Preparation and study of core shell Fe3O4/Au nanoparticles for

traceability of blood vessels and biosensing by surface enhanced

Raman spectroscopy
A-M. Iordache1, C. Rizea2, C. Giuglea3, C.N. Zoita1, I. Stamatin4, S. M. Iordache1, C. R. Stefan (Iordanescu)1,,
M. I. Rusu1, L. Tortet5, A. Tonetto4, R. Notonier5 C. E. A. Grigorescu1*

1
National Institute of Research and Development for Optoelectronics INOE 2000, Magurele, Romania
2
ROXY VETERINARY S.R.L. Magurele, Romania
3
University of Medicine and Pharmacy Carol Davila, Bucharest, Romania
4
Faculty of Physics,University of Bucharest, Romania
4
Aix-Marseille Universitè, MADIREL & 5 Centrale Marseille, CNRS, Federation Sciences Chimiques
Marseille – PRATIM, France
ABSTRACT

Fe3O4/Au core-shell nanoparticles have been prepared using reverse micelle synthesis with the aim of studying
the traceability of blood vessels and allow biosensing through surface enhanced Raman spectroscopy. The shell
provides protection against oxidation, plasmon properties and prevents agregation of the magnetic nanoparticles.

A solution of Fe3O4/Au core-shell nanoparticles in fluoresceine has been injected into fresh ex-vivo animal
tissues to check the traceability of blood vessels. Optical properties of the tissues in the UV-Vis-NIR have been
explored on both fresh and injected tissues to prove the penetration of Raman excitation sources through the
dermis and the entire sequence of the skin layers. Postbariatric surgery as well as rapid diagnosis on circulatory
issues are examples of future applications.

Keywords: magnetite synthesis, magnetite/Au core shell, traceability of blood vessels, optical transmissivity of
skin, surface enhanced Raman scattering, label free biosensing

1. INTRODUCTION
Raman spectroscopy is a particularly attractive technique owing to its high sensitivity and specificity to the
vibrations of intramolecular bonds thus finding applications in many fields such as biosensing, diagnosis and
monitoring of treatment in medicine, characterization of tissue in vivo, ex vivo or in vitro. In addition, its
nondestructive character and the needless of sample preparation enhance the rapidity of evaluation,
classification, and discrimination of tissue type in biopsy, margin assessment, therapeutic evaluation etc., based
on the “finger prints” of Raman-active functional groups composing the molecules in a sample. The frequencies
at which the Raman bands are found in a spectrum correspond to the vibrational modes of the bonds in a
molecule whereas their intensities increase with the number of bonds in the probed area. Although Raman and
infrared (IR) spectroscopy are complementary they might not always perform simultaneously since Raman
modes and IR ones are not necessarily active in every samples. Also, there might be strong enough differences
between the intensities of the IR and Raman effects like for instance in the case of water molecules. The IR
radiation is strongly absorbed by water above ~1800 cm-1 whereas Raman scattering can be observed up to
higher than that frequencies making possible to assess even O-H stretching modes (2400 to 3500 cm-1) vibrations
in biological samples. Therefore, Raman spectroscopy proves better capabilities than IR for tissue and water
containing biosamples. [1, 2]. The small scattering cross section (∼10 -30 cm2 per molecule) of biologic samples
is however a drawback in using spontaneous Raman scattering as a spectroscopic technique in the clinique
because of the long acquisition and integration times required to obtain an exploitable spectrum [3]. Research to
enhance the Raman signal in these particular cases resulted in development of several techniques among which
surface enhanced Raman scattering (SERS) and surface plasmon resonance (SPR), quite close to each other,
made a serious progress in using Raman spectroscopy in biological sensing and various medical applications[4].
SERS is funded on the interaction between incident light from a laser beam that excites surface plasmons, i.e.
collective oscillations of conduction electrons in a single electron metal (e.g. Au, Ag, Cu), metal. This leads to
an enhanced electric field around the metal nanostructures localized at a few nanometers above the metal
surface. If biomolecules are attached or adsorbed on the metal surface the enhanced electric field enables an
amplification of the vibrations of the intramolecular component bonds thus leading to a spectacular increase in
the Raman signal [5]. Localisation of light at the subwavelength limit would yield in a even stronger
enhancement of electromagnetic fields in spin-plasmonic nanostructures. The reactivity of magnetic
nanoparticles increases with decreasing size and thus surface effects are more intense than in bulk [6].
Drawbacks on using them in active sensing parts are the low conductivity, poor optical properties and the
tendency to form aggregates when dispersed in particular solvents.[2].
Therefore, we propose nanostructured magnetic surfaces and plasmon generating coatings (Au) in a core shell
sequence to produce substrates suitable for surface enhanced Raman spectroscopy. The surfaces of the
nanoparticles are the substrates in this work where we tempt the traceability of blood vessels in various tissues
(skin, lymphnodes, etc.) exciting the Raman effect through the entire scaffold of the skin.

2. EXPERIMENTAL SECTION
2.1 Synthesis and characterization of core shell Fe3O4 /Au nanoparticles
2.1.1 Synthesis, size and dispersity
Core shell Fe3O4 /Au (Au@Fe3O4) nanoparticles find many applications in analytical chemistry, electrochemical
and optical sensors, biomedicine (e.g. contrast agents in MRI), targeted drug delivery and others. This wide
range of Au@Fe3O4 applications is due to their special properties, which can be adjusted to fit the applications
by tailoring their size, shell thickness, shape, charge, and surface [6].
Usually the synthesis of core shell magnetite/Au nanoparticles comprises two processes: 1) the synthesis of
Fe3O4 nanoparticles 2) the gold capping. The preparation of magnetite nanoparticles through reverse micelle
synthesis is detailed in one of our previous works [7]. For this study the synthesis of the Au@Fe3O4 occurred in
one step by mixing two solutions in the ultrasonic bath: (a) solution A containing 200 µL Au nanoparticles
(7.14% dilution in lavender oil) dispersed in 20 ml lavender oil and (b) solution B containing 1 ml Tween 20 and
3,5 ml Fe3O4 in DI water (3.255 mg Fe3O4). Upon vigorous mixing, the final solution (solution F) became milky
pale pink. After 1 h in the ultrasonic bath 200 ml isopropyl alcohol has been added to the solution and the
particles were separated using a powerful magnet. The diameter of the Fe3O4/Au core-shell particles was
determined from dynamic light scattering (DLS) and Raman spectroscopy to be 221 nm, with a polydispersity
index (PDI) of 0.521, which indicates that the solution is monodisperse. The zeta potential value obtained was -
17 mV. The low polydispersion index can be correlated with the narrow zeta potential distribution of the
Fe3O4/Au core-shell particles proving that the synthesized particles are stable, do neither agglomerate nor
undergo sedimentation and exhibit a low inter-particle interaction thus having low ionic strength. Figure.1 a)
shows a schematic view of the synthesis of Au@Fe3O4 nanoparticles and b) DLS characteristic spectrum

a) b)
Figure 1. a) synthesis of Au@Fe3O4 nanoparticles; b) DLS characteristic spectrum

The particle size was determined by DLS using the Zetasizer Nano ZS from Malvern Instruments Ltd., U.K. The
scattering angle was 173° and the laser wavelength 532 nm. Zeta potential (ZP) measurements were carried out
on the same sample and same instrument by applying an electric field across the analysed aqueous suspension.
All data were expressed as mean value (presented as mean ± standard deviation, SD) of three individual
experiments.

2.1. 2 Optical properties

This study aims at using Au@Fe3O4 as SERS substrates to enhance the Raman signal of blood in blood vessels
which must be traced through the various layers of the skin. A section where fluorescein traces the blood vessels
and the schematics of the skin scaffold, in which Au@Fe3O4 were injected after dispersing them in an aqueous
solution of fluorescein are displayed in the figure 2 a) and b) respectively, where a) is a section where
fluorescein traces the blood vessels and b) shows the skin scaffold with Au@Fe3O4. Irradiation with UV light at
325 nm put in evidence traces of blood vessels. Exploring the activity of Au@Fe3O4 nanoparticles inside the skin
layers has also required the characterisation of the optical transmittance of the skin layers and the Raman activity
of the Au@Fe3O4 as shown in the figure 3, a, b, and c.

a b
Figure2. a)- a section where fluorescein traces the blood vessels; b) schematics of the skin scaffold with
Au@Fe3O4

UV-VIS-NIR transmittance of the samples was measured with a Perkin Elmer SPECTRUM 100 UV-VIS-NIR
spectrophotometer, at room temperature, over the range 400 to 2800 nm wavelength range. The fluorescein at a
concentration of 5% mg/ml in the blood sends photons through the vessel wall for visualization. The vessel wall
did not selectively absorb the light but within a short while the vessel wall absorbed the fluorescein and
contributed significantly to the detected fluorescence [8].

a b

514 nm
Au-Magnetite 633 nm
Intensity (a.u.)

200 400 600 800 1000 1200 1400 1600 1800

-1
Raman shift (cm )

c
Figure 3. a) & b) optical transmittance of the skin layers; c) the Raman spectra of the Au@Fe3O4 collected
with two irradiation sources

The Raman spectra in the figure 3 c were performed with a LABRAM HR 800 SPECTROMETER (Horiba
Jobin Yvon) using two laser wavelengths in the visible range of the electromagnetic spectrum, 514 and
respectively 632 nm. All measurements were carried out at room temperature. The reason of using excitation at
two different wavelengths lays in the penetration depth, i.e. the radiation energy provided by the two laser
sources. The typical vibration mode of magnetite is found at 661 cm-1 and corresponds to A1g mode in Fe-O [9].
As seen in the figure 3c, 633 nm excitation provides better resolution and enhancement of the Raman spectrum
than 514 nm. The peak at 1317 cm-1 can be attributed to a two-magnon scattering feature [10] rather than to
carbon presence [11]. The intensity of the spectra collected on noble metal coated magnetite might be attributed
to the magnetic – surface plasmon interaction under visible monochromatic light as a result of incoherent spin
pumping induced by the 514 and 632 nm laser irradiation at the magnetite-gold interface, knowing that a thermal
nonequilibrium state at a ferromagnetic/paramagnetic interface can make magnons in e.g. magnetite and
electrons in gold diverge from their equilibrium state and produce a spin current at the interface [3, 7].
Magnetite has been intensely studied by Raman spectroscopy [7, 10, 11] leading to a variable number of
vibration modes, peak positions and assignments.

3. CONCLUSIONS
Au@Fe3O4 nanoparticles were prepared by one step process with the aim to provide SERS substrates with spin-
plasmonic interfaces for blood vessels traceability in various tissues (skin, lymphnodes, etc.) exciting the Raman
effect through the entire scaffold of the skin. Transmittance of skin containing only Au@Fe3O4 nanoparticles is
higher than of the raw skin itself. The blood vessel walls absorbing the Au@Fe3O4 nanoparticles in fluorescein
aqueous solution at 5mg/ml are perfectly traceable thus allowing targeted Raman spectroscopy/SERS
measurements through the skin. Magnons in the magnetite and electrons in gold diverge from equilibrium and
generate a spin current at the magnetite/plasmonic metal interface, as proved by the shifts in the measured
Raman spectra A thermal nonequilibrium state is expected to be at the origin of this result, where.

ACKNOWLEDGEMENTS
This work has been performed within CORE Programme, Ctr. 18/N/2019 and Contract nr.19PFE/17.10.2018
financed by Ministry of Research and Innovation.
The pet patients and their owners that consented for the samples used in this are greatly acknowledged. Many
thanks to the technicians in all involved teams for their invaluable work.

REFERENCES

[1] Gregory W. Auner, et al: Applications of Raman spectroscopy in cancer diagnosis, Cancer and
Metastasis Reviews 37:691–717, https://doi.org/10.1007/s10555-018-9770-9 (2018)
[2] C. Kendall, J. Day, J. Hutchings et al.: Evaluation of Raman probe for oesophageal cancer diagnostics,”
Analyst, vol. 135, no. 12, pp. 3038–3041, 2010
[3] Sishan Cui, Shuo Zhang, and Shuhua Yue: Raman Spectroscopy and Imaging for Cancer Diagnosis,
Hindawi, Journal of Healthcare Engineering Volume 2018, Article ID 8619342,
https://doi.org/10.1155/2018/8619342.
[4] E. Darrigues, Z. A. Nima, W. Majeed et al., Raman spectroscopy using plasmonic and carbon-based
nanoparticles for cancer detection, diagnosis, and treatment guidance. Part 1: diagnosis, Drug
Metabolism Reviews, vol. 49, no. 2, pp. 212–252, 2017
[5] A. N. Dizaji, et al.: Silver or gold deposition onto magnetite nanoparticles by using plant extracts as
reducing and stabilizing agents, al.: Artificial Cells, Nanomedicine, and Biotechnology, 2015; Early
Online: 1–7, DOI: 10.3109/21691401.2015.1019672
[6] S. Moraes Silva et al. : Gold coated magnetic nanoparticles: from preparation to surface modification for
analytical and biomedical applications, Chem. Commun., 52, 7528, 2016.
[7] C. E. A. Grigorescu, A-M. Iordache, M. I. Rusu, C. R. Stefan (Iordanescu)1, S. M. Iordache, C. Rizea, et
al.: Spin Plasmonics and Surface Enhanced Raman Spectroscopy in Label Free Biomolecular Sensing,
IEEE Xplorer ICTON 2019, 181714, 2019
[8] John L. Fox, and M. Gazi Yasargil: Fluorescein Angiography in Microvascular Surgery: A Study Using
the Rodent Artery, Stroke, Vol. 5, March-April 1974
[9] A. Hirohata et al: Roadmap for Emerging Materials for Spintronic Device Applications, Advances in
Magnetics, ISSN 1941-0069, 2015.
[10] L.J. Swartzendruber, Properties, units and constants in magnetism, J. Magn. Magn. Mater. 100, 573,
1991.
[11] R. J. Soulen et al. : “Measuring the spin polarization of a metal with a superconducting point contact,
Science 282, 85, 1989.
[12] Y.Ji. et al. : Determination of the spin polarization of half-metallic CrO2 by point contact Andreev
reflection, Phys. Rev. Lett. 86, 5585, 2001. dedicated surgical microscope (Pentero, Carl Zeiss Meditec)