Conjugated polymer nanoparticles for biochemical protein kinase assay

Article abstract of DOI:10.1039/b710807a

Sensitive and reliable monitoring of kinase activity was reported by using highly efficient fluorescence resonance energy transfer of conjugated polymer nanoparticles (CPNs) to a rhodamine labelled peptide substrate. The Royal Society of Chemistry.

Full text of DOI:10.1039/b710807a

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Conjugated polymer nanoparticles for biochemical protein kinase
assay{
Joong Ho Moon,* Paul MacLean, William McDaniel and Lawrence F. Hancock*
Received (in Cambridge, UK) 16th July 2007, Accepted 5th September 2007
First published as an Advance Article on the web 14th September 2007
DOI: 10.1039/b710807a
quenching (FQ) and FRET. We demonstrated a biochemical
kinase assay for screening inhibitors in a multiwell format.
Combined with the advantage of the large spectral difference
between the excitation and emission wavelengths, ratiometric
analysis from fluorescence quenching (FQ) of CPN and FRET to
rhodamine allows for sensitive and reliable detection of the
substrate phosphorylation.
Sensitive and reliable monitoring of kinase activity was reported
by using highly efficient fluorescence resonance energy transfer
of conjugated polymer nanoparticles (CPNs) to a rhodamine
labelled peptide substrate.
Many diseases such as cancers, diabetes, and inflammation are
closely related to uncontrolled functions of certain protein kinases
that phosphorylate substrates in the signalling pathways.1
A
CPNs are easily fabricated by a simple solvent exchange process
and their sizes can be controlled by functionality of conjugated
polymers. We previously demonstrated a facile formation of
polymer particles and sensitive detection of Cy5-labeled oligonu-
cleotide using particles formed by a simple solvent exchange
process.6 The superior sensitivity was attributed to the efficient
energy transfer from the three-dimensional particles (donor) to the
labeled oligonucleotide (acceptor). While we observed very strong
fluorescence quenching of the particles by the Cy-5, a weak FRET
peak was observed at 635 nm. Considering the poor spectral
overlap between the donor (particle) and acceptor (Cy-5), the
FRET signals observed at the Cy-5 emission were somewhat
unusual. Despite the weak FRET signal at the Cy-5 emission
wavelength (635 nm), the extremely large difference between the
excitation wavelength (415 nm) and the emission wavelength
(635 nm) is very useful for the sensitive detection of analyte with
almost no background signals. This observation lead us to
optimize the system to exhibit better FRET by substantially
increasing the spectral overlap between the particle and dye, while
maintaining the large wavelength difference.
structural change resulting from the phosphorylation activates or
alters the substrate function in its various signaling pathways.
Because of their pivotal roles on the cellular process and diseases
consequences, these pathways are prime targets for drug
discovery.2 There have been extensive efforts to screen molecular
libraries for inhibitors or activators for a specific kinase.
Biochemical assays offer quantitative monitoring of the enzyme
activity in the presence of potential inhibitors using high replicate
and throughput formats.3 Among the biochemical assays,
fluorescence-based homogeneous assays are widely used because
they can detect phosphorylation events with a high degree of
sensitivity and because they avoid radioisotopic labels.4 One
common issue associated with fluorescence based detection is
interference from the background fluorescence of small molecule
compound libraries. These signals can lead to false negatives or
positives as a result of their inherent fluorescent or quenching
properties, thereby complicating screening results. Time resolved
fluorescence (TRF) is an excellent way to minimize the interfering
signals by adapting long lifetime (millisecond) lanthanide
reagents.5 Fluorescence signals will be collected at a delayed time
to remove fluorescence background from short lifetime (nano-
second) interferents. Fluorescence resonance energy transfer
(FRET)-based assay can also reduce the background if the
difference between the excitation wavelength of the donor and the
emission wavelength of the acceptor, is extremely large (i.e., more
than 150 nm). In this case, negligible fluorescence intensity is
expected at the long wavelength when the interferents are excited
at short wavelength.
Pentiptycene-containing poly(p-phenylene ethynylene (PPE) was
synthesized as reported previously (Fig. 1).6,7 To exploit the
outstanding photophysical properties of the pentiptycene-contain-
ing polymers for the detection of phosphorylated peptides in
aqueous phase, we co-polymerized the pentiptycene with a
monomer containing iminodiacetic acids (IDAs) (see ESI{). The
IDAs are widely used for metal ion immobilized chromatography
to purify various biological substances such as proteins or
peptides.8 The IDA allows the chelation of metal ions onto
Here we exploit the efficient energy transfer of the conjugated
polymer nanoparticles (CPNs) for the sensitive detection of
rhodamine-labeled peptides commonly used as substrates for
kinase enzymes. Highly efficient energy transfer from CPNs to
rhodamine allows for the quantitative monitoring of phosphoryla-
tion of the peptide substrates by monitoring both fluorescence
ICx Technologies, Inc., 215 First St., Suite 104, Cambridge, MA 02142,
USA. E-mail: Joongho.moon@icxt.com; Lawrence.hancock@icxt.com;
Fax: 1 617 441 8874; Tel: 1 617 441 8871
{ Electronic supplementary information (ESI) available: Experimental
details on synthesis, dynamic light scattering, and kinase assay. See DOI:
10.1039/b710807a
Fig. 1 Chemical structure of poly(p-phenylene ethynylene (PPE).
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CPNs for the selective detection of phosphopeptides. The viscous
PPE solution in a mixed solvent of DMSO–morpholine was
precipitated into an acetone solution and then re-dissolved in
DMSO. CPN was fabricated by transferring the PPE in DMSO
into an excess amount of water.6 The CPN solution was purified
by ultrafiltration through a membrane filter (molecular weight cut-
off: 10 000 Da) in water. Particle size was determined by dynamic
light scattering (DLS), indicating a z-average size of 28 nm with a
polydispersity index (PDI) of 0.254. PDI is a parameter calculated
from a cumulative analysis of DLS intensity measured via an
autocorrelation function. % PDI is the square of the coefficient of
variation (CV) and defined as (PDI)1/2 6 100. From this
relationship, particle size showed 50% of % PDI, indicating very
broad size distributions. Transmission electron microscopic study
supports formation of spherical nanoparticles (Fig. 2).9 Although
CPN showed poor contrast in the TEM, due to its low density,
formation of spherical nanoparticles was confirmed by the image.
For the detection of phosphopeptides, the CPN solution was
mixed with trivalent metal ions (i.e., GaCl3) solution in a buffered
media (pH 5.5) (see ESI{). The CPN/Ga solution was transferred
into rhodamine-labeled kemptide (Rh-kemptide) and rhodamine-
labeled phosphokemptide (Rh-p-kemptide).10 Fig. 3(a) demon-
strates the spectra of the CPN mixed with Rh-kemptide (dotted
line) and with Rh-p-kemptide (solid line). As shown in Fig. 3(a),
CPNs only exhibit efficient energy transfer to Rh-p-kemptides,
despite the poor spectral overlap between CPN and rhodamine.
Negligible spectral change was observed from the Rh-kemptide,
indicating the interaction is phosphopeptide-specific. Zheng and
Swager demonstrated that better stacking and orbital interaction
between the conjugated polymer backbone and acceptor dyes are
the main contributors to efficient energy migration.11 CPN’s
efficient energy transfer capability to an acceptor dye with longer
absorption wavelength has the direct benefit of reducing
unnecessary background signals. The background fluorescence
originated from the assay media or fluorescent molecules in the
library often interferes with assay readout resulting in uncertainty
and complexity in the interpretation of the assay results. The
difference between excitation wavelength (400 nm) and emission
wavelength (585 nm) was so large that unnecessary background
can be minimized.
Fig. 3 (a) Emission spectra of CPN mixed with rhodamine-labeled
kemptide (Rh-kemptide, dotted line) and with rhodamine-labeled
phosphokemptide (Rh-p-kemptide, solid line). Strong FRET occurred
from metal ion-assisted adsorption of phosphokemptides, while no
significant FRET was observed from nonphosphorylated kemptide. (b)
Ratiometric (FQ/FRET) calibration curve generated from standard
mixtures of Rh-kemptide and Rh-p-kemptide.
Fig. 3(b) indicates that the fluorescence quenching and FRET
intensity are proportional to the amount of phosphorylation.
Standard Rh-kemptide/Rh-p-kemptide solutions were prepared by
proportionally mixing Rh-p-kemptide and Rh-kemptide (i.e. 0, 5,
10, 25, 50 and 100% of Rh-p-kemptide). By taking the ratio of
fluorescence quenching to FRET signal, an accurate and
quantitative measurement at the low percent conversion of
phosphorylation was achieved. The ratiometric method can
eliminate fluorescence variations caused by the fluctuation of
excitation source or different assay concentrations.12 As seen in
Fig. 3(b), a significantly different signal change (5%) was observed
from the low phosphopeptide conversion.
To demonstrate the kinase assay, we monitored kinase activity
as a function of the inhibitor. The enzymatic reaction was
performed by incubating Rh-kemptide with protein kinase A
(PKA) for 60 min at room temperature in the presence of
adenosine triphosphate (ATP). PKA, is an important enzyme
involved in several key second-messenger signalling pathways, and
is implicated in a variety of cellular processes including cell cycle
progression, apoptosis, transcription, and cellular metabolism.13
After the enzymatic reaction, CPN/Ga solution was added and
incubated for 30 min. The amount of phosphorylated substrate
was measured by calculating the ratio between the FQ of the
CPNs at 455 nm and the FRET signal at 585 nm. Using the
Fig. 2 Transmission electron microscopic image of PPE in water.
Spherical shaped nanoparticles are observed. Poor contrast is attributed
to the low density of the CPNs.
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This work was partially supported by NSF grant (DMI-
0239285).
Notes and references
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Fig. 4 Inhibition curves of PKA in the presence of staurosporin. IC50
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9 The average particle size in the TEM image was about 70 nm. It was
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particles than 70 nm that are believed as a major population.
calibration curve [Fig. 3(b)], ratios from the assay were converted
to the amount of phosphorylation in the substrate (percent
phosphorylation). Therefore, the enzyme efficiency can be
measured as a function of percent phosphorylation. Enzyme
reactions were conducted in the presence of 10 mM ATP, 0.5 mM
Rh-kemptide substrate, and various amounts of inhibitor
(staurosporin). By plotting enzyme efficiency (percent phosphoryl-
ation) as a function of inhibitor concentration, 50% inhibitory
concentration (IC50) was calculated. Fig. 4 demonstrates that
CPNs can reliably and robustly detect y5% phosphorylation even
at the low enzyme concentration (0.03 U PKA mL21). Sigmoidal
dose response with acceptable error range indicates the sensitivity
and reliability of CPN assay. The IC50 value is well matched with
published values (3.6 nM).14
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Leu-Gly) specific substrate for protein kinase A (PKA): B. E. Kemp,
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In conclusion, we have fabricated a CPN with highly efficient
energy transfer to an acceptor dye and used it for sensitive
detection of kinase activity. Biochemical assay results show
promise for high throughput screening of small molecules with
improved sensitivity and reliability.
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