Magnetically AssistedSurface Enhanced RamanSpectroscopy(MA-SERS)

An Investigation into the clinical applications of nanocomposite particles

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

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

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

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

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

Principles of Raman Spectroscopy Radiation or incident light is scattered when it

passes through a source

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 -ΔE Anti-Stokes scattering – photons scattered with more energy than

incident radiation , E=hn +ΔE 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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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!

Raman Spectrum of Human Blood

* Raman spectroscopy results for real human blood sample

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 keep Thermo Crop. at the forefront of Raman Spectroscopy

Would allow Thermo to compete with companies such as Kaiser Optical Systems, the current leader in Raman Spectroscopy!

Leading Researchers/ Major Suppliers Researchers

Marián Hajdúch, M.D., Ph.D. Director of the Institute of Molecular and Translational 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 of Advanced Technologies 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 4337Fax: (+420) 58 563 4958