface needs to be specifically functionalized, i.e., appropriate
functional groups have to be formed to enable specific interactions with the protein molecules.
Various functionalization molecules can be used for the activation of the nanoparticle surfaces in
order to form strong covalent bonds with the biomolecules. Generally, the functionalization
molecule should consist of at least two functional groups, where one functional group is needed
for the attachment to the nanoparticle surface, while the other one provides an anchoring point
for the molecule bonding.
The most common approaches to conjugate proteins or antibodies to the amino-functionalized
nanoparticles are the following [18,38-40]: (i) conversion of the surface amine groups to aldehyde
groups with glutaraldehyde, a bifunctional crosslinker, and formation of a Schiff’s base between
the primary amine of the antibody and the free aldehyde groups present on the surface of the
functionalized nanoparticles [38,39], and (ii) conversion of the surface amine groups to carboxyl
groups using EDAC or EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] as cross-linking
agents and formation of an amide bond between the carboxylated nanoparticles and the amino
groups of the antibodies [29, 40].
In this study, highly functionalized superparamagnetic magnetite nanoparticles, well dispersed,
stable in aqueous solutions, are prepared, in order to be capable of covalently binding to specific
targeting ligands such as proteins and antibodies. The effect of the reaction parameters during
synthesis with a coprecipitation method, silica coating with TEOS and surface functionalization
with amino-groups is studied. The morphology and properties of prepared NPs are examined by
X-ray diffraction (XRD), N2 sorption, FTIR and HRTEM. Finally, surface amino groups are converted
either to aldehyde groups or to carboxyl groups and a series of experiments is carried out to prove
3
that the designed magnetic nanoparticles can effectively immobilize the proteins and optimize
their capability to conjugate by covalent bonding to proteins and/or antibodies.

2. EXPERIMENTAL
2.1. Materials
All chemicals are of analytical grade and used without any further purification. Ferric chloride
(FeCl3∙6H2O), ferrous chloride (FeCl2∙4H2O), ammonia (NH3, 25%), citric acid (C6H8O7),
tetraethylorthosilicate (TEOS), ethanol, methanol, glycerol, toluene, 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride and glutaric anhydride are purchased from Merck.
Aminosilane coupling agents, 3-aminopropyl-triethoxy-silane (APTES) and Bis[3-
(triethoxysilyl)propyl]amine, glutaraldehyde as well as 2-(N-morpholino)ethanesulfonic acid
(MES) are obtained from Sigma Aldrich. Phosphate buffer saline (PBS) solution is purchased from
Biochrom and Bovine Serum Albumin (BSA) from Sigma Aldrich. H3BO3 and Na2B4O7 used for the
preparation of borate buffer, are purchased from Merck. Vena human natural immunoglobulin
(Ig) is purchased from Kedrion S.p.A. Deionized water (18.2 MΩcm-1, MilliQ) is used in all the
reactions.
2.2. Synthesis of magnetite nanoparticles and silica coating
The Fe3O4 nanoparticles are synthesized by a chemical coprecipitation method. The synthesized
nanoparticles are then coated with a thin silica layer in order to become biocompatible and
hydrophilic.
Synthesis The preparation of the nanoparticles is carried out in a three-necked flat bottom flask
placed in a thermostat at 25 °C. Ferric chloride hexahydrate (FeCl36H2O) and ferrous chloride
tetrahydrate (FeCl24H2O) are dissolved in deionized water (concentration ratio Fe3+:Fe2+ =2:1)
under vigorous stirring. Nitrogen gas flow is used to prevent oxidation. The precipitation agent, a
1.5 Μ NaOH, or 1.5 Μ NH4OH solution is added dropwise until pH=12. Magnetite precipitates
according to the reaction:
Fe2+ + 2Fe3+ + 8OH- → Fe3O4+ 4H2O (1)
The pH is continuously measured with a WTW Inolab pH-meter. Vigorous mechanical stirring is
applied in order to achieve good mixing of the reactants and to prevent a possible agglomeration
of the formed nanoparticles. After the addition of the precipitating agent is completed, a citric
acid solution (0.5M) is added until pH=7, in some cases, and the stirring continues for at least
30 min. The suspension containing the resulting nanoparticles is ultra-sonicated for 30 min. After
that the nanoparticles are magnetically separated, washed with deionized water and finally
washed with ethanol.
During the nanoparticles synthesis by the “coprecipitation” method, the effect of the following
parameters is studied:
- total time of addition of the alkaline solution, which varies between 0.5 and 15 minutes.
- addition of a citric acid as dispersing agent, during the coprecipitation process
Silica Coating The Fe3O4 nanoparticles are coated with silica using a modified Stöbe, sol–gel
method [29,30]. The nanoparticles are dispersed, by sonication for about 15 min, in a solution of
ethanol/water (4:1) and the pH of the solution is adjusted to 9 by addition of ammonia. Then
under continuous mechanical stirring, the appropriate amount of the Si-source,
TetraEthylOrthoSilicate (TEOS) is added very slowly to the reaction mixture. The reaction is
allowed to proceed at room temperature for 10 h under continuous stirring. The resultant product
is obtained by magnetic separation; it is thoroughly washed with deionized water and finally
dispersed in water.
4
Different types of NPs are prepared with different silica layer thickness, by varying TEOS
concentration between 0.008% and 0.66% v/v.
2.3 Surface functionalization of silica coated NPs
Two different alkoxysilanes are used for the surface modification of the silica-coated
nanoparticles:
− Bis-3-triethoxy-silyl-propyl-amine
− 3 aminopropyl-triethoxy-silane (APTES)
The treatment of the NPs with the silane coupling agent is performed with two different methods:
Methanol – Glycerol method (APTA) 0.5 gr of the silica coated Fe3O4 nanoparticles are obtained
from the aqueous suspension by magnetic separation and resuspended in 80 ml of methanol
mixed with 2 ml of water. After ultrasonication for 30 min, 2.5 ml of the alkoxysilane are dissolved
in the suspension. The solution is mixed with 40 ml of glycerol and heated at 70°C. The reaction
is allowed to proceed for 3 h under continuous stirring [36].
Aqueous method (APTB) 0.5 gr of the silica coated Fe3O4 nanoparticles are obtained from the
aqueous suspension by magnetic separation and resuspended in a 150 ml of water. After
ultrasonication for 15 min, the appropriate amount of the alkoxysilane is dissolved in the
suspension. The reaction is allowed to proceed at room temperature for 5 h under continuous
stirring [32].
In both methods the resultant product is obtained by magnetic separation, it is thoroughly washed
with methanol and deionized water and resuspended in water.
2.4 Activation of nanoparticles for conjugation to proteins
Two different methods for protein conjugation to the functionalized nanoparticles are utilized.
Both methods include an activation step during which the amine groups, present on the surface
of the NPs are transformed, in order to allow the formation of covalent bonds with amino groups
from the protein molecules.
Formation of aldehydic groups (EN1): The activation is performed by the formation of aldehydic
groups on the surface of the nanoparticles. The amino modified Si-coated NPs of the previous step
are dispersed in a solution of 5% v/v glutaraldehyde in Phosphate Buffer Saline (PBS) and the
reaction is allowed to proceed at room temperature for 16 h under continuous stirring.
Subsequently the functionalized nanoparticles are separated magnetically, are thoroughly
washed with water and PBS and finally dispersed and stored in PBS. The protein conjugation is
performed in PBS buffer, either immediately after the activation or after several days.
Formation of carboxylic groups (EN2): The activation is performed by the formation of carboxylic
groups on the surface of the nanoparticles. Amino modified NPs are dispersed in toluene in which
10% w/v glutaric anhydride has been dissolved. The suspension is allowed to react for 2 hrs, at
room temperature, under N2 flow and continuous stirring. Subsequently the nanoparticles are
washed with methanol and water and finally dispersed and stored in water. The protein
conjugation is performed in borate buffer, either immediately or after several days, the same as
with method EN1. Prior to the proteins conjugation, the carboxyl-activated nanoparticles are
suspended in a MES (2-(Nmorpholino)-ethane-sulfonic acid) buffer containing (N-(3-
Dimethylaminopropyl)-N'-ethyl-carbodiimide hydro-chloride), under continuous stirring, for
15min, at room temperature. After that, the NPs are washed and dispersed in a borate buffer
solution.

2.5 Physicochemical Characterization

The crystalline phases present in the prepared nanoparticles are identified by X-ray diffraction
(XRD). Powder XRD patterns are recorded with a Siemens D 500 X-ray diffractometer, with an
5
autodivergent slit and graphite monochromator using CuKa radiation, with a scanning speed of
2°min-1. The characteristic reflection peaks (d-values) are matched with JCPDS data files and the
crystalline phases are identified. Since, from the appearance of the XRD diffractograms, it is
obvious that the examined samples have very small particle size (broad, low intensity peaks), the
Scherrer formula is used in order to get a rough estimate of the crystallite size of the materials,
based on XRD peak width.
Specific surface area, pore volume and pore size distribution are determined by nitrogen
adsorption-desorption isotherms at liquid nitrogen temperature (77 K) using a Micromeritics
Tristar apparatus. The samples are degassed at room temperature for at least 48 h prior to the N2
sorption measurement.
High-resolution transmission electron microscopy (HRTEM) images are obtained in a JEOL JEM
2010 microscope operating at 200 kV and equipped with an Oxford Instruments INCA EDS
detector. In order to prevent eventual agglomeration, the sample is mixed with pure ethanol in
an ultrasonic apparatus and superimposed on a lacey carbon film supported on a 3 mm Cu grid.
The presence of functional groups on the surface of the nanoparticles is studied by Fourier
transform infrared (FTIR) spectroscopy. The FT-IR spectra are obtained on a Bruker FTIR
spectrophotometer (Model Tensor 27) under ambient conditions. Typical scans are obtained at a
resolution of 4 cm-1 in the range 400-4000 cm-1. In order to record the spectra, the nanoparticles
are dispersed in KBr (ratio 10:90).
The detailed magnetization characterization of the prepared materials is performed by means of
a commercial superconducting quantum interference device (SQUID) magnetometer (Quantum
Design).
For the determination of the quantity of protein conjugated to the nanoparticles, aliquots are
obtained from the reaction vessels and the supernatant is assayed for remaining protein
concentration by UV-Vis spectroscopy. The UV-Vis spectra are obtained at a Hitachi U-3010 double
beam spectrophotometer, in the range 250-330 nm, using quartz cells with 1 cm optical path.

3. RESULTS AND DISCUSSION

3.1. Pure iron oxide nanoparticles

Figure 1: X-ray diffractogram of the synthesized magnetite nanoparticles

6
The X-ray diffractogram (XRD) of the synthesized pure NPs reveals peaks corresponding to pure
crystallites of magnetite (JCPDS 3-0863) with no impurities. No other phase such as Fe(OH)3 or
Fe2O3 is detected, which are usual co-products in a chemical co-precipitation procedure. The
broadening of the diffraction peaks denotes the presence of small crystallites with an average
particle size estimated to be 9-12nm by the Scherrer equation (Figure 1).

Figure 2: Effect of the rate of addition of the alkaline solution, during coprecipitation, to the
surface area of the nanoparticles.

The surface area of all the prepared samples is in the range 100-260 m2/gr, which indicates clearly
that the particles have diameters in the range 5-12 nm, in complete agreement with the results
of the Scherer analysis. Figure 2 shows the effect of the rate of addition of the alkaline solution
during the coprecipitation process, on the surface area of the obtained NPs. As mentioned in
paragraph 2.2, the total time of addition of the base varied between 0.5 and 15 minutes. As can
be seen in Figure 2, the surface area of the NPs is maximized when the total time of addition of
the alkaline solution is between 1 and 5 minutes. The results of Figure 2 denote that the total
surface area and thus the mean size of the nanoparticles can be adjusted by changing the addition
rate of the base.

Figure 3. TEM image of the NPs prepared (a) without a dispersing agent (b) with addition of citric
acid
7
A characteristic TEM image is presented in Figure 3a, obtained from the nanoparticles prepared
with the optimum addition rate of the alkaline solution (addition time 3 min). It can be observed
in the TEM image that the nanoparticles have sizes ranging from 8 to 12 nm. The particle sizes
measured with TEM are very close to the particle size values estimated from both XRD and surface
area measurements. However, agglomeration of some extent is observed due to the high surface
energy of the extremely small sized particles. The particles tend to aggregate to minimize their
total surface energy.
The effect of addition of citric acid, as a dispersing agent, during the coprecipitation process is an
increase in the dispersion stability of the magnetite nanoparticles thus less agglomeration is
observed. This was also evident in the TEM image, shown in Figure 3b, of nanoparticles prepared
in the presence of citric acid.
The FTIR spectrum obtained from pure magnetite nanoparticles is presented in Figure 4, curve a.
The two intense peaks observed between 580 cm-1 and 630 cm-1 are attributed to the stretching
vibration mode associated to the metal-oxygen Fe – O bonds in the crystalline lattice of Fe3O4.
They are characteristically pronounced for all spinel structures and for ferrites in particular [41,
42]. A band at 1629 cm-1 and the broad band centered at 3435 cm-1 are related to the presence
of hydroxyl groups and attributed to OH-bending and OH-stretching respectively.

Figure 4. FTIR spectra of (a) pure Fe3O4 magnetic nanoparticles (b) citric acid coated NPs (c) pure
citric acid

Curve c in Figure 4 is obtained by pure citric acid, while curve b is the FTIR spectrum of magnetite
nanoparticles synthesized in the presence of citric acid (CA-NPs). Since the NPs have been
thoroughly washed after synthesis, no mechanically deposited citric acid is expected.
New intense absorption peaks located at 1627 and 1390 cm-1 as well as lower intensity peaks at
1285 and 1085 cm-1 appear in the FT-IR spectra of citrate-treated Fe3O4. The 1730 cm-1 peak of
citric acid (curve c), assignable to the stretching vibration of C=O from the COOH group [43-46],
shifts to the intense band at about 1627 cm-1 for CA-NPs. This reveals that carboxylate groups of
citric acid should complex with the Fe atoms on the magnetite surface and render a partial single
bond character to the C=O bond, weakening it, and shifting the stretching frequency to a lower
8
value. Furthermore, the vibrational modes appearing at 1390, 1285 and 1085 cm-1 in CA-NP
correspond to the symmetric stretching of COO-, symmetric stretching of C–O, and OH group of
citric acid. It has been proposed [43] that citric acid binds to the magnetite surface by
chemisorption of the carboxylate, that is, citrate ions.
Magnetic measurements (Figure 5) obtained at a Superconducting Quantum Interference Device
(SQUID), prove that the prepared nanoparticles display superparamagnetic behaviour as the
magnetization curve passes through the origin of the axes. This means that when external field is
zero the magnetization is also zero. Saturation Magnetization measured at 300 K is 60 emu/g. It
is worth noting that this value is comparable to previously published reports measuring saturation
magnetization of Fe3O4 nanoparticles [15,24,29].

Figure 5: Magnetization curve of pure Fe3O4 nanoparticles at 300K obtained by SQUID

3.2 Silica coated nanoparticles

The coating of magnetite NPs with a thin silica (SiO2) layer offers many advantages since it
prevents electrostatic attraction and agglomeration of the NPs and also the nanoparticles become
biocompatible, non-toxic and hydrophilic. Moreover, the hydroxyl groups present on the silica
surface can be easily functionalized [29].
The presence of silica around NPs is verified by FTIR Spectroscopy. In Figure 6 the FTIR spectra of
pure and silica coated nanoparticles are compared. In all spectra the main bands of the magnetite
core between 580 cm-1 and 630 cm-1 [41,42] can be observed. In the IR spectrum of all the Si-
coated NPs (curves b-d of Figure 6) there is, additionally, a broad high-intensity band centered at
1100 cm-1, typically attributed to the Si-O-Si asymmetric stretching. Moreover a band emerging at
460 cm-1 is characteristic of Si-O-Si and or Si–O–Fe bending [47].
The morphology of silica around nanoparticles was examined by HRTEM combined with X-ray
microanalysis (EDS). Figure 8d shows a typical high resolution TEM image of the silica-
encapsulated iron oxide nanoparticles. The image of Figure 8d, reveals highly crystalline
structured iron oxide core surrounded by an amorphous silica coating.

TEOS concentration during the coating of the nanoparticles with a thin silica layer The
concentration of TEOS during the Silica coating of the Nanoparticles, which determines the
thickness of the Silica layer [30], was varied during the experiments, in order to examine the effect
of the coating thickness to the agglomeration of the NPs as well as their conjugation capacity. A
series of silica coated NP samples is prepared with TEOS concentrations, 0.008, 0.016, 0.066, 0.16
and 0.66% v/v.
9
The amount of silica deposited on the nanoparticles was examined by FTIR spectroscopy. Figure 6
shows the FTIR spectra of the first 4 samples of the series, prepared with 0% v/v TEOS (curve a),
0.008% v/v TEOS (curve b), 0.016% v/v TEOS (curve c) and 0.066% v/v TEOS (curve d). The two
most intense bands at 1100 cm-1 and 470 cm-1, attributed to silica are marked by an asterisk. The
band at 1100 cm-1 is clearly visible in the spectra of all three samples prepared with non-zero TEOS
concentration (curves b-d), with its intensity increasing with increasing TEOS concentration. The
second, less intense band can be distinguished only in the spectrum of the sample prepared with
the highest TEOS concentration, presented in curve d.

Figure 6. FTIR spectra of NP samples coated with different TEOS concentration (a) no TEOS (b)
0.008% v/v TEOS (c) 0.016% v/v TEOS and (d) 0.066% v/v TEOS. The peaks attributed to the silica
layer are marked with an asterisk

Figure 7 presents TEM images obtained from the series of NP samples coated with different TEOS
concentrations. As can be observed, the lowest TEOS concentration used, 0.008% v/v is very low
and thus the silica layer of the obtained nanoparticles (Figure 7a) can barely be distinguished,
indicating either that is very thin, or that it does not exist at all. For TEOS concentrations 0.016%
(Figure 7b) and 0.066% (Figure 7c & 7d) a nearly uniform coating around the nanoparticles is
obtained with thickness 1-2 nm. When the TEOS concentration is increased to 0.16% and 0.66%
v/v, the quantity of the deposited silica is much more than the required for a thin coating of the
NPs. Thus large agglomerates are formed while pure silica particles can be also observed.

10
 Figure 7: TEM images of NP samples coated with different TEOS concentration
(a) 0.008% (b) 0.016% (c) & (d) 0.066% (e) 0.16% (f) 0.66%

3.3. Protein Conjugation

Bovine-Serum-Albumin (BSA) and Ig human natural immunoglobulin are used in order to examine
whether the prepared magnetic nanoparticles can be covalently bonded to proteins and to
measure the protein immobilization capability of the NPs, as a function of the different synthesis
parameters studied.
11
Bovine Serum Albumin (BSA) has been one of the most extensively studied proteins, particularly
for its structural homology with human serum albumin. It has been used as the conjugation
protein in several previous works [27,36,42,48- 50]. BSA is used as a control protein in the first
experiments of this study, which aim in the optimization of the reaction parameters during the
synthesis and functionalization of the nanoparticles.

Figure 8: Typical UV-Vis spectra obtained from the starting protein solution (dashed line) and the
supernatant after reaction with NPs (solid line).

In a typical experiment, activated NPs with surface aldehydic groups (method EN1) or carboxylic
groups (method EN2), are added into protein solutions with different initial concentrations, in PBS
or borate buffer respectively. For the determination of the quantity of protein conjugated to the
nanoparticles, aliquots are obtained from the reaction vessels and the supernatant is assayed for
remaining protein concentration by UV-Vis spectra, in the range 250-330 nm. After the
measurement, the obtained aliquots are remixed and thrown back to the reaction vessel. The
maximum absorption at 278nm, which is mainly attributed to the aromatic amino acid residues
of the protein [51], is compared to the corresponding absorption obtained from solutions with
known initial concentrations (Figure 8). The amount of protein conjugated to the nanoparticles is
determined by the difference between the initial concentration of the protein and the measured
protein concentration in the supernatant.

(a) (b)

Figure 9. Quantity of BSA conjugated to NPs versus (a) time of reaction (b) initial concentration
of BSA.
In order to determine the time necessary to complete the reaction, a diagram of the protein
concentration in the supernatant as a function of reaction time is plotted (Figure 9a). In most
experiments the time required for equilibration is between 2-4 hrs.
12
For the determination of the protein immobilization capability of the NPs, the quantity of
conjugated protein is plotted as a function of the initial protein concentration (Figure 9b).
The presence of protein conjugated to the NPs is confirmed by FTIR spectroscopy. In Figure 10 the
spectra obtained from nanoparticles conjugated with protein (curve c and d) is compared with the
corresponding spectra of the aldehyde activated nanoparticles (curve b) and of the pure protein
BSA (curve a). The nanoparticles have been thoroughly washed with de-ionized water prior to the
FTIR measurement, in order to remove any physically deposited protein molecules. It is observed
that the 2 most intense peaks of the protein, located at 1529 cm-1 and at 1661 cm-1, which are
attributed to the amide bond of the protein [27,50], appear in the spectra of conjugated
nanoparticles (curve c and d) while they are absent in spectrum b, which is obtained from the
aldehyde modified NPs.

Figure 10: FTIR spectrum of silica coated nanoparticles conjugated with antibody (a) pure
protein (b) amino modified, aldehyde activated nanoparticles, (c) and (d) protein conjugated
nanoparticles In curve (d) the initial protein concentration is higher than in (c).

In Figure 11, the intensity of the most intense FTIR band of the protein, located at 1661 cm-1, as
measured in the spectra of the conjugated nanoparticles (Figure 10), is plotted as a function of
the initial protein concentration. In the same plot, the amount of protein conjugated to the NPs
as determined by the UV-Vis spectra of the supernatant is also plotted vs. the initial Protein
Concentration. The comparison of the 2 curves shows that, within experimental error, the
measured area of the protein FTIR peaks is analogous to the amount of protein conjugated to the
NPs.

13
Figure 11: :Intensity of the most intense FTIR band of the protein (1661 cm-1) in the spectra of
the conjugated nanoparticles and :amount of protein conjugated to the NPs as determined by
UV-Vis of the supernatant.

3.4 Effect of synthesis and surface functionalization parameters on the binding capacity of the
nanoparticles

3.4.1 TEOS concentration during the coating of the nanoparticles with a thin silica layer
The variation of the conjugation capacity of the series of NP samples coated with Silica layer with
different thickness is presented in Figure 12. It can be observed that the maximum quantity of
protein is conjugated to the nanoparticles prepared with TEOS concentration between 0.016%
and 0.066% v/v. As shown in the TEM images of Figure 7, the silica layer obtained in this TEOS
concentration range is the optimum, providing a nearly uniform coating around the nanoparticles
without causing their agglomeration. Therefore it is not unexpected that the conjugation capacity
would be maximum for TEOS concentration in this range.

Figure 12. Effect of TEOS concentration during the coating of the nanoparticles with silica, on
their protein conjugation capacity

14
3.4.2 Source of amino groups and silanization reaction conditions
The hydroxyl groups on the surface of the silica layer are converted to amino groups, via
silanization reactions. In this work two silane coupling agents with a terminal amino group are
used, 3-aminopropyltriethoxysilane (APTES) which is the most commonly used alkoxysilane [33,
36] and Bis-3-triethoxy-silyl-propyl-amine which is a dipolar silane.
The effect of the type of alkoxysilane used is presented in Figure 13a. The results presented in
Figure 13 have been obtained from experiments in which a wide range of parameters has been
also varied, as will be presented in the next paragraphs, and this is the reason for their big
divergence. However, it can be clearly seen that, in nearly all cases, the conjugation capacity of
the nanoparticles which are surface modified with APTES is much higher compared to the capacity
of the NPs to which the silanization is performed by the dipolar silane.
The effect of the reaction variables such as solvent type, temperature, and time is studied by
examining the effect of the two different silanization methods used, APTA (Methanol – Glycerol
method) and APTB (Aqueous method) on the experimentally determined conjugation capacity of
the nanoparticles. The results, presented in Figure 13b, have been obtained from experiments in
which a wide range of parameters has varied, as was the case in Figure 13a, therefore they also
show big divergence. However, in contrary to Figure 13a, there is not a clear trend indicating a
significant difference in the conjugation capacity of the NPs obtained by the two methods,
especially when high initial protein concentrations are used. Ιt can therefore be concluded that
the two methods are equally successful in NPs functionalization.

Figure 13: (a) Conjugation capacity of nanoparticles functionalized with : bis[3-(triethoxysilyl)

propylamine] and : 3-aminopropyltriethoxysilane (APTES) (b) Conjugation capacity of
nanoparticles functionalized with APTES in by reaction methods : APTA and : APTB

3.4.3. Effect of the type of surface active groups

The proteins are conjugated to the nanoparticles by covalent bonding of their amine groups to
the aldehydic or carboxylic surface groups of the activated NPs, as described in paragraph 2.4. The
conjugation capacity of the different NP preparations is studied and presented in Figure 14. Both
types of nanoparticles, activated with either aldehydic or carboxylic groups display significant
conjugation capacity as presented in Figure 14.

15
 Figure 14: Conjugation capacity of nanoparticles activated with : carboxyl groups and :
aldehyde groups
The results presented in Figure 14 are obtained from experiments in which a wide range of
parameters also varied, similar to Figures 13a and 13b. Although the dispersion of the
experimental points is again significant, there is a clear trend indicating that conjugation through
aldehyde groups (method EN1) is more effective than protein conjugation through carboxylic
groups.

3.5 Stability of surface groups

As mentioned in paragraph 2.4 the protein conjugation of the nanoparticles activated with either
aldehyde or carboxyl groups can be performed, either immediately following activation or after
several days.

Figure 15: Effect of time after activation on conjugation capacity of nanoparticles

The stability of surface groups was investigated by measuring, under identical conditions, the
conjugation capacity of NPs immediately and several days after their activation. Figure 15 presents
the amount of BSA protein conjugated to NPs that are activated with aldehyde groups during days
16
1, 4 and 5. As demonstrated in the graph the conjugation capacity of the NPs decreases 20% in
day 4 and 35% in day 5 after activation, indicating reduced stability of the surface groups. Similar
results are obtained with nanoparticles activated with carboxyl groups.

3.6 Effect of the protein type

After the completion of the first stage of the experiments aiming in the optimization of the NPs
preparation and surface activation parameters, the BSA protein, which, as mentioned in
paragraph 3.1, is used as a control protein, is replaced by the Ig human natural immunoglobulin
for the evaluation of the conjugation capacity of the optimized nanoparticles under more realistic
conditions. The procedure for the antibody conjugation experiments is similar to that described
in paragraph 3.3. No pre-treatment of the antibody was applied.
The conjugation capacity of NPs with aldehyde surface groups towards the antibody is compared
in Figure 16 to the conjugation capacity of the same NPs, under identical experimental conditions,
towards protein BSA. It is evident that nanoparticles’ conjugation capacity is greater towards
antibody than towards protein BSA. With the same type of nanoparticles and under similar
reaction conditions, the quantity of antibody conjugated to the NPs is more than 5x the
corresponding quantity of BSA.

Figure 16: Conjugation capacity of nanoparticles with aldehyde surface groups towards : Ig
human natural immunoglobulin and : Bovine Serum Albumin (BSA)

4. CONCLUSIONS
Superparamagnetic Fe3O4 nanoparticles with diameters in the range 8-12 nm and Saturation
Magnetization 60emu/g are synthesized by a chemical coprecipitation method. The surface area
of the NPs is maximized when the total time of addition of the alkaline solution during
coprecipitation is between 1 and 5 minutes. As revealed by FTIR and TEM study, the addition of
citric acid, which binds to the magnetite surface, increases the dispersion stability of the
magnetite nanoparticles.
The synthesized nanoparticles are coated with silica using a modified Stöbe method. The thickness
of the silica layer is varied by using different TEOS concentration. In TEOS concentrations 0.016%
17
- 0.066% v/v, a nearly uniform coating around the nanoparticles is obtained with thickness 1-2 nm
and the maximum quantity of protein is conjugated to the nanoparticles.
Silanization with 3-aminopropyltriethoxysilane (APTES) achieves higher conjugation capacity,
while both Methanol – Glycerol (APTA) and Aqueous (APTB) reaction environments are equally
good for NPs functionalization.
Covalently bonding of the NPs to proteins through surface aldehyde groups is more effective that
through surface carboxylic groups. The conjugation capacity of the NPs decreases by 35%, within
5 days after activation.
The conjugation capacity the optimized nanoparticles is greater towards Ig human natural
antibody than towards Bovine Serum Albumin (BSA). The quantity of conjugated antibody is more
than 5x the corresponding quantity of BSA, under identical reaction conditions, with the same
type of nanoparticles.

AKNOWLEDGMENTS
Dr Michalis Pissas, Research Director at the Institute of Materials Science, National Center for
Scientific Research "Demokritos", in Athens, Greece is kindly acknowledged for providing
Magnetic measurements at SQUID.
Professor Angeliki Lemonidou of the Department of Chemical Engineering, Aristotle University of
Thessaloniki, Greece is kindly acknowledged for providing access to the FTIR facility.

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