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D.C. Hill et al.
of biomolecular interactions can then be achieved using competition methods,
making this technique useful as a high throughput screening method [169].
Fluorescence polarization and SPA assays both allow monitoring of 'real time'
kinetics of association or dissociation of the receptor-ligand/protein-protein
complex as they do not require a separation step.
One possible alternative as a detection system for heterogeneous assays is to
use time resolved fluorescence (TRF). TRF utilizes lanthanide chemistry to
overcome problems such as quenching and background flourescence which are
often observed with standard fluorescence systems. Under appropriate conditions, lanthanides produce a high fluorescence intensity with a sharp emission
peak and long decay time, and exhibit a large Stoke's shift (i.e. difference
between the excitation and emission wavelength). In practice this means that
fluorescence associated with the target interaction can be measured after background has decayed and that assays are highly sensitive and have a wide
dynamic range [170]. In addition, a stable signal is generated making the
technology ideal for automation [171]. Streptavidin, antibody and proteinlanthanide complexes are available commercially and most proteins can be
readily labelled to take advantage of this versatile technology which is easily
adaptable to natural products drug screening [172].
Time resolved fluorescence technology can also be used in delayed fluorescence energy transfer DEFRET techniques which enable homogeneous assays
to be carried out, as fluorescence is generated by the close proximity of the
lanthanide to an acceptor molecule. The acceptor molecule used in this technique may either transmit or quench fluorescence, depending on the choice of
assay [173].
A range of techniques including solution phase assays [174, 175], heterogeneous assays [176, 177] and SPA have also been used in enzyme inhibitor
screening programmes as shown in Fig. 11.
Emerging assay technologies other than cell-based SPA and DEFRET
include electrochemiluminescence (ECL), surface plasmon resonance (SPR), and
rnicrophysiometry (see Fig. 12).
Electrochemi/uminescence utilizes labelling of one of the assay reagents. In
this case, diffusion of a precursor molecule onto an electrode surface is followed
by rapid electron transfer, which initiates the excitation of the reporter molecule
and results in the emission of a photon of light at a specific wavelength. Use of
an electrode allows reactions to take place in a variety of solvents, and gives the
capability of increasing assay sensitivity to the fmoll-1 range by applying
different electrode potentials [178].
Surface plasmon resonance is a label free, real time optical detection technique which facilitates quantification of molecular interactions. The basis of
SPR is that a metal (usually gold) coated sensor chip is used to give rise to
electronic oscillations, or surface plasmons, at a metal surface. These decay
exponentially as a function of distance and their refractive index will change if
a complex is formed on the sensor chip surface. This change can be detected and
recorded using appropriate instrumentation [179].
D.C. Hill et al.
of biomolecular interactions can then be achieved using competition methods,
making this technique useful as a high throughput screening method [169].
Fluorescence polarization and SPA assays both allow monitoring of 'real time'
kinetics of association or dissociation of the receptor-ligand/protein-protein
complex as they do not require a separation step.
One possible alternative as a detection system for heterogeneous assays is to
use time resolved fluorescence (TRF). TRF utilizes lanthanide chemistry to
overcome problems such as quenching and background flourescence which are
often observed with standard fluorescence systems. Under appropriate conditions, lanthanides produce a high fluorescence intensity with a sharp emission
peak and long decay time, and exhibit a large Stoke's shift (i.e. difference
between the excitation and emission wavelength). In practice this means that
fluorescence associated with the target interaction can be measured after background has decayed and that assays are highly sensitive and have a wide
dynamic range [170]. In addition, a stable signal is generated making the
technology ideal for automation [171]. Streptavidin, antibody and proteinlanthanide complexes are available commercially and most proteins can be
readily labelled to take advantage of this versatile technology which is easily
adaptable to natural products drug screening [172].
Time resolved fluorescence technology can also be used in delayed fluorescence energy transfer DEFRET techniques which enable homogeneous assays
to be carried out, as fluorescence is generated by the close proximity of the
lanthanide to an acceptor molecule. The acceptor molecule used in this technique may either transmit or quench fluorescence, depending on the choice of
assay [173].
A range of techniques including solution phase assays [174, 175], heterogeneous assays [176, 177] and SPA have also been used in enzyme inhibitor
screening programmes as shown in Fig. 11.
Emerging assay technologies other than cell-based SPA and DEFRET
include electrochemiluminescence (ECL), surface plasmon resonance (SPR), and
rnicrophysiometry (see Fig. 12).
Electrochemi/uminescence utilizes labelling of one of the assay reagents. In
this case, diffusion of a precursor molecule onto an electrode surface is followed
by rapid electron transfer, which initiates the excitation of the reporter molecule
and results in the emission of a photon of light at a specific wavelength. Use of
an electrode allows reactions to take place in a variety of solvents, and gives the
capability of increasing assay sensitivity to the fmoll-1 range by applying
different electrode potentials [178].
Surface plasmon resonance is a label free, real time optical detection technique which facilitates quantification of molecular interactions. The basis of
SPR is that a metal (usually gold) coated sensor chip is used to give rise to
electronic oscillations, or surface plasmons, at a metal surface. These decay
exponentially as a function of distance and their refractive index will change if
a complex is formed on the sensor chip surface. This change can be detected and
recorded using appropriate instrumentation [179].