This article describes novel data analysis of fluorescence lifetime-based protein kinase assays to identify and correct for compound interference in several practical cases. This ability, together with inherent advantages of fluorescence lifetime technology (FLT) as a homogeneous, antibody-free format indepen- dent of sample concentration, volume, excitation intensity, and geometry, makes fluorescence lifetime a practical alternative to the established ‘‘gold standards’’ of radiometric and mobility shift (Caliper) assays. The analysis is based on a photochemical model that sets constraints on the values of fluorescence lifetimes in the time responses of the assay. The addition of an exponential component with free floating lifetime to the constrained model, in which the lifetimes are constants predetermined from control mea- surements and the preexponential coefficients are ‘‘floating’’ parameters, allows the relative concentra- tion of phosphorylated and nonphosphorylated substrates to be calculated even in the presence of compound fluorescence. The method is exemplified using both simulated data and experimental results measured from mixtures of dye-labeled phosphorylated and nonphosphorylated kinase substrates. A change of the fluorescence lifetime is achieved by the phosphorylated substrate-specific interaction with a bifunctional ligand, where one binding site interacts with the phosphate group and the other interacts with the dye.

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Use of fluorescence lifetime technology to provide efficient protection from
false hits in screening applications
Dmitry M. Gakamsky ⇑
, Richard B. Dennis, S. Desmond Smith
Edinburgh Instruments, Kirkton Campus, Livingston, West Lothian EH54 7DQ, UK
a r t i c l e i n f o
Article history:
Received 5 August 2010
Received in revised form 12 October 2010
Accepted 13 October 2010
Available online 21 October 2010
Keywords:
Fluorescence lifetime technology
Fluorescence lifetime data analysis
Screening
False hits
Kinase/phosphatase assay
a b s t r a c t
This article describes novel data analysis of fluorescence lifetime-based protein kinase assays to identify
and correct for compound interference in several practical cases. This ability, together with inherent
advantages of fluorescence lifetime technology (FLT) as a homogeneous, antibody-free format indepen-
dent of sample concentration, volume, excitation intensity, and geometry, makes fluorescence lifetime
a practical alternative to the established ‘‘gold standards’’ of radiometric and mobility shift (Caliper)
assays. The analysis is based on a photochemical model that sets constraints on the values of fluorescence
lifetimes in the time responses of the assay. The addition of an exponential component with free floating
lifetime to the constrained model, in which the lifetimes are constants predetermined from control mea-
surements and the preexponential coefficients are ‘‘floating’’ parameters, allows the relative concentra-
tion of phosphorylated and nonphosphorylated substrates to be calculated even in the presence of
compound fluorescence. The method is exemplified using both simulated data and experimental results
measured from mixtures of dye-labeled phosphorylated and nonphosphorylated kinase substrates. A
change of the fluorescence lifetime is achieved by the phosphorylated substrate-specific interaction with
a bifunctional ligand, where one binding site interacts with the phosphate group and the other interacts
with the dye.
Ó 2010 Elsevier Inc. All rights reserved.
Many biotechnological applications, including drug discovery,
require screening of compounds of large chemical libraries on
interaction with target proteins. Among the most important tar-
gets are protein kinases (PKs)1
involved in many cell signaling path-
ways [1,2]. Traditional methods for PK assay were based mainly on
radiometric technology, which provided high accuracy and was con-
sidered as a ‘‘gold standard.’’ However, such applications are limited
by health and safety concerns, the need to dispose of the radioactive
waste, the short shelf life of radioisotopes, and the inhomogeneous
format requiring a fraction separation. Fluorescence techniques have
been suggested as an alternative to radiometry to avoid the above
shortcomings and to miniaturize and accelerate screening of vast li-
braries of chemical compounds [3,4]. Fluorescence intensity and
polarization methods based on steady-state measurements suffer
from a relatively large number of ‘‘false hits’’ that are caused, in part,
by a significant number of fluorescent compounds in the libraries
[5]. In addition, some compounds can have a significant optical den-
sity at either or both of the excitation and emission wavelengths or
can be fluorescence quenchers.
Background fluorescence or absorption of screening compounds
can be partially accounted for by spectroscopic profiling of com-
pounds’ libraries. However, it is difficult to account for quenching
properties of screening compounds because this would require
their interactions with different fluorescence probes used in fluo-
rescence intensity applications to be investigated.
Within the limits set by assay conditions, interference of fluo-
rescent compounds can be reduced by increasing the concentration
of fluorescence probes. A significant reduction in the number of
false hits is expected by using fluorescence probes with emission
in the red spectral range where the number of fluorescent com-
pounds is significantly smaller [6]. However, fluorescence probes
with red-shifted emission usually have shorter fluorescence life-
times than those emitting in the blue–green spectral range, leading
to a reduction in sensitivity of fluorescence polarization (FP) mea-
surements. In addition, steady-state polarization (anisotropy) is a
function of both the rotation correlation time and fluorescence life-
time. Therefore, FP measurements can be affected by a change in
fluorescence lifetime caused by dynamic quenching of screening
compounds.
In certain cases, the presence of interference fluorescence in
intensity and polarization assays can be ‘‘flagged’’ when values of
the assay parameter deviate significantly from the margins set by
0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.10.017
⇑ Corresponding author. Fax: +44 0 1506 425320.
E-mail address: dgakamsky@edinst.com (D.M. Gakamsky).
1
Abbreviations used: PK, protein kinase; FP, fluorescence polarization; TR-FRET,
time-resolved fluorescence resonance energy transfer; FLT, fluorescence lifetime
technology; 9AA, 9-aminoacridine; DECCA, 7-diethylaminocoumarin-3-carboxylic
acid; PMA, phenylmalonic acid; DTT, dithiothreitol; FAST, Fluorescence Analysis
Software Technology; cpm, counts per maximum.
Analytical Biochemistry 409 (2011) 89–97
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

3. Author's personal copy
control samples. However, this method cannot reveal false hits
when interfering fluorescence changes the assay parameter within
assay limits. Moreover, flagging suspicious assay samples does not
allow assay parameters to be determined.
Time-resolved fluorescence resonance energy transfer (TR-
FRET) assays are based on ratiometric intensity measurements of
the sensitised emission of a fluorescence acceptor (e.g., XL665)
and luminescence of a long lifetime donor emitting in the micro-
or millisecond range (e.g., europium cryptate) [7]. Although the
long lifetime emission of the donor allows discrimination of a
nanosecond background, screening compounds may still affect re-
sults of these assays by changing the parameters of the donor or
acceptor emission or the conditions of operation of the resonance
energy transfer. In addition, a micro- or millisecond background
can also affect results of TR-FRET measurements. Serious short-
comings of this technology include the availability of specific
monoclonal antibodies and their cost. The latter becomes a severe
problem for screening of large libraries.
All of these drawbacks significantly hinder steady-state fluores-
cence application in drug research. In contrast, fluorescence life-
time measurements, being significantly less dependent on
different experimental parameters and background fluorescence,
have been suggested for screening applications [8–10]. In addition,
fluorescence lifetime assays can be designed in antibody-free for-
mats. Significant progress achieved in the measurement of fluores-
cence lifetime has prompted the emergence of this method from
state-of-the-art to routine experiments. Moreover, fluorescence
lifetime plate readers (e.g., the NanoTaurus from Edinburgh Instru-
ments, Livingston, UK) operating in 96- and 384-well formats and
providing advanced analysis of fluorescence time responses have
now became available. This success has stimulated development
of lifetime-based applications [10,11] employing new classes of
high luminosity and long lifetime fluorescence probes such as acri-
dones [12] and acridines [13].
The potential of fluorescence lifetime technology (FLT) for
screening applications has not yet been fully exploited. In this
work, we present both simulated and experimental data to show
that the use of FLT allows quantitative determination of the rela-
tive concentration of phosphorylated and nonphosphorylated sub-
strates and can, in many cases, provide effective protection from
interference of screening compounds.
Materials and methods
Unless otherwise stated, all general chemicals used were pur-
chased from Sigma–Aldrich (Dorset, UK). A PKBa peptide substrate
(GRPRTSSFAEG) labeled at the N terminal by either 9-aminoacri-
dine (9AA) with a natural lifetime of approximately 17 ns or Pure-
time 14 (AssayMetrics, Cardiff, UK) with a lifetime of 14 ns was
synthesized by Almac Sciences (Scotland). The dyes TG404-NHS
(Seta Biomedicals, Urbana, IL, USA) and 7-diethylaminocoumarin-
3-carboxylic acid (DECCA), (Invitrogen, Carlsbad, CA, USA) were
used to test compound interference.
Fluorescence lifetime assay
The fluorescence lifetime of the dye-labeled peptide substrate
was changed by reacting it with a ligand that specifically interacts
with the phosphate group. The ligand, composed of an equimolar
phenylmalonic acid (PMA):iron(III) complex, was prepared by
diluting PMA in a 50% (v/v) acetic acid solution and FeCl3 in water
to a 0.32-M concentration and mixing their equal volumes to get a
0.16-M stock solution. The stock was diluted in cold water to a 5-
mM concentration and kept refrigerated at 4 °C before measure-
ments. Peptide substrate solutions (2 lM) were dissolved in
50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5 mM ATP, 5 mM MgCl2,
and 1 mM dithiothreitol (DTT). Next, 100-ll volumes of the sub-
strate mixtures (2 lM total concentration) with different propor-
tions of the phosphorylated and nonphosphorylated fractions
ranging from 0% to 100% with 20% steps and the ligand were mixed
before measurements to get 1-lM peptide and 2.5-mM ligand final
concentrations.
Fluorescence lifetime experiments were carried out in a Nano-
Taurus plate reader based on the technique of time-correlated sin-
gle-photon counting (Edinburgh Instruments). The excitation
source was a picosecond semiconductor diode laser (Edinburgh
Instruments) emitting at 405 nm with a repetition rate of 5 MHz.
The fluorescence was collected through a 470-nm emission filter
with a 25-nm half-width. The fluorescence time responses were
measured in the ‘‘reverse’’ photon counting mode in the 200-ns
time range binned into 512 channels. The measurement time
was typically 1–5 s to acquire high-quality data with 1 to 5 Â 104
counts per maximum.
Data simulation and analysis
In our simulations, we suggest that fluorescence time response,
R(t), of a mixture of phosphorylated and nonphosphorylated sub-
strates is given by an exponential model function with two compo-
nents when the measurement is not influenced by the excitation
pulse width or the limited response time of the detection system:
RðtÞ ¼
Xn
i¼1
BieÀt=si ; ð1Þ
where the number of components n is equal to 2, Bi are preexponen-
tial coefficients proportional to concentrations of the phosphory-
lated and nonphosphorylated substrates, respectively, and si are
their lifetimes. A ‘‘real’’ experimental time response, I(t), is given
by a convolution of the ‘‘ideal’’ time response function, R(t), and
an instrument response function, IRF(t), determined largely by the
limited temporal resolution of the detection system and the nonin-
stantaneous excitation pulse:
IðtÞ ¼
Z 1
0
IRFðtÞRðt À sÞds: ð2Þ
IRF(t) can be determined by measuring Rayleigh or Raman scatter-
ing in the instrument [14]. Poissonian noise was then added to the
simulated fluorescence responses.
The average fluorescence lifetime hsi of an n-component time
response is given as follows [14]:
hsi ¼
Pn
i¼1Bis2
i
Pn
i¼1Bisi
: ð3Þ
hsi can also be used as a readout parameter in a fluorescence life-
time plate reader.
Simulated data were generated in Matlab. All simulated and
experimental data were evaluated using discrete component anal-
ysis of Edinburgh Instruments’ Fluorescence Analysis Software
Technology (FAST) with up to four lifetimes model. The FAST expo-
nential model fitting routine is based on a modified Levenberg–
Marquardt algorithm for solving a nonlinear least squares problem
by searching for the global minimum of the v2
function. The itera-
tion process was stabilized by using a singular value decomposi-
tion of the Jacobian matrix [15].
Results
Fluorescence lifetime-based assays use the sensitivity of life-
time to different factors such as the polarity and viscosity of micro-
environment of the probe, pH, FRET, and collision quenching. For
90 Fluorescence lifetime technology / D.M. Gakamsky et al. / Anal. Biochem. 409 (2011) 89–97

4. Author's personal copy
example, the lifetime of a fluorescence probe conjugated to a ki-
nase peptide substrate can be affected by phosphorylation.
To illustrate the potential of FLT, we assume that a dye-labeled
substrate emits with lifetimes of 5 and 15 ns in the phosphorylated
and nonphosphorylated states, respectively. (Results of analysis of
a more complex case when a fluorescence time response of a phos-
phorylated peptide is given by a biexponential lifetime decay pat-
tern are shown in Figs. 1–5 in the supplementary material.)
Fig. 1A shows simulated fluorescence time responses with 104
photon counts per maximum (cpm) for mixtures of dye-labeled
substrates with different fractions of the phosphorylated compo-
nent from 0% to 100% with 20% steps. In this example, all time re-
sponses have the same lifetimes independent of the proportion of
the phosphorylated and nonphosphorylated substrates. Their val-
ues can be determined most accurately from control samples with
0% and 100% phosphorylation because their time responses can be
evaluated by a model comprising only a single exponential compo-
nent and, hence, having the minimal number of parameters. We
calculate values of these lifetimes using the discrete component
model of the FAST software. These values are then used as con-
stants in the evaluation of time responses with unknown fractional
concentrations of the phosphorylated and nonphosphorylated sub-
strates. Fig. 1A shows results of evaluation of the simulated time
responses (dots) by a two-exponential model (Eq. (1)) with 5-
and 15-ns lifetime constants (solid lines). Fig. 1B shows residual
functions for each time response:
Fi ¼
Ni À Ii
ffiffiffiffiffi
Ni
p ; ð4Þ
where Ni are simulated data and Ii are values of a fitted function at
time ti. These functions show random scattering around the zero le-
vel, indicating satisfactory fits. The inset in Fig. 1A shows graphi-
cally the calculated s1 and Bi parameters. The normalized B1 and
B2 coefficients as functions of the relative concentration of the phos-
phorylated substrate are plotted in Fig. 1C by circles, and the ‘‘true’’
values of the parameters used in the simulations are shown by solid
lines. In this case, the values of the normalized preexponential coef-
ficients are calculated with an accuracy of better than 1% over the
whole concentration range, as shown by deviations of the calcu-
lated Bi values from the straight lines.
Ability of FLT to effectively protect from interferences of screening
compounds
Screening compounds can cause several types of interference.
First, a significant optical density of a compound at the excitation
or emission wavelength of the assay would attenuate the emission
intensity of a probe. Second, a static quenching caused by the inter-
action of a screening compound with a fluorescence probe in the
ground state to form a nonfluorescent complex would change the
efficient concentration of the fluorescent molecules. These two
mechanisms may result in false hits in assays based on steady-
state fluorescence measurements but do not affect results of life-
time measurements because they are independent of fluorescence
intensity and concentration. The third type of interference comes
from compounds that affect the lifetime of the probe in both phos-
phorylated and nonphosphorylated substrates in a nonspecific
manner (e.g., by collision quenching). The fourth type is caused
by background fluorescence of screening compounds. Whereas
the last two mechanisms affect both steady-state and time-re-
solved measurements, we show that the latter are less susceptive
because they can be identified and often discriminated by analysis
of fluorescence time responses.
To illustrate this point, we again analyze the above assay model
where the fluorescence time responses can have only two life-
times. Therefore, the presence of an additional interfering lifetime
component can be identified by the value of the v2
parameter,
when it deviates from the range of 1.0 to 1.4 where the fit is con-
sidered as satisfactory, or by the observation of a nonrandomly
fluctuating residuals function.
Consider a situation where a compound affects the lifetimes of
both phosphorylated and nonphosphorylated dye-labeled peptides
Fig.1. Simulation of a fluorescence lifetime phosphorylation assay of mixtures of dye-labeled phosphorylated (s1 = 5 ns) and nonphosphorylated (s2 = 15 ns) peptide
substrates with different proportions of the phosphorylated fraction ranging from 0% to 100% with 20% steps. Fluorescence time responses were simulated by convolution of
the assay model functions (Eq. (1)) with experimental IRF and following the addition of Poissonian noise. (A) Evaluation of the simulated time responses (dots) by FAST
discrete component analysis using the constrained two-exponential model with fixed 5- and 15-ns lifetimes (solid lines). Inset: Graphic representation of the si–Bi
parameters. (B) Residual functions for all evaluated time responses shown in panel A. (C and D) Assay standard curves plotted as normalized preexponential coefficients B1
and B2 (C) and average lifetimes hsi (D) versus phosphorylated fraction concentration.
Fluorescence lifetime technology / D.M. Gakamsky et al. / Anal. Biochem. 409 (2011) 89–97 91

5. Author's personal copy
by changing their values by 10% to give s1 = 4.5 nm and
s2 = 13.5 ns. The constrained model with fixed 5- and 15-ns life-
times now yields an unsatisfactory fit with v2
> 6 (Fig. 2A) and
clear nonrandom shapes of the residual functions (Fig. 2B). These
are obviously due to a significant restriction of the pool of possible
two-exponential functions available for the fitting. Assuming that
the reason for the unsatisfactory fit is unknown, we can first sug-
gest a presence of background fluorescence and add an additional
exponential component, with ‘‘floating’’ B3 and s3 parameters, to
the above model. Results of evaluation by this model are shown
in Fig. 2C. It gives a better fit with v2
< 1.2 to all time responses;
however, it does so with s3 values too close to s2, indicating a
degeneracy of the model parameters. In addition, B3 parameters
have negative values, indicating the presence of a ‘‘lighting-up’’
process associated with this component. All of these indicate that
the results formally obtained with this three-exponential model
do not have any physical sense. The failure to fit the data with
the modified model indicates fatal interference in the assay sam-
ples, and a compound with such a property cannot be assayed by
this method.
The above data can, of course, be fitted by a two-exponential
model if the s parameters are ‘‘floating’’ and the program yields
4.5- and 13.5-ns lifetimes. If we use average lifetime calculated
with the use of the nonconstrained model as the readout and plot
them as a function of relative concentration of the phosphorylated
substrate (Fig. 2E, dashed line) we see that this standard curve sig-
nificantly differs from that calculated for the unaffected measure-
ments (Fig. 2E, solid curve). An average lifetime of 13.5 ns obtained
now for the nonphosphorylated substrate in the presence of the
quencher would be interpreted as approximately 32% phosphory-
lation. In other words, ignoring the fact that the lifetimes deter-
mined for a sample are significantly different from those
expected in the assay fatally leads to a false result.
A major cause of false hits occurs when the screening com-
pound fluoresces. In Figs. 3–5, we investigated cases where com-
pound interference occurs with short (Fig. 3), intermediate
(Fig. 4), and long (Fig. 5) lifetimes.
Simulated time responses with s1 = 5 ns, s2 = 15 ns, and an
additional interfering component with s3 = 1 ns and B3 = B1 + B2
are shown in Fig. 3A (dots). As expected, the evaluation of such
data by the two-exponential model with fixed s1 and s2 parame-
ters gives poor fits, as indicated by significantly distorted patterns
of the residual functions (Fig. 3B) and v2
P 2.4. The addition of a
third component with floating parameters provides satisfactory
fits to all time courses (Fig. 3C and D) with the third lifetime values
grouped around 1 ns (Fig. 3C, inset). Importantly, the preexponen-
tial coefficients B1 and B2 exhibit the expected linear dependences,
as shown in Fig. 3F. These results show that the background fluo-
rescence can be extracted from the experimental data and the rel-
ative concentrations of the phosphorylated and
nonphosphorylated substrates can be determined. However, if we
use average lifetime calculated by a formal evaluation by a two-
exponential model with free floating lifetimes as a readout param-
eter (Fig. 3E, dashed line), the presence of the 1-ns component
again gives a false negative hit because the sample with 0% phos-
phorylation now gives a 14-ns lifetime that corresponds to approx-
imately 26% phosphorylation of the assay standard curve (Fig. 3E,
solid line).
Fig. 4 shows simulated data using the same two-exponential
model with the presence of a background fluorescence with life-
times between s1 and s2, s3 = 10 ns, and B3 = B1 + B2. As above,
the constrained lifetime analysis can detect the interference
(Fig. 4A and B), and the addition of a third exponential component
with floating parameters allows the interference component to be
separated (Fig. 4C and D). The method yields relative concentra-
tions of the substrates that are shown in Fig. 4F. In contrast, the
average lifetime readout gives 42–86% phosphorylation for any
proportion of phosphorylated substrate (i.e., a false hit) (Fig. 4E).
This method can also be used when the background’s lifetime is
larger than the largest assay’s lifetime. In our simulations, we used
Fig.2. Effect of a nonspecific 10% quenching of lifetime by a screening compound on the results of the assay shown in Fig. 1. The time responses were simulated as described
in Fig. 1 using 4.5- and 13.5-ns lifetimes for the phosphorylated and nonphosphorylated substrates, respectively. (A and C) Evaluation of the simulated time responses (dots)
by FAST using the constrained two-exponential model, with fixed s1 = 5 ns and s2 = 15 ns (solid lines) (A), and a three-exponential model, with fixed s1 = 5 ns, s2 = 15 ns, and
free floating s3 (solid lines) (C). Insets: Graphic representation of the si–Bi parameters. (B and D) Residual functions of all time responses shown in panels A and C, respectively.
(E) Assay standard curves plotted as average fluorescence lifetime versus phosphorylated fraction concentration for the standard conditions as in Fig. 1 (solid line) and for the
quenched conditions (dashed line).
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6. Author's personal copy
s3 = 20 ns and B3 = B1 + B2 (Fig. 5A, dots). The constrained two-
exponential model does not allow satisfactory fits to be achieved
(Fig. 5A, solid lines, and Fig. 5B), but the addition of a third expo-
nential component with floating parameters allows satisfactory fit-
ting of the data (Fig. 5C and D). The inset in Fig. 5C shows that
values of the third lifetime calculated by the FAST software are
Fig.3. Effect of a short lifetime (s3 < s1) fluorescence background of a screening compound on the results of the assay shown in Fig. 1. The time responses were simulated as
described in Fig. 1, using 5 and 15 ns for the phosphorylated and nonphosphorylated dye-labeled substrates, respectively, and 1 ns for the background component. (A and C)
Evaluation of the simulated time responses (dots) by FAST using the constrained two-exponential model, with fixed s1 = 5 ns and s2 = 15 ns (solid lines) (A), and a three-
exponential model, with fixed s1 = 5 ns, s2 = 15 ns, and free floating s3 (solid lines) (C). Insets: Graphic representation of the si–Bi parameters. (B and D) Residual functions of
all time responses shown in panels A and C, respectively. (E) Standard curves plotted as average fluorescence lifetime versus phosphorylated fraction concentration for the
assay without (solid line) and with (dashed line) fluorescence background. The latter was calculated using results of data evaluation by a two-exponential model with floating
parameters. (F) Assay standard curves plotted as normalized B1 and B2 coefficients versus phosphorylated fraction concentration.
Fig.4. Effect of an intermediate lifetime (s1 < s2 < s3) fluorescence background of a screening compound on the results of the assay shown in Fig. 1. The time responses were
simulated as described in Fig. 1 using 5 and 15 ns for the phosphorylated and nonphosphorylated dye-labeled substrates, respectively, and 10 ns for the background
component. (A and C) Evaluation of the simulated time responses (dots) by FAST using the constrained two-exponential model, with fixed s1 = 5 ns and s2 = 15 ns (solid lines)
(A), and a three-exponential model, with fixed s1 = 5 ns, s2 = 15 ns, and free floating s3 (solid lines) (C). Insets: Graphic representation of the si–Bi parameters. (B and D)
Residual functions of all time responses shown in panels A and C, respectively. (E) Standard curves plotted as average fluorescence lifetime versus phosphorylated fraction
concentration for the assay without (solid line) and with (dashed line) fluorescence background. The latter was calculated using results of data evaluation by a two-
exponential model with floating parameters. (F) Assay standard curves plotted as normalized B1 and B2 coefficients versus phosphorylated fraction concentration.
Fluorescence lifetime technology / D.M. Gakamsky et al. / Anal. Biochem. 409 (2011) 89–97 93

7. Author's personal copy
consistently grouped around 20 ns. Normalized B1 and B2 coeffi-
cients again allow determination of relative concentrations of the
substrates (Fig. 5F). The values of average lifetimes in this case
are always higher than the values of the standard curve, suggesting
interference. Thus, both the average lifetime and the restricted
model analysis indicate the presence of interference, but only the
latter allows determination of the substrate relative
concentrations.
PMA/iron(III) assay
To experimentally prove the method described earlier, we used
a lifetime-based assay for PKs developed previously by the Univer-
sity of Dundee and Edinburgh Instruments [11]. A PKBa substrate
(GRPRTSSFAEG) [16] and its product labeled by 9AA at the N termi-
nal was used. Similar results obtained with PT 14 are shown in the
Supplementary material. To affect fluorescence lifetime of the
probe, we used an equimolar PMA/iron(III) complex. This molecule
is a bifunctional ligand that can specifically bind to the negatively
charged phosphate group of the substrate by the coordinated pos-
itively charged metal ion and interact with the dye by the phenyl
group, leading to accelerated deactivation of the excited state. This
ligand also quenches emission of the substrate by a nonspecific
collision mechanism at high concentration. The largest difference
between the lifetimes of the probe on the nonphosphorylated
(0%) and phosphorylated (100%) substrates was found at a 2.5-
mM concentration of the ligand. At this ligand concentration, the
emission spectrum of the 9AA- and PT 14-labeled phosphorylated
substrates remained homogeneous, as was confirmed by time-re-
solved emission spectra (not shown). The lifetime of the 9AA-
and PT 14-labeled nonphosphorylated substrates at this ligand
concentration (16.8 and 13.8 ns) also remained close to the values
observed without the ligand (17.0 and 14.1 ns), suggesting that
most of the lifetime change in the phosphorylated substrate is
caused by the specific substrate–ligand interaction.
The lifetime pattern of the time response of the 9AA probe con-
jugated to the phosphorylated substrate was given mostly by two
components, s1 = 1.9 ± 0.2 ns (B1 = 0.34) and s2 = 5.8 ± 0.4 ns
(B2 = 0.64), with a residual emission of a third component,
s3 = 17 ± 3 ns (B3 = 0.02), which is attributed to emission of the un-
quenched probe (Fig. 6A). A time response of the nonphosphory-
lated substrate at the above conditions was nearly single-
exponential, with s3 = 16.8 ± 0.2 ns (B3 = 0.93) in the presence of
two residual short lifetime components, s1 and s2 (when the data
were evaluated by a three-exponential model), with
B1 + B2 = 0.07. This short lifetime decay was apparently caused by
a nonspecific quenching of 9AA with the bifunctional ligand or
by a probe–substrate interaction. Because assay samples differ
only by the proportions of the phosphorylated and nonphosphory-
lated substrates, all experimental time responses must be com-
posed of exponential components with 1.9-, 5.8-, and 16.8-ns
lifetimes. This concept sets a basis for using a three-exponential
model for data evaluation and attribution of the normalized
(B1 + B2) and B3 preexponential coefficients to relative concentra-
tions of the phosphorylated and nonphosphorylated substrates
(Fig. 6F, circles). These coefficients can be used to determine the
relative concentrations of the phosphorylated and nonphosphory-
lated fractions.
A next example illustrates application of this method for analy-
sis of assay samples when measurements were interfered by an
8.5-ns emission of TG404 dye (Fig. 6C, dots). The presence of the
interference component makes it impossible to obtain satisfactory
fits with the use of the three-exponential model when the lifetime
parameters are fixed (results not shown). However, the addition of
a fourth exponential component with floating parameters allows
the data to be fitted with v2
< 1.3 for all time courses and to ac-
count for the interference (Fig. 6C, solid lines, and Fig. 6D). The nor-
Fig.5. Effect of a long lifetime (s3 > s2) fluorescence background of a screening compound on the results of the assay shown in Fig. 1. The time responses were simulated as
described in Fig. 1 using 5 and 15 ns for the phosphorylated and nonphosphorylated dye-labeled substrates, respectively, and 20 ns for the background component. (A and C)
Evaluation of the simulated time responses (dots) by FAST using the constrained two-exponential model, with fixed s1 = 5 ns and s2 = 15 ns (solid lines) (A), and a three-
exponential model, with fixed s1 = 5 ns, s2 = 15 ns, and free floating s3 (solid lines) (C). Insets: Graphic representation of the si–Bi parameters. (B and D) Residual functions of
all time responses shown in panels A and C, respectively. (E) Assay standard curves plotted as average fluorescence lifetime versus phosphorylated fraction concentration
without (solid line) and with (dashed line) fluorescence background. The latter was calculated using results of data evaluation by a two-exponential model with floating
parameters. (F) Assay standard curves plotted as normalized B1 and B2 coefficients versus phosphorylated fraction concentration.
94 Fluorescence lifetime technology / D.M. Gakamsky et al. / Anal. Biochem. 409 (2011) 89–97

8. Author's personal copy
malized B1 + B2 and B3 coefficients again can be used for determi-
nation of relative concentrations of the phosphorylated and non-
phosphorylated fractions (Fig. 6F, asterisks). Thus, the
preexponential coefficients allow reliable determination of the
substrate concentrations even when the assay model is given by
a three-exponential function and an interference component
Fig.6. Fluorescence lifetime-based phosphorylation assays carried out in a NanoTaurus plate reader. (A) Fluorescence time responses of 9AA-labeled phosphorylated and
nonphosphorylated crosstide peptide mixtures (0–100% with 20% steps of the phosphorylated component) reacted with 2.5 mM PMA/iron(III) ligand (dots) and their fits
(solid lines) by a constrained three-exponential model with fixed s1 = 1.9 ns, s2 = 5.8 ns, and s3 = 16.8 ns. Insets: Graphic representation of the si–Bi parameters. (B and D)
Residual functions of all time responses shown in panels A and C, respectively. (C) Fluorescence time responses of the above peptide mixtures with the addition of an
‘‘interfering compound,’’ TG404 (2 lM), emitting with an 8.5-ns lifetime (dots) and their fit by a four-exponential model with fixed s1 = 1.9 ns, s2 = 5.8 ns, and s3 = 16.8 ns and
free floating s4 (solid lines). (E) Assay standard curves plotted as average fluorescence lifetime versus phosphorylated fraction concentration without (solid line) and with
(dashed line) fluorescence background. (F) Assay standard curves plotted as normalized B1 + B2 and B3 coefficients versus phosphorylated fraction concentration for assay
samples without (circles) and with (asterisks) fluorescence background.
Fig.7. Fluorescence lifetime-based phosphorylation assays with the presence of a short lifetime interfering compound. (A) Fluorescence time responses of the 9AA-labeled
phosphorylated and nonphosphorylated crosstide peptide mixtures (0–100% with 20% steps of the phosphorylated component) with the addition of 2.2 lM DECCA were
reacted with 2.5 mM PMA/iron(III) ligand (dots). The time responses were fitted by a four-exponential model (fixed s1 = 1.9 ns, s2 = 5.8 ns, and s3 = 16.8 ns and free floating s4
[solid lines]). Inset: Graphic representation of the si–Bi parameters. (B) Residual functions of all time responses shown in panel A. (C) Assay standard curves plotted as
normalized B1 + B2 and B3 coefficients versus phosphorylated fraction concentration for assay samples without (circles) and with (asterisks) fluorescence background. (D)
Assay standard curves plotted as average fluorescence lifetime versus phosphorylated fraction concentration without (solid line) and with (dashed lines) fluorescence
background.
Fluorescence lifetime technology / D.M. Gakamsky et al. / Anal. Biochem. 409 (2011) 89–97 95

9. Author's personal copy
overlaps on the probe emission. If average lifetime would be used
here as a readout, it would give the concentration of phosphory-
lated substrate in the range of 34–87% independent of its actual
concentration (i.e., false negative) (Fig. 6E).
Fig. 7 illustrates an application of the method to the assay in the
presence of 2.2 lM DECCA with an 80-ps lifetime. 9AA time re-
sponses shown in Fig. 7A cannot be properly fitted by the restricted
three-exponential model (data not shown). As before, the data
were successfully fitted by the addition of a fourth exponential
component with free parameters to the model. Results of the data
evaluation are shown by solid lines in Fig. 7A and by residual func-
tions in Fig. 7B. The lifetime of the interfering component was cal-
culated to be in the range of 0.07–0.2 ns for all experimental data.
The normalized B1 + B2 and B3 coefficients shown in Fig. 7C yield
relative concentrations of phosphorylated and nonphosphorylated
peptides (shown with asterisks) with nearly the same accuracy as
they were determined in the experiment without the interference
(shown with circles). In this case, the average lifetime shown in
Fig. 7D gives a concentration of the nonphosphorylated peptide
of approximately 49% (i.e., false negative).
Discussion
Fluorescence as a process determined by emission of photons in
the course of electron transition from the excited state to the
ground state of a fluorescence probe is characterized by several
parameters. One of them, fluorescence intensity (i.e., the number
of emitted photons over a time unit), has found many applications
in life sciences and biotechnology. However, fluorescence intensity
is an arbitrary characteristic dependent on excitation and emission
wavelengths, excitation intensity, experimental geometry, concen-
tration, quantum yield, and the like.
Another characteristic of fluorescence is the rate of depopula-
tion of the energetically lowest excited state, S1, or its reciprocal
value—the lifetime, s. Fluorescence lifetime is predominantly
determined by the molecular electronic structure, which in same
cases can be affected by parameters of the assay. Fluorescence life-
time offers a better option for circumventing the shortcomings
associated with fluorescence intensity.
We have demonstrated that an advanced analysis for fluores-
cence lifetime screening applications has the potential to indicate,
and in many cases to eliminate, false hits caused by compound
fluorescence. We showed that the readout based on average life-
times calculated by a model with free floating parameters is more
susceptible to interferences than a readout based on the results of
the restricted model. In addition, hsi is a nonlinear function of
phosphorylated fractions concentration and gives smaller relative
changes ($7%) than the normalized Bi parameters in the range of
0–20% applicable for PK assays.
The method was illustrated by analysis of results of the PK assay
[11] where the difference in fluorescence lifetime of the phosphor-
ylated and nonphosphorylated substrates is achieved by the inter-
action of the substrate with the bifunctional ligand, which
specifically changes lifetime of the probe on the phosphorylated
substrate. Fluorescence time responses in such an assay must be
formed by only two functions corresponding to emissions of the
dye-labeled phosphorylated and nonphosphorylated substrates.
The first function is given by two exponential terms with s1 and
s2 lifetime constants, and the second is given by a term with a s3
constant. At any proportion of the phosphorylated and nonphos-
phorylated substrates, experimental time responses must contain
only these lifetime components.
Predetermining the assay lifetimes and using them as constants
in data evaluation (i) allows linearization of the model function,
leading to a dramatic simplification of calculations and increasing
the accuracy, and (ii) provides examination of experimental data
on the presence of interference. In other words, the analysis repre-
sents a ‘‘procrustean bed’’ for experimental time responses that can
be fitted only by a restricted pool of functions determined by the
constrained model. In some cases, this allows an account for back-
ground emission by introducing an additional exponential compo-
nent with floating parameters and the assay parameters to be
calculated.
This analysis does not depend on a method of modulation of
fluorescence lifetime and requires only that a substrate and its
product emit with different fluorescence lifetime patterns. We re-
cently applied this method to evaluation of a protease assay where
two types of fluorescence responses corresponding to emission of
whole and cleaved substrates are used. The method exhibited a
successful correction of data affected by background emission
(not shown). Successful application of the method to analysis of re-
sults of two different lifetime assays and to the assay designed
with the use of different fluorescence probes suggests its general
significance.
The examples presented here do not exhaust all possible situa-
tions with regard to variations in fluorescence lifetimes and con-
centrations of interference compounds or their quenching
properties. For example, if a screening compound emits signifi-
cantly more strongly than the probe, the method accuracy will
be compromised and its application may become impossible. An-
other unfavorable situation could be when a background’s lifetime
is close to one of the assay’s lifetimes. In such a case, the con-
strained model will give a good fit, but the concentrations deter-
mined for the substrate will be wrong. However, the method can
still indicate interference if the normalized preexponential coeffi-
cients will significantly deviate from the values expected for the
assay.
In the limited situations where the background emission con-
tains more than one lifetime or, more generally, can only be de-
scribed as a continuous distribution of lifetimes and where a
screening compound does not emit itself but affects the lifetime
of the assay, this method will not determine concentration of the
phosphorylated and nonphosphorylated substrates but will pro-
vide a clear warning about the presence of interference.
In a high-throughput screening application, where plate mea-
surement time becomes crucial, lifetime data are often collected
only to a peak count of approximately 103
to 3 Â 103
cpm.
Although this is sufficient to identify a problem well with com-
pound interference, a stable and reliable multiexponential analysis
requires higher quality data with P104
cpm, which in turn re-
quires a longer data acquisition time. However, it should be
remembered that the likely rate of occurrence of interfering com-
pounds will be a small fraction of total wells measured (typically
1–2%) and extended measurement time on the problem wells only
will not significantly lengthen the plate readout time.
Hence, the method presented here can be used to design a new
generation of expert lifetime analysis software for screening appli-
cations that will be able to determine inhibition efficiency of
screening compounds exhibiting background emission and to warn
about unreliable measurements in situations where the interfer-
ence cannot be ‘‘filtered out’’ while maintaining all of the benefits
of fluorescence lifetime as a measurement modality.
Acknowledgments
Funding for this project was provided by The UK Technology
Strategy Board (TSB) under a Fluorescence Lifetime-based Assays
and Sensors for Healthcare (FLASH) project (TP M1537F) and a
Royal Society Industry Fellowship (to D.M.G.).
96 Fluorescence lifetime technology / D.M. Gakamsky et al. / Anal. Biochem. 409 (2011) 89–97

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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ab.2010.10.017.
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