Raman tags: Novel optical probes for intracellular sensing and imaging

1. RAMAN TAGS: NOVEL OPTICAL PROBES FOR
INTRACELLULAR SENSING AND IMAGING
Zulquernain Haider (19)
Muhammad Ikram kalyar (08)

2. LETS MAKE A DECISION!
Fluorescence labels & tags
• Provide high sensitivity
but
• Investigate only the targeted
object
• Limited Information
• Fluorescent tags are relatively
bulky
• Alter biological activity when used
to tag small biomolecules
• Not suitable for multiplex
• Cell death occur
Optical labels
• With enhanced information
content,
• Minimal perturbation,
• High sensitivity
• Specificity
• Non-intrusive
• Living cell

3. RAMAN TAG
• emerging strategy
• permits quantitative, qualitative, and multiple analyses of molecules
• intracellular pharmacokinetics,
• drug location
• drug concentration in subcellular regions
• intracellular kinetics,
• nature of the interaction between drugs and their pharmacological targets.

4. RAMAN EFFECT
• The inelastic scattering of a photon by molecules which are excited to higher
vibrational or rotational energy levels.
• The sharp, molecularly specific spectra obtained from endogenous biomolecules
and exogenous reporters make it possible to specifically identify individual
components from a mixture
• .

5. BASIC ASSUMPTIONS
• The Raman signals of the molecules, which work as a Raman probe, should have
unparalleled strong Raman intensity or a unique Raman shift.
• Which enables recognition of the targeted molecule in a complex cell environment.

6. TYPES OF THE RAMAN TAG
1. SERS probe
2.Bioorthogonal
Raman probe
3. RR probe

7. SURFACE-ENHANCED RAMAN SPECTROSCOPY
PROBES (SERS)
• the most attractive Raman probes.
• capable of marking specific molecules
• SERS probe consists of four parts:
1. Nano-structured substrates,
2. Raman reporter,
3. Targeting molecule,
4. Protection shell

8. NANO-STRUCTURED SUBSTRATES
Au and Ag are most popular SERS substrates
Because they are air-stable
They have localized surface plasmon resonances
that cover most of the visible wavelength ranges
where most Raman measurements occur

9. AU AND Ag NANOSPHERES
• The most broadly used nanostructures for Raman enhancing because
1. Easily controlled sizes
2. Good stability
3. Outstanding biocompatibility
• The SPR range of all NSs is 400–700 nm, which fits the wavelength of
commonly used visible lasers

10. Comparison of Ag
and Au
• Extinction (the sum of scattering and
absorption) spectra of gold/silver NSs:
• (a) Gold NSs with diameters ranging
from 10 to 100 nm in aqueous
(Concentration: 0.05 mg/mL);
• (b) 50 nm gold NSs in air, water, and
silica;
• (c) Silver NSs with diameters ranging
from 10 to 100 nm in aqueous
(Concentration: 0.02 mg/mL);
• (d) 50 nm silver NSs in air, water, and
silica.

11. As the medium refractive index increases, the nanoparticle extinction spectrum shifts to longer wavelengths.
For SERS probes application in complicated environments such as intracellular detection or imaging, the surrounded medium
should be considered in selecting the proper excitation wavelength.
Silver NS is more efficient for SERS enhancement because of less damping of the plasmon modes,
But the biocompatibility and stability of silver is much poorer than that of gold.
Thus, currently, gold is the most commonly nano-structured substrate used for SERS tags,,
especially for live biological application

12. . RAMAN REPORTERS
• Raman reporter molecules, which have a characteristic Raman spectral signature are
conjugated to the substrates.
• Slelction based on following aspects
1. The Raman cross section of reporter molecules should be relatively large, producing
strong SERS signals.
2. The reporter molecule structure should be designed properly for attachment on the
surface of gold or silver because SERS enhancement is strongly dependent on the
distance between the reporter molecule and the NPs..
3. To avoid signal overlap of tags for multiplex labeling, the Raman spectrum of
reporters should have few characteristic peaks

14. COATING MATERIALS
• the naked probe's reliability and efficiency are poor because of the ligand
dissociation of reporters and the bare metal surface that absorbs other molecules in
the complex biochemical environment
• biotoxicity is also a considerable problem for live cell application of SERS labeling
• Bovine serum albumin is the most popular material

15. TARGETING MOLECULES
• The dominant molecular recognition method in biology is immuno-labeling, in
which antibodies are conjugated with the designed probe with the SERS signal in
the previous three steps
• Small-molecule ligands are also applied for specific biological targets, which should
be designed for particular applications
• Immunoassay-based microarray detection
• Cell imaging
• . In vivo (a living mouse)

16. IN VIVO (A LIVING
MOUSE)
SERS spectra upon incubation with
scFv-conjugated SERS nanotags in:
a) HER-2 positive SKBR-3 cells,
b) HER-2 negative MDA-MB231
cells,
785 nm and a laser power of 60
mW.

17. THE BIOORTHOGONAL RAMAN PROBES
• All natural biomolecules possess no Raman vibrational signal in the range from 1800
cm− 1 to 2800 cm− 1, called the cell's Raman-silent region
• The bioorthogonal Raman reporters consist of chemical bonds, such as alkyne,
nitrile, and C – D, that show Raman signals in this specific region
• Alkyne compounds are the most favorable bioorthogonal Raman reporters because
they possess desirable spectroscopic and chemical features as tags
• They are composed of two atoms small enough for labeling small molecules.
Moreover, almost no C triple bondC stretch can be found in cell components
• It is inactive to cells' endogenous biomolecules

18. PRINCIPLES OF THE
BIOORTHOGONAL
Raman probe (the Raman
spectrum of a live HeLa cell, the
naturally existing biomolecules
produce no Raman signal ranging
1800 cm− 1 to 2800 cm− 1).

19. ALKYNE PROBE CONSTRUCTION
• The alkyne probe consists of two parts:
1. Raman reporter
In particular, the Raman reporter's design takes Raman spectroscopic
bioorthogonality and design principles of chemical reporters into consideration.
Within this region, it possesses a Raman band with which Raman signals from
the cellular background do not interfere.
2. Targeting molecule
 The targeting molecule works as a biorecognition component for a specific
biomolecular binding

21. ADVANTAGES AND PROSPECTIVE
• The probe is ultra-small and can easily penetrate into the cell nucleus.
• The bioorthogonal probe provides almost no fluorescence background.
• Through one-time cell sample scanning, we can obtain distribution information of
labeled and unlabeled biomolecules from the sample.
• Repeating laser illumination does not affect Raman intensities
• The quantitative analyses of tagged molecules are feasible.
• multicolor imaging can be achieved by using 13C isotopically edited C ≡ C

22. RESONANCE RAMAN (RR) PROBES
• RR probes are based on the resonance Raman mechanism
• The excitation wavelength is carefully chosen to be close in energy to the Raman
reporter molecule's electronic transition.
• Raman reporter molecules provide greatly enhanced scattering intensities because
of such overlap, while the Raman intensities of all other molecules remain
unchanged
• With the special targeting molecules, the RR probe can mark specific molecules
• Using Raman microscopy indirectly, map the target molecules in a biological
sample.

23. PRINCIPLES OF THE
RESONANCE RAMAN
PROBE
(the Raman spectrum of the cytoplasm
from treated and untreated HeLa cell).

24. RR PROBE CONSTRUCTION
1. RR reporter
A specially designed chromophore, displays a Raman signal strong enough to cover up
Raman scattering from all other molecules
2. Targeting moiety.
An active bound ligand that recognizes the biomolecules of interest.

26. ADVANTAGES AND PROSPECTIVE
• RR reporters are especially advantageous for intracellular Raman imaging.
• The RR reporter's Raman signal is selectively enhanced based on a resonance
mechanism that allows for high-contrast Raman imaging with high sensitivity and
velocity.
• The sizes of RR probes are at molecular scale, which enable easy access to dense
intracellular structures and eliminate artificial interference with cells.