A step toward simplified detection of serum albumin on SDS-PAGE using an environment-sensitive flavone sensor

Article abstract of DOI:10.1039/c5cc03516c

In this study, we report a series of novel flavone-based sensors that exhibit a superior fluorescence response when interacting with serum albumin in real serum samples and in acrylamide gels. The detection limit of probe 4 for serum albumin solution is 0.09 μg mL^-1, and the detectable volume for monkey serum reaches as low as 0.03 μL.

Full text of DOI:10.1039/c5cc03516c

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A step toward simplified detection of serum albumin on SDS-
PAGE using an environment-sensitive flavone sensor
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
a
a
a
Bin Liu, Yi Pang,* Rachida Bouhenni, Ernest Duah, Sailaja Paruchuri Lucas McDonald
DOI: 10.1039/x0xx00000x
www.rsc.org/
2
6, 27
In this study, we report a series of novel flavone-based sensors dye (30 min).
It is thus highly desirable to develop a simplified
that exhibit superior fluorescence response when interacting with procedure that allows direct staining without the need of the protein
serum albumin in real serum samples and in acrylamide gel. The fixing (prior to staining) and washing out the nonꢀbound staining
detection limit of probe 4 for serum albumin solution is 0.09 reagent. It should be noted that the current staining methods are
μg/mL, and the detectable volume for monkey serum reaches as displaying all proteins without discrimination.
low as 0.03 μL.
Serum albumin is the most abundant protein in blood plasma and
plays a key role in the disposition and transport of various
28ꢀ31
The Detection and discrimination of different proteins with high
sensitivity and selectivity are very important for biological studies
and clinical diagnosis.
endogenous ligands, fatty acids, and drugs.
Some biological
functions of serum albumin are associated with its natural
1
ꢀ5
3
2ꢀ34
For decades, sodium dodecyl sulfateꢀ
hydrophobic pocket, which can be used to bind flavone dyes.
polyacrylamide gel electrophoresis (SDSꢀPAGE) followed by
western blotting has formed the core technology for protein
Our recent study shows that a large fluorescence turnꢀon can be
induced when a weakly fluorescent flavone derivative in water is
moved to the hydrophobic pocket of mitochondrial cells, thereby
6
ꢀ9
analysis.
Western blotting method has been widely used to
3
5
separate, detect, and identify specific proteins in a complex mixture.
In order to achieve the selective detection of a specific protein, the
process is time consuming and often involves the use of expensive
reagents and complicated multistep processes, which include the
protein transfer, blocking, reacting with the primary and secondary
antibodies followed by fluorescent detection of the bound
allowing the washꢀfree mitochondrial imaging. Reasoning that a
similar effect could be generated when a properly tailored flavone
dye enters a hydrophobic pocket of other biomacromolecules, we
decide to examine the flavone dyes 1ꢀ6. Remarkably, the sensors
exhibited a dramatic fluorescence turnꢀon upon selectively binding
to serum albumin. Since the nonꢀbound flavone dye is nonꢀ
fluorescent in a hydrophilic environment, the dramatic fluorescence
turnꢀon enabled direct coloration of serum albumin on the
polyacrylamide gel without the need of “washing off” the free dye.
The finding thus provides an attractive approach to achieve selective
protein staining for the technologically important SDSꢀPAGE.
8
antibodies. An essential step in Western blotting is to stain the
proteins following the separation by SDSꢀPAGE, where the postꢀ
stain washing is often necessary to remove the excess dyes.
Therefore, it is highly desirable to develop new strategies that can
facilitate protein visualization on a gel without going through a
multiple steps process.
1
0ꢀ20
Among the known protein sensors,
only few are capable of
In addition, these dyes either exhibit
low selectivity in responding to different proteins, or suffer from
21ꢀ24
detecting proteins in gels.
4
a
4
b,4c
strong interference from SDS.
The requirement and demand for
improved staining can be clearly seen from SYPRO Ruby, which is
25
widely used for staining the proteins in SDSꢀPAGE. The standard
process using SYPRO Ruby requires washing out the SDS (prior to
staining), long time staining (overnight), and removing the excess
a.
Department of Chemistry, University of Akron. Akron, Ohio 44325, United State.
E-mail: yp5@uakron.edu
b.
Department of Ophthalmology, Summa Health System, Akron, Ohio 44325, United
State.
†
Electronic Supplementary Information (ESI) available: Synthesis and
Fig. 1 (a) Illustration of the fluorescence turn-on for selective
protein detection; (b) Chemical structure of flavone dyes 1-6
characterization of compounds, experimental methods and procedures, supporting
figures. See DOI:10.1039/x0xx00000x
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Flavone dyes 1-6 with different substituent (Fig. 1) were the relative solvent polarity. Interestingly, the fluorescence of 1 was
prepared, in order to examine the impact of the fine structures on nearly 100% quenched by water due to intermolecular electron or
3
7
their response properties. The full synthesis details of the compounds proton transfer. Nonꢀfluorescence in water raises the possibility of
38
1-6 are shown in the experimental section and Fig. S1ꢀS17 in the eliminating the washing process in the protein detection procedure,
electronic supplementary information (ESI†). The absorbance and since the free dye molecules gives negligible background signal in
fluorescence spectra of compounds 1-6 in dimethyl sulfoxide water.
(
DMSO) were investigated and the results were summarized in
The fluorescence of flavone 1 was found to exhibit remarkable
fluorescence enhancement upon addition of bovine serum albumin
Table S1 and Fig. S18ꢀS19 (ESI†).
The fluorescence of the compound
1
revealed positive (BSA) (Fig. 2), showing a great potential for sensor applications. In
solvatochromism (i.e. emission redꢀshifted with increasing solvent the aqueous buffer (0.5% DMSO, 10 mM of PBS, pH =7.4), the
polarity) (Fig. S20, ESI†). As shown in Fig. 2, the flavone 1 gave BSA bindingꢀinduced fluorescence turnꢀon was so large that it could
two fluorescence bands corresponding to the excited normal form be easily seen by naked eye. Improved response was observed from
(
N*) and the tautomeric form (T*) associated with excited state flavone derivatives 2, 3, and 4, as they gave almost no fluorescence
intramolecular proton transfer (ESIPT, Fig. S21, ESI†). Upon in the absence of BSA (Fig. 2, Fig. 3 & Fig. S22 in ESI†). Higher
excitation via photon irradiation, the dipole moment increased fluorescence response from 2-4 indicated that the alkyl substituent at
dramatically because of the charge transfer from DꢀA structure in 1. the 6ꢀposition of flavone derivatives could have a large impact on
An increase in the solvent polarity could relax the excited molecules the flavoneꢀwater and flavoneꢀprotein interactions.
Higher
more efficiently, thus decreasing the energy of the excited state of fluorescence response from 2-4, in comparison with 6, also indicated
36
fluorophore and resulting in a redꢀshifted emission. Fig. 2c showed that the amino group should be covered with the alkyl groups. The
that both N* and T* emission wavelengths of 1 correlated well with intramolecular charge transfer along the DꢀA conjugated backbone
plays very important role in the environmentꢀsensitivity of flavone.
All the evidences consistently pointed to that the flavone dye was
entering the hydrophobic site of BSA. In addition, the fluorescence
intensities of flavone dyes revealed good linear correlation with BSA
concentration ranging from 0 to 1.0 mg/mL (Fig. S23, ESI†).
Generally, the detection limit can be defined as the analyte
concentration at which a 10% increase in fluorescence emission
3
9
can be measured. In this case, the fluorescence enhancement
of compounds in the presence of 1.0 mg/ml of BSA was 7 ~
100 fold (Table S2, ESI†). Therefore, the detection limits of
1ꢀ6
1
compounds 1-6 were 2.6, 0.14, 0.59, 0.09, 14, and 3.4 ꢁg/ml,
respectively. For Off/On type fluorescent sensor, the Signalꢀtoꢀ
Noise (S/N) ratio above 3:1 is considered reliable for visual
evaluation. The visual recognizable detection limits of
compounds 1-6 were calculated as 78, 4.2, 17.7, 2.7, 420, and
1
02 ꢁg/mL, respectively. The reference range for serum
albumin concentration in human blood plasma is approximately
4
0
3
5ꢀ50 mg/ml, even in fish, the concentration of serum
4
1
albumins can reach 0.5ꢀ5 mg/mL. The detection limits of our
Fig. 2 Fluorescence spectra changes of flavone derivatives 1(a) and
(b) (10 μM, 10 mM of PBS buffered, containing 0.5% DMSO as a
cosolvent) upon addition of different concentration of BSA (0~1.0 Fig. 3 Fluorescence enhancements (
mg/ml). Fluorescence spectra of flavone derivatives were excited at 1-6 (10 μM, 10 mM PBS buffer containing 0.5% DMSO, pH=7.4)
00 nm. Inset: the photographs of dye solutions in the absence in the absence and the presence of 0.1 mg/ml of various
left) and the presence of BSA (right) were all excited by handheld proteins. Analytes from left to right in each group: none,
4
F/Fcontrol-1) of compounds
4
(
UV lamp (365 nm).
collagen, gelatin, fibrinogen, lysozyme, trypsin, lipase, and
2
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BSA.
F: Fluorescence intensity of dye in the presence of (0~0.025 mL). Fluorescence spectra of flavone derivatives were
analytes;
F
control: fluorescent intensity of free dye in water
excited at 400 nm.
compounds were much lower than serum albumin concentration,
indicating its capability for actual blood sample testing.
Serum albumin is known as the most abundant protein in blood
serum. The probe’s fluorescence response to serum albumin in real
The photostability of flavone dyes 1-6 in the presence of BSA blood samples would be considered as an ideal sensing tool. In this
was also examined to evaluate its application for longꢀterm imaging. study, we prepared 10 ꢁM of 4 dissolved in 1 mL of PBS buffer as a
The dyeꢀBSA complexes (10 ꢁM of compounds 1-6, 1.0 mg/ml of test solution. Upon addition of initial monkey serum to the test
BSA in 10 mM PBS buffer containing 0.5% DMSO, pH=7.4) were solution, the fluorescence of 4 was rapidly increased (Fig. 4), then
placed in cuvette cells, then were continuously irradiated under a gradually reached equilibrium with the 5 ꢁL of serum, and
2
handꢀheld UV lamp (λ=365 nm, 500ꢁW/cm ). As shown in Fig. S24 eventually became very intense blueꢀgreen emission. The
(
ESI†), although the obvious fluorescence quenching of compound 5 fluorescence intensity of 4 at 505 nm was enhanced up to 135ꢀfold in
and 6 was observed, the fluorescence intensities of compounds 1-4 the presence of 25 ꢁL of serum sample and showed linear response
could retain above 80% of their original intensities after one hour to monkey serum concentration ranging from 0 to 1 ꢁL/mL. The
continuous UV excitation.
detection limit (S/N>3) for monkey serum was calculated as 0.03
42
In real blood sample or proteins extraction from tissues, there ꢁL. The result demonstrated that the probe was capable of estimating
are many different proteins which will cause serious interference the albumin concentration in monkey serum samples.
with the detection result. Thus, the high selectivity towards the target
To demonstrate the feasibility of a washꢀfree method for protein
protein over the other competitive proteins is of great importance for imaging in polyacrylamide gels, compound 4 was directly used to
43
fluorescence probes. The fluorescence response of compounds 1ꢀ6 stain the electrophoresis gel. Various proteins were run on the 1ꢀD
to various proteins in PBS buffer was investigated and the results SDSꢀPAGE minigels with running buffer (25 mM Tris, 192 mM
were shown in Fig. 3. Under the same conditions, the fluorescence glycine, 0.1% SDS pH 8.3) under 100 volts for 1.5 hrs. The minigels
enhancement in the presence of BSA was much larger than that in were then placed in the dye solution (10 mM of compound 4, 0.3%
the presence of other proteins: collagen, gelatin, fibrinogen, SDS, and AcOH/DMSO/H O = 5:10:85). Fig. 5 showed the
2
lysozyme, trypsin and lipase (Fig. 3). For all these flavone dyes, the fluorescent images of the gel containing BSA (0.1 ꢁg, 1 ꢁg, 10 ꢁg,
fluorescence response to other proteins was lower than 20% of that and 100 ꢁg), 10 ꢁg of human serum albumin (HSA), fibrinogen,
to BSA. The high selectivity could be attributed to the specific lysozyme, and trypsin after 2 hours staining by 4. It was found BSA
binding of flavone dyes to the hydrophobic pockets in the BSA was efficiently stained and clearly observed at very low
structure.
concentration (~1 ꢁg) under a UV lamp. The serum albumin proteins
were selectively stained because HSA had similar folding structure
4
4
as BSA (seen in Fig. S25ꢀS26 ESI†).
Interestingly, the
fluorescence pattern of proteins in the gel exhibited acceptable
contrast ratio without washing process. Although the fluorescence
background could be reduced by washing, the free dyes showed
much weaker fluorescence in an aqueous medium and in gels, in
comparison with commercial SYPRO Ruby. Therefore, the
discovered proteinꢀstaining reagent had the following advantages:
(
1) low interference of SDS to fluorescence, thereby eliminating the
pretreatment; (2) very weak fluorescent in water and gels to
eliminate the need for the washing process; (3) strong interaction
with the proteins (serum albumin) to reduce the staining time; (4) a
high signalꢀtoꢀnoise (S/N) ratio for the high contrast ratio of images.
Fig. 4 (a) The fluorescence spectra changes and (b) fluorescent
intensities (λ = 505 nm) of compound 4 (10 μM, in 1 mL of PBS Fig. 5 Fluorescence images of several proteins in electrophoresis gel
buffer solution) upon addition of different volume of monkey serum stained by compound 4 before and after washing. 1. BSA at 0.1 μg;
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