The prognostic value of HistidineRich Glycoprotein RNA in Breast tissue using Unmodified Gold NanoparticlesAssay

Running title: Prognostic value of tissueHistidineRich Glycoprotein RNA using AuNPs

SanaaEissa1 ,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.

3General 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..

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 γ-Fe2O3 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 mMHAuCl4(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 λmaxfor 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 3- aminopropyltriethoxysilane (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, MalvernZetasizer3000HSA, Malvern Instruments, UK).

Figure 1A

Figure 1B

Figure 1C

Figure 2A

Figure 2B