The prognostic value of Histidine Rich Glycoprotein RNA in Breast
tissue using Unmodified Gold Nanoparticles Assay
Running title: Prognostic value of tissue Histidine Rich Glycoprotein RNA using AuNPs
Sanaa Eissa1 , Hassan M.E. Azzazy2#,Marwa Matboli1, Sherif M. Shawky2,
Hebatallah Said1, Fatin A.Anous3.
1
Oncology Diagnostic Unit, Medical Biochemistry and Molecular biology Department,
Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O. box 11381.
2
Department of Chemistry & Youssef Jameel Science & Technology Research Center,
the American University in Cairo, P.O. Box 74 New Cairo 11835, Egypt.
3
General Surgery Department, Faculty of Medicine, Ain Shams University, Abbassia,
Cairo, Egypt, P.O. box 11381.
#Corresponding
author:
Sanaa Eissa1, 1Oncology Diagnostic Unit, Medical Biochemistry and Molecular biology
Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt, P.O.
box 11381. Dr_sanaa_eissa@yahoo.com. Drsanaa_mohamed@med.edu.eg
Magnetic nanoparticles synthesis, functionalization and characterization
Synthesis of Fe2O3 Magnetic Nanoparticles(19-20)
Magnetic nanoparticles Fe2O3 were prepared by the hydrothermal co-precipitation of
ferric and ferrous ions in basic media as described elsewhere(19-20). The synthesis of γFe2O3 nanoparticles involved the following steps: (1) a designated molar ratio of iron
chloride and urea was dissolved in deionized water; (2) the mixture was continuously
stirred for 45 min at 90°C in a water bath before it was cooled to room temperature (3)
the produced precipitation was centrifuged and washed by deionized water and followed
by ethanol .During washing, the magnetic nanoparticles were separated from the
supernatant using a magnet, and the particles were finally dried in a vacuum oven at
70°C.
Magnetic nanoparticles surface functionalization with amino groups(21)
Amino functionalization of the prepared magnetic nanoparticles was done by amino
propyl Trimethoxysilane (APMS) as described elsewhere(21). Briefly, magnetic
nanoparticles (1 g) were washed with 99.5% methanol and twice with Nanopure water
and soaked in 10 mL of 3 mM APTMS solution in a toluene/methanol (1:1 v/v) mix. The
suspension was then transferred into a three-necked flask with a water-cooled condenser
and temperature controller with a nitrogen gas flow at 80°C for 20 h under vigorous
stirring. Silanization was bound to occur at the surfaces of the particles bearing hydroxyl
groups, which in the presence of an organic solvent results in the formation of an APTMS
coating with a large density of amines. The particles were recovered by applying an
external magnetic field after the silanization process and washed three times with
methanol and dried at 50°C in a vacuum oven.
Characterization of Magnetic nanoparticles
The prepared magnetic nanoparticles were characterized by SEM for size and particles
distribution determination. Moreover, Fourier transform infrared spectroscopy (FT-IR)
was used to record the IR spectra of the samples using potassium bromide (KBr) pellet
technique.
Gold nanoparticles synthesis(26)
Spherical gold colloid was prepared using citrate reduction method as described
elsewhere (30). Briefly, In a 500 mL round bottom flask, 250 mL of 1 mM
HAuCl4(99.99% Sigma Aldrich, Germany) were brought to a boil while vigorously
stirring. While in reflux, 25 mL of 38.8 mM sodium citrate were quickly added and the
mixture was kept refluxing for 15 minutes with continuous stirring. The colloidal solution
was left to cool to room temperature while keeping the continuous stirring. The resulted
spherical gold nanoparticles were filtered using a 0.25 µm syringe filter (Sigma) then
transferred to a 250 mL Erlenmeyer flask with a ground glass cap, covered with
aluminium foil and stored in the dark at room temperature.
Characterization of AuNPs(27)
Morphological characterization of the prepared AuNP was performed by field emission
scanning electron microscopy (Model: Leo Supra 55). One drop of the AuNPs solution
was added onto a silicon slide that was allowed to air dry before examination. The λmax
for AuNPs was detected by scanning the colloidal AuNPs from 400-700 nm using UV
spectrophotometer (Jenway 6800). The concentration of the prepared AuNPs was
determined by the Lambert–Beer law assuming a calculated molar absortivity for the
plasmon resonance band maximum (526 nm) of 2.33×108 M−1 cm−1 (31).
Results
Size distribution and FT-IR spectra of amine functionalized magnetic
nanoparticles
Scanning
electron
image
of
amine
functionalized
magnetic
nanoparticles
(Supplementary Figure 1A) was analyzed using 1.41 J software package (Wayne
Rasband, National Institutes of health, USA. Http//:rsb.info.nih.gov/ij/Java1.6.0_05). The
magnetic nanoparticles were well dispersed and the majority of the measured
nanoparticles had a mean diameter of 90 nm (Supplementary Figure 1B). The magnetic
nanoparticles were functionalized by amino groups through their reaction with 3aminopropyltriethoxysilane (APTES) in a silanization reaction which includes hydrolysis
and condensation steps(26,27). The FT-IR spectra (Supplementary Figure 1C) of the
functionalized magnetic nanoparticles confirmed the synthesis and the functionalization
procedures. The band at 583cm-1 corresponds to the Fe-O bond, the one at 1050 cm-1 and
1380 cm-1 corresponds to the vibrations of SiOCH2 structure and Si-CH2 scissoring
vibrations respectively, while the N-H bending mode and stretching vibrations of the free
amino groups appeared at bands 1625 cm-1 and 3436 cm-1 respectively. Also, the
anchored propyl group of the APTES was present at band 2923 cm-1.
Size distribution and surface Plasmon band of the prepared AuNPs
Scanning electron microscope image of AuNPs (Supplementary Figure 2A) was
analyzed. The AuNPs were well dispersed and the mean diameter was found to be 15 nm
(Supplementary Figure 2B). The absorption spectrum of the prepared AuNPs displayed
a single peak in the visible region with λmax at 518–520 nm.
Figures
Supplementary Figure 1: (A) Scanning electron monograph (SEM) of the prepared
magnetic nanoparticles, functionalized with amino groups. (B) Analysis of magnetic
size distribution. The SEM image was analyzed using the Image 1.41 J software
package (Wayne Rasband, National Institutes of health, USA,
Http//:rsb.info.nih.gov/ij/Java1.6.0_05). As shown, the majority of the analyzed
particles have a mean diameter of about 90 nm. (C) FT-IR spectra of the prepared
magnetic nanoparticles after functionalization with amino-propyl tri-ethoxysilane
(APES). The peaks showed by the arrows are described in the text.
Supplementary Figure 2: (A) Scanning electron monographs of the prepared AuNPs.
One drop of AuNPs was placed on silicon slide and left to dry then examined using field
emission scanning electron microscopy (Model: Leo Supra 55). (B) Analysis of AuNPs
size distribution. The mean particle size of the prepared AuNPs was measured by
dynamic light scattering (DLS, Malvern Zeta sizer 3000HSA, Malvern Instruments, UK).
Figure 1A
Figure 1B
Figure 1C
Figure 2A
Figure 2B