Fluorescence lifetime based pH sensing using Resorufin.
Alan G. Ryder
∗a
, Sarah Power
a
, and Thomas J. Glynn
a
.
a Department of Physics, NUI- Galway, Ireland.
ABSTRACT
Accurate, non-contact pH sensing is of particular importance in the biological and clinical sciences. Fluorescence lifetime
based pH sensing is potentially more useful than intensity based methods because of the reduced sensitivity to excitation
source intensity variations, scattering effects, and photobleaching. In this work, we investigate the variation of fluorescence
lifetime with pH for resorufin. The intensity averaged lifetime (
τ
) of resorufin sodium salt in 0.1M phosphate buffer shows
an increase of > 3 ns over the 2 – 10 pH range, with 90% of the signal change occurring between pH 4 and 8. The
fluorescence is not quenched by chloride or oxygen and was unaffected by the ionic strength of the buffer. Resorufin is
relatively insoluble in non-alkaline phosphate buffered solutions, but
τ
was estimated to increase by ~2 ns between pH 6
and 8. Resorufin and its sodium salt were both incorporated into sol-gels by either acid or base hydrolysis of tetramethoxysilane (TMOS). Various surfactants were also added to the sol-gels in an attempt to optimise the fluorescence
properties and pH sensitivity of the dyes, and to prevent cracking. The sols were then cast from petri-dishes or dip-coated
onto acrylic and glass slides. The dyes retained their pH sensitivity, with
τ
showing an increase of approximately 2 ns over
the pH range 6 – 8. However, leaching of the dye is observed at higher pH and attempt to minimise dye leaching and solgel cracking, poly(vinyl alcohol) (PVA) was cross-linked to the silica gel to form a more flexible matrix.
Keywords: Fluorescence, lifetime, pH, sensors, sol-gel, resorufin.
1. INTRODUCTION:
Accurate, non-contact pH sensing is of particular importance in the biological and clinical sciences and optical methods
have significant advantages over more traditional electrochemical methods methods. These advantages include: reduced
sensitivity to electrical interference, rapid response times, miniaturised fibre-optic probes.
1 Measuring pH by fluorescencebased methods is well established for both imaging and sensing applications, and offers significant advantages over other
optical methods due to its high sensitivity, high specificity, and wide range of indicator dyes.
2 Fluorescence lifetime based
techniques have several advantages over more traditional fluorescence intensity methods which are susceptible to changes
in excitation light intensity, to photobleaching, and to variation in light scattering and absorption of the sample.
3
In lifetime based pH sensing a number of different approaches have been demonstrated: 1) indicator acid and base forms
have different lifetimes, 2) a mixture of fluorophores only one of which is sensitive to pH, resulting in a change in the
measured lifetime due to a change in the fractional contributions,
4 and 3) Fluorescence Resonant Energy Transfer (FRET).
5
The most common sensing method is that based on acid/base forms of the indicator having different lifetimes, examples of
which are: carboxy SNAFL-1 for intracellular microscopy based pH measurements,
6 fibre optic based systems using
carboxy-SNAFL-2,
7 and carboxy SNARF-6.
8, 9 In each case the indicator dye or the dye/matrix sensing layer should have
an apparent pKa* (excited state pKa) close to the desired pH sensing range.
∗ Corresponding author: alan.ryder@nuigalway.ie; phone: 353-91-750469; fax: 353-91-750584;
http://www.physics.nuigalway.ie/People/ARyder/index.html ; Department of Physics, National University of Ireland-Galway, Galway,
Ireland.
Full Citation: Fluorescence lifetime based pH sensing using Resorufin. A.G. Ryder, S.
Power, and T.J. Glynn. Proc SPIE Int. Soc. Opt. Eng. , 4876, 827-835, (2003).
Alan G. Ryder
∗a
, Sarah Power
a
, and Thomas J. Glynn
a
.
a Department of Physics, NUI- Galway, Ireland.
ABSTRACT
Accurate, non-contact pH sensing is of particular importance in the biological and clinical sciences. Fluorescence lifetime
based pH sensing is potentially more useful than intensity based methods because of the reduced sensitivity to excitation
source intensity variations, scattering effects, and photobleaching. In this work, we investigate the variation of fluorescence
lifetime with pH for resorufin. The intensity averaged lifetime (
τ
) of resorufin sodium salt in 0.1M phosphate buffer shows
an increase of > 3 ns over the 2 – 10 pH range, with 90% of the signal change occurring between pH 4 and 8. The
fluorescence is not quenched by chloride or oxygen and was unaffected by the ionic strength of the buffer. Resorufin is
relatively insoluble in non-alkaline phosphate buffered solutions, but
τ
was estimated to increase by ~2 ns between pH 6
and 8. Resorufin and its sodium salt were both incorporated into sol-gels by either acid or base hydrolysis of tetramethoxysilane (TMOS). Various surfactants were also added to the sol-gels in an attempt to optimise the fluorescence
properties and pH sensitivity of the dyes, and to prevent cracking. The sols were then cast from petri-dishes or dip-coated
onto acrylic and glass slides. The dyes retained their pH sensitivity, with
τ
showing an increase of approximately 2 ns over
the pH range 6 – 8. However, leaching of the dye is observed at higher pH and attempt to minimise dye leaching and solgel cracking, poly(vinyl alcohol) (PVA) was cross-linked to the silica gel to form a more flexible matrix.
Keywords: Fluorescence, lifetime, pH, sensors, sol-gel, resorufin.
1. INTRODUCTION:
Accurate, non-contact pH sensing is of particular importance in the biological and clinical sciences and optical methods
have significant advantages over more traditional electrochemical methods methods. These advantages include: reduced
sensitivity to electrical interference, rapid response times, miniaturised fibre-optic probes.
1 Measuring pH by fluorescencebased methods is well established for both imaging and sensing applications, and offers significant advantages over other
optical methods due to its high sensitivity, high specificity, and wide range of indicator dyes.
2 Fluorescence lifetime based
techniques have several advantages over more traditional fluorescence intensity methods which are susceptible to changes
in excitation light intensity, to photobleaching, and to variation in light scattering and absorption of the sample.
3
In lifetime based pH sensing a number of different approaches have been demonstrated: 1) indicator acid and base forms
have different lifetimes, 2) a mixture of fluorophores only one of which is sensitive to pH, resulting in a change in the
measured lifetime due to a change in the fractional contributions,
4 and 3) Fluorescence Resonant Energy Transfer (FRET).
5
The most common sensing method is that based on acid/base forms of the indicator having different lifetimes, examples of
which are: carboxy SNAFL-1 for intracellular microscopy based pH measurements,
6 fibre optic based systems using
carboxy-SNAFL-2,
7 and carboxy SNARF-6.
8, 9 In each case the indicator dye or the dye/matrix sensing layer should have
an apparent pKa* (excited state pKa) close to the desired pH sensing range.
∗ Corresponding author: alan.ryder@nuigalway.ie; phone: 353-91-750469; fax: 353-91-750584;
http://www.physics.nuigalway.ie/People/ARyder/index.html ; Department of Physics, National University of Ireland-Galway, Galway,
Ireland.
Full Citation: Fluorescence lifetime based pH sensing using Resorufin. A.G. Ryder, S.
Power, and T.J. Glynn. Proc SPIE Int. Soc. Opt. Eng. , 4876, 827-835, (2003).