pH changes in biology
- pH changes are important to several biological processes:
- muscle contraction, endocytosis, cell proliferation, apoptosis,
ion transport
- Biological enzymes function at an optimal pH
- Folded protein is stablized by the specific ion concentration
- Protein enzyme ATPase uses a proton gradient for energy production
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
- Fluorescent dyes can be
encapsulated by phospholipid bilayer vesicles (lipsomes).
- Water soluble while retaining same reactivity
- Protect dye from quenchers
- Liposome vesicle fuses with cellular lipid bilayer and delivers
dye
inside cell for reactions
- Also used for drug or gene delivery
Liposomes containing Fluorescein
(McNamara et al 2001)
|
Liposmome is
phospholipid bilayer vesicle
|
Cellular Data
(McNamara
et. al. 2001)
- Mice macrophages were incubated with
fluorescent pH-sensing
lipobeads
- Lipobead is a membrane on a
polystyrene bead
- Lipobeads filled with fluorescein and
tetramethylrhodamine
- Lipobeads were removed using a buffer
wash
- Cells were analyzed with
fluorescence-imaging microscopy
- Exposure to detection light
- Intracellular pH is determined from
emission peaks
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
