The document discusses magnetically assisted surface enhanced Raman spectroscopy (MA-SERS) and its potential clinical applications. It describes how MA-SERS uses a magnetic nanocomposite containing iron oxide and silver nanoparticles to isolate analytes from complex matrices and enable highly sensitive label-free detection. As an example, MA-SERS was used to detect human immunoglobin G at concentrations 1000 times lower than standard levels in whole blood samples. The document concludes that developing MA-SERS capabilities could help companies like Thermo Scientific better compete in the Raman spectroscopy market and support important medical research.

1. MagneticallyAssisted
Surface Enhanced Raman
Spectroscopy(MA-SERS)
An Investigation into the clinical applications of nanocomposite
particles

2. The Basics: Comparison of Raman and IR
Spectroscopy
 A vibrational spectroscopy
 IR and Raman are the most common vibrational spectroscopies
for assessing molecular motion and fingerprinting species
 Raman is based on inelastic scattering of a monochromatic
excitation source
 Routine energy range: 200 - 4000 cm–1
 Complementary selection rules to IR spectroscopy
 Raman spectroscopy complements IR spectroscopy because, as
we have seen before, not all vibrations result in an IR
absorbance due to lack of dipole moment

3. The Basics: Comparison of Raman and IR
Spectroscopy
 Raman spectroscopy is a powerful and non-invasive tool for:
 studying molecular vibrations by light scattering
 determining chemical species
 Instead of examining the wavenumber at which a functional group has a vibrational
mode, Raman observes the shift in vibration of the molecule from an incident light
 It complements IR absorption spectroscopy which only results in absorptions if there is a
change in the dipole moment during vibration, symmetric stretches as shown below are
Raman active
 A change in dipole moment is required for a vibrational mode to be IR active, only then
can photons of the same energy as the vibrational state interact

4. What does Raman Spectroscopy
Measure?
 A change in the polarizability of a bond is required for a vibrational mode to
be Raman active
 Symmetric vibrations give rise to intense Raman lines
 Raman activity depends on the polarizability of a bond and how easily
electrons can be displaced from the bond, or conversely how tightly they are
held to the nuclei
 Distortion of electrons is easier as the bond becomes longer and harder
when it shortens thus polarizability changes with vibration– and this
vibrational mode scatters Raman light

5. What does Raman Spectroscopy
Measure?
 In an asymmetric stretch the electrons are more easily polarized in the
bond that expands & less easily in the bond that compresses, thus there is
no overall change in the polarizability of the bond in it is Raman inactive
 In general if there is a large number of loosely held electrons the Raman
signal will be strong
 Raman spectroscopy is generally more sensitive to the overall geometry
and framework of the molecule rather than specific functional groups

6.  Polarizability trend decreases going across a period as the effective
nuclear charge increases as electrons are held closer to nucleus and thus
are not easily deformed
 Increases going down a group as atomic radius increases and effective
nuclear charge decreases
 Inorganic and organic species can be analyzed
 Metals in coordination complexes and their corresponding ligands
generally have many loose electrons and provide strong Raman signals
 It can be used to predict structure and stability of these complexes
 No two compounds ever give the exact same Raman spectra and the intensity
of scatted light is proportional to the amount of analyte present thus is is both
qualitative and quantitative

7. Principles of Raman Spectroscopy
 Radiation or incident light is scattered when it passes
through a source

8. Principles of Raman Spectroscopy
 Radiation or incident light is scattered when it passes through a source
 When light is scattered an incident photon( E=hn) raises the vibration
state to any one of an infinite number of states between the ground
and first excited state , called virtual or imaginary states
 3 main types of scattering result
 Rayleigh scattering- photon leaves with its original E, E=hn & molecule
relaxes to original state
 Stokes scattering- photon scattered with less energy than incident
radiation, E=hn -E
 Anti-Stokes scattering – photons scattered with more energy than incident
radiation , E=hn E
 The change in E between the incident light from source and scattered
photons is measured as change wavenumber (cm-1)– thus any source
wavelength may be used ( 400-2000cm-1)

9. * 3Types of Scattering in Raman Spectroscopy, most common shown in
bold- filters used to reduce Raleigh scattering reaching detector
*E = frequency of IR vibration- if sample is IR active there would be a peak in IR
spectrum at frequency equal to change in E

10. *Rayleigh scattering is most common/intense transition as no change occurs in
Vibrational state, anti-stokes is the least frequent because molecule must be excited
before incident light strikes

11. What is Surface enhanced Raman
spectroscopy (MA-SERS)?
 Raman measurements are inherently weak at only .001% of source
intensity because only 1 photon in a million will scatter with a shift in
wavelength
 The main drawback to this techniques is that a very large sample quantity is
necessary for a reliable signal, and low quantities of analyte cannot be
detected
 Surface enhanced Raman spectroscopy requires absorption of species
to be studied on a prepared rough metal surface- the Raman laser
produces electron oscillations on the surface which interact with the
analyte to enhance the signal

12. What is Surface enhanced Raman
spectroscopy (SERS)?
 Using SERS increases in the intensity of Raman signal have been regularly
observed on the order of 104-106, and can be as high as 108 and 1014 for
some systems
 SERS works best with coinage (Au, Ag, Cu) or alkali (Li, Na, K) metal
surfaces
 The importance of SERS is that the surface selectivity and sensitivity
extends RS applications to a wide variety of interfacial systems previously
inaccessible to RS because RS is not surface sensitive
 These include in-situ and ambient analysis of electrochemical, catalytic,
biological, and organic systems
 An novel technique called magnetic assisted surface enhanced Raman
spectroscopy has made an even greater improvement on SERS

13. What is magnetically assisted-surface
enhanced Raman spectroscopy (MA-SERS)?
 Magnetically assisted surface enhanced Raman spectroscopy is an
innovative approach which employs a magnetic nanocomposite
and and efficient SERS enhancement inferred by Fe3O4 and silver
nanoparticles
 A magnet is then used to magnetically separate the analyte of
interest from surrounding complex matrix which is immediately
analyzed using SERS
 This technique has many advantages over other preparation
techniques such as sandwich methods because (I)only one
nanoparticle is needed(I)the methods is simpler (III)there is no
possibility of non-specific interaction from other matrix elements

14.  In previous immunoassay SERS detection techniques Raman
labels have been required to provide a strong Raman Signal
 Indirect analysis has been performed by measuring the signal of
the Raman label present as a linker between the antibody and
metal surface of the SERS active substrate
 In one previous approach gold nanoparticles were labeled with
Raman active 4-mercaptobenzoic acid
 These particles were attached to sandwich complexes as shown in
the below figure
4-mercaptobenzoic acid

15.  In such methods magnetic substrates are used to efficiently extract a
target from a complex matrix
 a selective bond between an analyte and immunorecognition molecule
(previously immobilized on the surface) binds only to the analyte of
interest
 magnetic particles are then attached to the surface and the target
analyte is extracted from its surrounding matrix by application of
external magnetic force

16.  The SERS-active silver or gold nanoparticles are added after purification
and selectively attached to the target using the same set of
immunorecognition molecules present on the metal surface
 The Raman label, 4-meraptobenzoic acid, serves as a linker between
the antibody of interest and the SERS active metal substrate
 This techniques has a high LOD of 1-10 ng/mL, however two sets of
nanoparticles must be synthesized, each with limited stability
 Experimental design is highly complicated
 There is a high risk of false positive signals due to non specific
interactions between particles and non targeted compounds
attracted from matrix
 The immobilization of anti-IgG via a bond with streptavidin did not
influence its total activity, in contrast to the approaches mentioned
above, which utilized unspecific direct immobilization on the metal
surface

17. Label-Free Determination of Human Immunoglobin G
(IgG) in Blood using Fe3O4@Ag Nanocomposite

18. Fe3O4@Ag@streptavidin@anti-IgG
Synthesis
 The novel
Fe3O4@Ag@streptavidin@anti-IgG
nanocomposite allows for the first
label-free SERS analysis
 Fe3O4@Ag@streptavi-din@anti-
IgG is composed of a magnetic
core(Fe3O4) modified by O-
carboxymethylchitosan
 O-carboxymethylchitosan is used to
encapsulate the magnetic
nanoparticle (MNP) to avoid the
agglomeration & to make the MNPs
monodisperse in suspension
 It can be seen clearly in Raman
spectra that the 680 cm−1 peak of
Fe–O–Fe in Fe3 O4 shifts to 672cm−1
after covering the MNP by OCMCS

19. Fe3O4@Ag@streptavidin@anti-IgG
Synthesis
 The silver surface was
subsequently modified by
streptavidin and finally anti-
immunoglobulin G
 Streptavidin immobilizes the
anti-IgG( antibody which binds
specifically to IgG) without
affecting the total activity of
the metal surface– meaning
SARS signal will not be
affected
 Streptavidin

20. *Individidual modification steps in formation of Fe3O4@Ag@streptavidin@anti-
IgG

21. Potential Applications
 Development of analytical methods to determine ultralow levels of
immunoglobulins in various clinical samples including whole human
blood and plasma is a scientific challenge
 Many essential discoveries in the fields of immunology and
medicine in the past few decades have made this a prominent field
as intravenous immunoglobins have been found to have multiple
clinical applications:
 diopathic thrombocytopenic purpura (ITP)
 Kawasaki disease
 Guillain–Barré syndrome
 other autoimmune neuropathies
 myasthenia gravis
 Dermatomyositis

22. Potential Application
 As Immunoglobins play an essential role defending the human body against
viruses and disease, they are indispensible to clinical and pharmaceutical
industries
 MA- SERS was successfully used to isolate IgG from whole human blood using
the Fe3O4@Ag@streptavidin@anti-IgG nano-composite
 IgG has a considerable smaller diameter than the other immunoglobins (A,M,
E, and D) and is able to enter the placenta of an unborn baby to protect it
during pre-natal development
 IgG is usually present in human blood samples at 10 g·L−1 which changes
when a disease interrupts the body’s pathological processes
 MA-SERS is capable of detecting human IgG at 1000× lower concentration
level
 The results of the analysis show that samples from two patients 9 g·L−1 of IgG
and 10 g· L−1 of IgG --- what a healthy human should have!

23. Raman Spectrum of Human Blood
* Raman spectroscopy results for real human blood sample

24. Conclusion
 Thermo Corp. already offers a DXR Raman spectroscope capable of SERS
and an accompanying SERS analysis package, however the package is very
basic and only contains 70nm gold colloids( gold nanoparticles suspended
in solution)
 Investment in a package containing the necessary nanoparticles and and
magnetic materials needed to perform MA-SERS would be a prudent
investment and would keepThermo Crop. at the forefront of Raman
Spectroscopy
 Would allowThermo to compete with companies such as Kaiser Optical
Systems, the current leader in Raman Spectroscopy!

25. Leading Researchers/ Major Suppliers
 Researchers
 Marián Hajdúch, M.D., Ph.D.
 Director of the Institute of Molecular andTranslational Medicine, Faculty of
Medicine and Dentistry, Palacky University in Olomouc (Czech Republic)
 Email: marian.hajduch@upol.cz
Phone: +420 585632082, +420 585632083
 Prof. Radek Zbořil, Ph.D.
 General director of the Regional Centre ofAdvancedTechnologies and Materials
 Professor at the Palacky University in Olomouc (Czech Republic)
 Email: radek.zboril@upol.cz
Address: Šlechtitelů 11, Olomouc, Czech Republic, 78371
Phone: (+420) 58 563 4337
Fax: (+420) 58 563 4958