Development of a Fluorimetric Assay for Fluorescence Quantification of Streptavidin Concentrations
in Solution and on Surfaces
Barrett J. Nehilla1,2 and Joyce Wong2
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine,
2
Department of Biomedical Engineering, Boston University, Boston, MA 02218
1
Abstract
A recently published fluorimetric assay for fluorescence quantification of streptavidin in solution was
studied to ascertain its feasibility for applications in Dr. Wong’s laboratory. The Wong lab is interested in
the binding of streptavidin-coated beads to different surfaces, but there is no method to quantify the
streptavidin concentration on beads. The motivation of this study was to determine if the published
fluorimetric assay could be applied to quantify the concentration of SA on the surfaces of beads. The
intrinsic tryptophan fluorescence assay was performed in an effort to reproduce previously published
results. Several different concentrations of unmodified d-biotin were used in the titration of streptavidin
solutions. There was no breakpoint in the fluorescence data at any of the tested d-biotin concentrations.
Quantitative measurements are not possible without a breakpoint. Biotin-4-fluorescein was used in all
subsequent titrations in hopes that the saturation of binding sites would be noticeable with a bright
fluorescent compound. Several experiments were conducted to work out the details of the biotin-4fluorescein protocol. It was determined that .10 mg/mL BSA in buffer A should be used to prevent nonspecific streptavidin binding to cuvette walls. Also, a 1-minute delay between the biotin-4-fluorescein
addition and the fluorescence measurement was used to allow the reaction to reach equilibrium.
Unfortunately, this assay is not yet accurate enough for fluorescence quantification of 4.81µM streptavidin
in solution.
Introduction
The binding of biotin to streptavidin is a wellstudied system in bioengineering. Streptavidin
(SA) is a tetramer with 4 biotin-binding sites.
The dissociation constant of the biotin/SA
complex is very low, and the binding kinetics are
fast. Biotin’s affinity and nearly irreversible
binding with SA is the basis for fluorimetric
assays.
The intrinsic trp fluorescence assay was the first
method used to quantify SA concentrations in
solution. Trp is an aromatic amino acid that
absorbs UV light. The trp residues in SA are
located near but not within the d-biotin binding
sites (4). When d-biotin binds to SA, the SA
undergoes a conformational change that blocks
the trp residues near the d-biotin binding site.
Therefore, the intrinsic trp fluorescence of SA
decreases as more d-biotin is added to solution
until all of the binding sites on SA are filled.
Once all of the sites are filled, the SA
fluorescence should remain constant even with
further additions of d-biotin. There should be a
well-defined breakpoint in the fluorescence
graph, and this point is the amount of d-biotin
added for complete biotin-binding site saturation.
The concentration of the stock SA solution can
be calculated from the breakpoint.
Unfortunately, a breakpoint in the fluorescence
was not observed after several trp fluorescence
assay trials in the lab.
There are several limitations to the intrinsic trp
fluorescence assay. The trp fluorescence in
solution is weak and nearly impossible to
monitor in the presence of other proteins (1).
Obviously, other proteins would contribute
fluorescence to the solution. Additionally, the
trp fluorescence assay can not detect less than
20nM SA in solution (1).
Biotin-4-fluorescein is one of the many
commercially available biotin-fluorophore
conjugates. The fluorescence of biotin-4fluorescein is much stronger than trp, and its
binding kinetics are nearly identical to that of
unmodified d-biotin. Biotin-4-fluorescein’s
fluorescence is completely quenched when it
binds to SA. In a SA solution titration with
biotin-4-fluorescein, the fluorescence increases
slowly as the biotin-fluorophore binds to free
binding sites on SA. After all of the biotinbinding sites are filled, the fluorescence should
increase quickly because additional biotin-4fluorescein remains unbound in solution. In fact,
it has been shown that the biotin-4-fluorescein
fluorimetric assay can detect .2nM SA in
solution so it is much more sensitive than the trp
fluorescence assay (3). Biotin-4-fluorescein is
an ideal candidate for fluorimetric assays for
quantitative measurement of SA.
Fluorimetric assays are preferable to other
quantification methods because they are fast,
easy and inexpensive (3). They also eliminate
the use of radio-labeled ligands, an especially
important consideration in this lab. A protocol
was recently optimized over the course of several
papers (1, 2, 3) for a reliable, quantitative,
fluorimetric assay using biotin-4-fluorescein.
The applications of fluorimetric assays in the lab
are far-reaching. In the near future, this biotin-4fluorescein assay may be used to quantitatively
measure the concentration of SA on coated
beads. With quantitative values, the effect of SA
concentration on the bead binding specificity to
biotin-labeled surfaces can be tested in a flowchamber experiment.
Materials and Methods
Solutions
SA, d-biotin and bovine serum albumin (BSA)
were obtained from Sigma. Buffer A (1mM
EDTA, 50mM NaH2PO4, 100mM NaCl) was
adjusted to pH 7.5 with NaOH pellets and stored
at 4˚C. The .5mM biotin-4-fluorescein was
prepared in DMSO, and aliquots were stored at
–70˚C. SA solutions (4.81µM) were prepared
with buffer A and stored at -70˚C until the day of
use. Similarly, stock solutions of d-biotin and
biotin-4-fluorescein were diluted in buffer A to
concentrations suitable for titration (usually
16µM). The titration solutions were kept at 4˚C
for up to one week.
Perkin-Elmer Spectrometer and DOS program
A Perkin-Elmer LS50B Luminescence
Spectrometer was used for all fluorescence
measurements. There is a protocol for this
instrument attached to the report. Although there
are simple spectra manipulation tools in the DOS
program, all data was exported to MATLABv6.1
for graphing and detailed data analysis.
Tryptophan Fluorescence
A cuvette was filled with 1920µL of cold buffer
A and 80µL of 4.81µM SA. Then, 5µL of 16µM
d-biotin was added to the cuvette at 90-second
intervals and the fluorescence was monitored
immediately after the d-biotin addition.
Excitation was at 295nm, and the slit widths
were 5nm and 7.5nm for excitation and
emission, respectively. After the fluorescence
spectra’s dependence on temperature and volume
was discovered, 120µL of 4.81µM SA was
added to 2880µL of room temperature buffer A.
Then, 7.5µL of 16µM d-biotin was added to the
cuvette at 90-second intervals.
Biotin-4-fluorescein titration
The same procedure as above was followed
except 7.5µL of 16µM biotin-4-fluorescein was
added to the cuvette at 90-second intervals. The
fluorescence was monitored immediately after
the biotin-4-fluorescein was added. Excitation
was at 490nm and both the excitation and
emission slits were 5nm.
Biotin-4-fluorescein titration with .10mg/mL
BSA in buffer A
Buffer A with BSA in solution was prepared by
adding 10mg BSA to 100mL buffer A. This
solution was heated at 60˚C for 30 minutes to
denature the BSA. A cuvette was filled with
2880µL of room temperature .10 mg/mL BSA in
buffer A and 120µL of 4.81µM SA. Then,
7.5µL of 16µM biotin-4-fluorescein was added
to the cuvette. There was a 1-minute delay
before the fluorescence was monitored to allow
the reaction to reach equilibrium. Basically, the
biotin-4-fluorescein was added in 2-minute
intervals.
Results and Discussion
Before obtaining results with the intrinsic trp
fluorescence assay, several variables were tested.
The most important variables were the volume
and temperature of the solution in the cuvette. In
all trials with cold solutions, the fluorescence
spectrum of buffer A decreased over time. This
indicates that the fluorescence spectrum of buffer
A must depend on its temperature. Furthermore,
when both very hot and very cold solutions are
added to the cuvette, water condenses on the
walls. ‘Foggy’ walls obviously have an effect on
the light path in the spectrometer. It is important
to assure that only solutions at or near room
temperature are added to the cuvette in the
spectrometer. Otherwise, the fluorescence
spectrum will change until the solution in the
cuvette reaches room temperature.
The maximum volume of cuvettes is 4mL,
however previous investigators performed trp
assays with only 2mL (1). If the fluorescence
spectra depend on the volume of solution in the
cuvette, fluorescence assays would be unreliable
(Figure 1). The buffer A spectrum has three
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120
Volume dependence
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average fluorescence of peaks
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Intrinsic trp fluorescence spectra
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fluorescence
distinct peaks when excited at 295nm (refer to
Figure 2). A quick method to relate different
spectra is to average the ‘fluorescent height’ of
the three peaks. Between 3mL and 4 mL, there
is much less variability in the fluorescent
spectrum of buffer A than between 1mL and
3mL. Therefore, it is important to maintain a
cuvette solution volume of at least 3mL so that
volume changes will not affect fluorescence
readings.
330
340
350
360
nm
370
380
390
Figure 2: Four spectra for intrinsic trp fluorescence assay.
Data comes directly from the DOS program. The thick line
is the baseline. The other lines are fluorescence spectra at 3
different d-biotin concentrations.
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1
2
3
v olume(mL)
4
Figure 1: The effect of changes in volume on the
fluorescence of a solution.
Tryptophan fluorescence assay
It was difficult to analyze the spectra measured
by the DOS program in the trp fluorescence
assay. Figure 2 shows the baseline (thick line)
and 1 spectrum for 3 consecutive additions of dbiotin. It is obvious from Figure 2 that the
fluorescence decreases as more d-biotin is added
to the solution. The only difference between
these spectra appears to be the area under the
curves between about 320nm-390nm. The DOS
program can calculate the area under a spectrum
curve. In the data analysis for this assay, the
spectrum of interest was subtracted from the
baseline. The result was a very noisy
‘difference’ spectrum. It is probable that this
noisy spectrum is more indicative of machine
variability than fluorescence trends during a
titration.
In order to estimate machine variability and
binding kinetics, 3 consecutive fluorescence
measurements were recorded after each addition
of d-biotin (Figure 3). The y-axis is fluorescence
difference because the spectrum of interest is
subtracted from the baseline spectrum. The
spectrum of interest is the fluorescence spectrum
measured for each d-biotin addition. The error
bars are indicative either of machine error in
reproducing spectra or the kinetics of biotin/SA
binding. If the reaction is slow, the fluorescence
after each d-biotin addition would decrease as
the d-biotin slowly binds to SA. Therefore, the
error bars would all be approximately the same
size. The error bars do not display this trend so
the spectrometer is probably the source of error.
Intrinsic tryptophan fluorescence
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fluorescence difference
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100
0
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300
400
pmol d-biotin
500
600
Figure 3: Intrinsic trp fluorescence, titration with 6µM dbiotin. The error bars are standard deviation. The dot in the
middle is due to overlap of error bars.
In the titration of SA with 6µM d-biotin, there
was no apparent breakpoint in the fluorescence
(Figure 3). There was also no breakpoint in the
titrations with 4µM d-biotin (data not shown).
After all the biotin binding sites on SA are filled,
Overall, the trp fluorescence assay was
inconclusive. Because trp fluorescence is so
weak, the spectrometer may not have the
sensitivity to detect the small changes in
fluorescence over the course of a titration with dbiotin.
Biotin-4-fluorescein assay
The biotin-4-fluorescein assay was initiated in
hopes of elucidating the problems of the trp
assay. Several experiments were performed to
confirm that this assay could at least predict a
breakpoint. A new solution of 4.81µM SA was
made to ensure that all lyophilized SA was
dissolved in buffer A.
Several experiments with 16µM biotin-4fluorescein were conducted (Figure 4). The
fluorescence spectra were recorded immediately
after the biotin-4-fluorescein addition (0-second
delay). All graphs have a clear breakpoint,
unlike the trp fluorescence assay. The (•) graph
differs from the others only in the age of the
biotin-4-fluorescein (<1 hour vs. 6 days).
Previous research indicates that biotin-4fluorescein slowly degrades after it is dissolved
in buffer A (1). However, because the
breakpoint of the (•) curve lies within the range
of the breakpoints of the other graphs, it appears
that the biotin-4-fluorescein has not degraded
noticeably after 6 days.
Unfortunately, there is considerable variability in
the breakpoints (Figure 4, arrows). In fact, the
breakpoints range from 1680pmol in the (x)
curve to 1920pmol in the (+) curve. The (x) and
(+) trials were conducted less that 1 hour apart
from each other, so the temperatures of the
buffer A, SA, and biotin-4-fluorescein, and the
age of the biotin-4-fluorescein, were the same.
This assay is unreliable because the breakpoint
varies considerably between trials.
Biotin-4-fluorescein assay
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1000
fluorescence
the SA trp fluorescence is completely blocked.
Thus, the addition of more d-biotin should not
affect the fluorescence of the solution. To
confirm that d-biotin has no intrinsic
fluorescence, large volumes of d-biotin were
added to a cuvette in the absence of SA and the
fluorescence was measured. It was shown that dbiotin has no intrinsic fluorescence.
800
600
400
200
0
0
500
1000
1500
2000
pmol biotin-4-fluorescein
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Figure 4: Biotin-4-fluorescein (16µM) assay, 90-second
intervals, 0-second delay. The (•) trial is the titration with
<1h biotin-4-fluorescein, the other curves are titrations with 6
day old biotin-4-fluorescein. Trendlines are displaced
vertically so they do not overlap. The arrows point to the
breakpoints.
The breakpoint variability could be due to nonspecific binding of SA or reaction kinetics. If
SA binds to the cuvette walls, some biotinbinding sites are blocked. With fewer biotinbinding sites, the breakpoint (indicative of
biotin-binding site saturation) would be lower
than if all SA was free in solution. Thus, the
degree of non-specific binding between trials
would obviously affect the breakpoints. The
kinetics of the biotin/SA reaction must also be
considered. With a 0-second delay (Figure 4),
the reaction might not have reached equilibrium
before the fluorescence was measured. If the
fluorescence is measured before the reaction
reaches equilibrium, too much unbound biotin-4fluorescein would be free in solution. The result
is an artificially high fluorescence measurement.
It is important to give the reaction enough time
to reach equilibrium before measuring the
fluorescence.
BSA is a protein that blocks non-specific
binding, so a solution of .10mg/mL BSA in
buffer A was made. It is known that BSA does
not affect fluorescence measurements in the
biotin-4-fluorescein assay (3). A SA solution
with BSA was titrated with 16µM biotin-4fluorescein. Three fluorescence measurements
were recorded 1 minute after adding the biotin-4fluorescein (Figure 5). The standard deviation at
most of these points is negligible. Not only does
the machine reproduce the spectra with high
accuracy, but the reaction also seems to be at
equilibrium. If the reaction was not at
equilibrium, the spectra would have decreased
with each measurement and the error bars would
have been larger. Thus, a 1-minute delay should
be adequate for the reaction to reach equilibrium.
biotin-4-fluorescein assay, 1 minute interv al
1000
Biotin-4-fluorescein assay with BSA
fluorescence
1000
fluorescence
800
600
800
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400
400
200
200
0
0
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1000
1500
pmol biotin-4-fluorescein
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0
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1000
1500
pmol biotin-4-fluorescein
2000
Figure 5: Average and standard deviation of three
consecutive fluorescence measurements at each biotin-4fluorescein addition, 1-minute delay. Note that the upper and
lower limits of some of the error bars are so close together
that they are interpreted by the printer as dots.
Several experiments were performed to try to
quantify SA in solution. Buffer A with
.10mg/mL BSA was used to block non-specific
binding and room temperature solutions were
used to eliminate any temperature effects on the
fluorescence. The biotin-4-flourescein was less
than 2 days old for all trials, and a 1-minute
delay between biotin-fluorophore addition and
fluorescence measurement was used to account
for kinetics (Figure 6). The control line (*)
shows the rapid increase of fluorescence when
biotin-4-fluorescein is added to a cuvette in the
absence of SA. Among the 4 trials, the
breakpoints vary from 1320pmol to 1560pmol of
biotin-4-fluorescein.
Previous research indicated that in a SA solution
titration with biotin-4-fluorescein, a 1-minute
delay was not adequate for the biotin-4fluorescein/SA reaction to reach equilibrium.
SA solution titrations with a 10-minute delay
were performed to ensure that the reaction
reached equilibrium (1). It appears that the 10minute interval assay is unnecessary because it
did not improve the accuracy of the titration
(Figure 7). The 2 breakpoints in the 10-minute
delay assay are within the range of the
breakpoints in the 1-minute delay assay.
BSA does not appear to increase the assay
accuracy, either. However, it is important to
ensure that there is no non-specific binding of
SA in the cuvette, so the .10mg/mL BSA buffer
A should be used as a precaution.
Figure 6: Biotin-4-fluorescein assay, 1-minute delay
(interval). The (*) is the control, all other graphs are
different trials with the same procedure. The graphs are
vertically displaced so they do not overlap. Arrows point to
breakpoints.
Biotin-4-fluorescein assay, 10 minute delay
1000
800
fluorescence
0
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200
0
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1000
1500
pmol biotin-4-fluorescein
2000
Figure 7: Biotin-4-fluorescein assay, 10-minute delay.
Arrows point to breakpoints.
At this time, the most consistent assay is the
biotin-4-fluorescein titration of SA in .10mg/mL
BSA buffer A with a 1-minute delay (Figure 6).
The breakpoint of these trials is at 1480+/98.0pmol biotin-4-fluorescein. This breakpoint
corresponds to the addition of 92.5µL of 16µM
biotin-4-fluorescein. Assuming that the
concentration of the stock SA was correct, 577.2
pmol (4.81µM *120µL) SA should be present in
the cuvette. The effective concentration of the
biotin-4-fluorescein concentration is 6.24µM
(577.2pmol SA/92.5µL biotin-4-fluorescein),
60% less than the 16µM nominal concentration.
Conclusions
Fluorimetric assays have the potential to allow
easy, fast and quantitative measurements of SA
in solution and on surfaces. More
experimentation is needed before the titration of
SA with biotin-4-fluorescein can be used for
quantitative analysis of SA solutions. It is
important to ensure that all solutions are near
room temperature and that the solution volume
in the cuvette is at least 3mL before conducting
any fluorimetric assay.
Unfortunately, the spectrometer may not have
the sensitivity to measure the weak trp
fluorescence in SA. In the biotin-4-fluorescein
assay, it was determined that a 1-minute delay
between ligand addition and fluorescence
measurement was adequate for the biotin/SA
reaction to reach equilibrium. As a precaution
against non-specific binding, .10mg/mL of BSA
was added to buffer A with no effect on the
fluorescence. At this time, the standard
deviation of the breakpoint calculation across
several trials is too high for this assay to be used
in fluorescence quantification of SA. After more
experiments are performed to elucidate the
variables affecting this assay, the error in the
breakpoint prediction should decrease.
probably enough time for all biotin-binding sites
to be filled with biotin-4-fluorescein.
Acknowledgements
The authors would like to thank Kelley Burridge
for the guidance and insight throughout the lab
rotation.
References
[1] Gruber, Hermann J., Gerald Kada, Markus
Marek, and Karl Kaiser. “Accurate titration of
avidin and streptavidin with biotin-fluorophore
conjugates in complex, colored biofluids.”
Biochimica Biophysica Acta 1381 (1998) 203212.
[2] Kada, Gerald, Karl Kaiser, Heinz Falk and
Hermann J. Gruber,. “Rapid estimation by
fluorescence quenching or fluorescence
polarization.” Biochimica Biophysica Acta 1427
(1999) 44-48.
Future Work
In the trp fluorescence assay data analysis, the
areas under very noisy ‘difference’ spectra were
calculated. Rather than calculating the area
under the ‘difference’ spectrum, only the area
under the spectrum of interest should be
calculated. This modification may result in more
accurate trp fluorescence data.
The large error reported in the biotin-4fluorescein assay might be eliminated with more
experiments. More experiments should be
conducted simply to improve the accuracy of the
mean and standard deviation. The fluorescence
behavior around the breakpoint might be better
understood with a more sensitive titration.
Instead of 7.5µL additions of 16µM biotin-4fluorescein, 3.75µL increments might increase
the assay’s precision in measuring the
breakpoint.
In previous research, the binding kinetics for the
first 2 biotin-4-fluorescein molecules was
reported to be much faster than the kinetics for
the last 2 molecules (1). Thus, the first
breakpoint in the titration might be due to the
binding of only 2 biotin-4-fluorescein molecules.
Because a titration with a 10-minute delay was
performed (Figure 7), the breakpoint in these
experiments is probably due the binding of 4
biotin-4-fluorescein molecules. Ten minutes is
[3] Kada, Gerald, Heinz Falk and Hermann J.
Gruber. “Accurate measurement of avidin and
streptavidin in crude biofluids with a new,
optimized biotin-fluorescein conjugate.”
Biochimica Biophysica Acta 1427 (1999) 33-43.
[4] Kurzban, Gary P., Gerry Gitlin, Edward
Bayer, Meir Wilchel, and Paul Horowitz (1990).
“Biotin binding changes the conformation and
decreases tryptophan accessibility of
streptavidin.” J. Protein Chemistry 9:673-682.