Fluoresecent pH-dependent Lipobeads in vivo

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pH changes in biology
Standard pH meter

pH meter
pH electrode: glass electrode filled with electrolyte and Ag/AgCl reference electrode
thin glass membrane in contact with solution

Potential difference builds up over the thin glass membrane due to differences in H+ concentration

Potential measured against reference electrodes and pH calculated

Too big to be implanted in a cell!


Fluorescense
As seen in the Energy Level Diagram (left):

1.  Energy Absorption
excites the molecule to excited state
some molecules may be in vibrationally or rotationally excited states 

2.  Vibrational relaxation
molecule transitions to lowest energy excited state 

3.  Fluorescence
molecule returns to ground state by emitting a photon  

4.  Non-radiative relaxation
molecule returns to ground state but does not emit radiation

Energy Level Diagram depicting fluorescense. 

pH-sensitive Dyes
Fluorophores are aromatic or conjugated with delocalized electrons
pH sensitive fluorophores: emission differs at different pH values
Fluorescein and tetramethylrhodamine (pH insensitive) are used frequently in cellular applications
They have high absorbance and emission wavelength in the visible light range


Structures of pH-sensitive Dyes


pH insensitive Dyes

pH insensitive dyes, such as tetramethylrhodamine (above), are used as a control


Experimental Results
(McNamara et. al. 2001)





Fluorescence spectra of individual lipobeads containing fluorescein at varying pH levels:
(a )pH = 5, (b) pH = 6 (c) pH = 7,  (d) pH = 8. 

Dye Delivery

Liposomes containing Fluorescein (McNamara et al 2001)

Liposmome
is phospholipid bilayer vesicle


Cellular Data
(McNamara et. al. 2001)


pH change of a single liposome of fluorescent marker:
A sharp drop in fluorsecence is observed (t=9 sec)
when the cell ingests the dye into a more acidic environment. 
The more acidic environment causes fluorescein to fluoresce less.



Mice macrophages loaded with fluorescent lipobeads under
bright field (left) an fluorescent imaging (right) under x40. 

Advantages
  • No leaking like other methods (polymer matrix)
  • High chemical stability in solution
  • Protection of dye from quenching species
Disadvantages
  • Biocompatability/Cytotoxicity

References

http://accessscience.com/content.aspx?id=504050


Kerry P. McNamara,, Thuvan Nguyen,, Gabriela Dumitrascu,, Jin Ji,, Nitsa Rosenzweig, and, Zeev Rosenzweig.  "Synthesis, Characterization, and Application of Fluorescence Sensing Lipobeads for Intracellular pH Measurements”  Analytical Chemistry 2001 73 (14), 3240-3246