Fluorescent semiconducting polymer conjugates of poly(N-isopropylacrylamide) for thermal precipitation assays

Article abstract of DOI:10.1021/ma035373+

New thermally responsive fluorescent polymers conjugated with poly(N-isopropylacrylamide) (polyNIPA) have been synthesized. A nonionic water-soluble poly(phenylene-ethynylene) (PPE) was end-capped with a di-tert-butylnitroxide derivative, and subsequent n

Full text of DOI:10.1021/ma035373+

716
Macromolecules 2004, 37, 716-724
Fluorescent Semiconducting Polymer Conjugates of
Poly(N-isopropylacrylamide) for Thermal Precipitation Assays
Ken ich i Ku r od a a n d Tim oth y M. Sw a ger *
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received September 13, 2003; Revised Manuscript Received December 8, 2003
ABSTRACT: New thermally responsive fluorescent polymers conjugated with poly(N-isopropylacrylamide)
(polyNIPA) have been synthesized. A nonionic water-soluble poly(phenylene-ethynylene) (PPE) was end-
capped with a di-tert-butylnitroxide derivative, and subsequent nitroxide-mediated radical polymerization
of NIPA afforded PPE-polyNIPA block copolymers. These copolymers phase-separate from aqueous
solutions upon heating, and the resultant precipitates are efficiently collected by filtration. The fluorescent
spectra of the precipitates indicate the absence of strong associations between the PPE π-systems.
Furthermore, the fluorescent intensities of the collected solids have a linear correlation with the polymer
concentrations in the solutions of origin. When copolymers are thermally coprecipitated with dye-labeled
(rhodamine B) polyNIPA materials, the dye is localized to the PPE segments, inducing fluorescent
resonance energy transfer from the PPE segment (donor) to the dye (acceptor).
In tr od u ction
advantageous to develop new nonionic systems that are
capable of analyzing complex unprocessed biological
fluids that contain a variety of analytes and charged
biomolecules.
Fluorescent semiconducting polymers (FSPs) have
emerged as a powerful class of sensory materials1 and
have enabled the formation of ultrasensitive sensors for
explosivemolecules,2-4 metalions,5-8 and biomolecules.9-13
The delocalized π-electrons in the polymer’s backbone
create energy bands and facilitate the transport of
excited states (excitons).14 This transport allows excitons
to visit many potential binding sites that may be
occupied by an analyte molecule that is capable of
inducing fluorescence quenching or a new emission
feature. This long-distance exciton transport produces
the signal amplification that allows for ultrasensitive
sensory responses.15
Considering the anticipated limitations of ionic FSP
biosensors, we are seeking to develop a new transduc-
tion scheme which would (1) make use of water compat-
ible FSPs to allow biomolecular recognition events take
place in solution, (2) employ nonionic FSPs that are
immune to indiscriminate electrostatic interactions, (3)
maximize the signal amplification resulting from energy
migration in FSPs, and (4) allow selective collection of
sensory FSPs from analyte solutions in order to remove
interferants and facilitate fluorescence measurements.
To this end we report herein a thermally responsive
block polymer of a nonionic water-soluble poly(phe-
nylene-ethynylene)19 (PPE) and poly(N-isopropylacry-
lamide) (polyNIPA), P -I (Chart 1). The polyNIPA block
produces phase separation upon heating (>30 °C) due
to a hydrophobic collapse mechanism.20 To investigate
the potential of P -I for use in sensory schemes, we have
demonstrated that thermal coprecipitation with dye-
polyNIPA conjugate P -II (Chart 1) induces FRET from
the PPE segment to the dye.
A variety of different detection mechanisms have been
developed to translate analyte binding to fluorescent
sensory responses. These include electron-transfer
quenching by paraquat bound to receptor sites,16 π-com-
plexation with nitroaromatics such as trinitrotoluene
(TNT),2,3 energy transfer to activated indicators,17 ana-
lyte-initiated aggregation of FSPs,7 and coordination to
pyridyl ligands by transition metal ions.6,18 To extend
the utility of FSPs as sensory materials, new transduc-
tion schemes making use of conjugated polyelectrolytes
have been investigated.9-13 DNA detection has been
recently demonstrated9,10 utilizing FSP amplified fluo-
rescence resonance energy transfer (FRET).17 This DNA
detection scheme makes use of the well-known distance
dependence of FRET that is the basis of many biosen-
sory transduction schemes with cationic FSPs electro-
statically binding to DNA.13 The localization of the FSP
and a fluorescent DNA probe results in efficient FRET.
However, few other biosensory schemes can rely on such
strong ionic associations. A concern is that those sys-
tems having weaker molecular recognition elements
may suffer from nonspecific electrostatic associations.
Hence, in ionic FSP biosensory systems, the sensory
responses will likely be limited to conditions wherein
solutions contain only a few species of analytes, thereby
producing less interference in analysis. Clearly, it is
Resu lts a n d Discu ssion
Syn th esis a n d Ch a r a cter iza tion of a n F SP -
P olyNIP A Con ju ga te. The FSP-polyNIPA conjugate
(P -I) was synthesized from a water-soluble PPE end-
capped with an alkoxyamine capable of initiating ni-
troxide-mediated radical polymerization (NMRP) of
NIPA to give a block copolymer. The synthesis of the
end-capping/NMRP initiator 3 (Scheme 1) begins with
compound 1 obtained from treatment of 4-iodobenzoyl
chloride and an excess amount of 2,2′-(ethylenedioxy)-
bis(ethylamine), and a subsequent reaction with 2-chlo-
ropropionyl chloride produces 2. We have adapted
methods developed by Matyjaszewski to afford 3,21
wherein compound 2 reacts with CuCl to produce a
radical that is captured by the di-tert-butyl nitroxide
radical to give 3. A competitive process is the back-
reaction of the radical with CuCl2/Me6TREN to regener-
ate 2. This process is attenuated by the addition of
* Corresponding author: e-mail tswager@mit.edu.
10.1021/ma035373+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/16/2004

Macromolecules, Vol. 37, No. 3, 2004
Polymer Conjugates of Poly(N-isopropylacrylamide) 717
Ch a r t 1
Sch em e 1
(GPC) analysis indicated that Mn, Mw, and polydisper-
sity index (PDI) of purified 6 are 23 556, 34 411, and
1.46, respectively.
The NMRP of NIPA was carried out using 6 as an
initiator in DMF or DMSO at 120 °C. The resultant
polymer was purified by precipitation in diethyl ether
and dialysis against water. Generally, in the NMRP
mechanism, the weak C-O bond of alkoxyamine initia-
tor homolyzes to give a nitroxide radical and a carbon
radical.22 The carbon radical then initiates radical
polymerization, and in an ideal case, a polymer chain
grows without termination reactions, thereby enabling
the synthesis of multiblock copolymers.23,24 Previous
studies on the NMRP process have revealed the impor-
tance of the nitroxide’s (alkoxyamine’s) structure to
control reactivity in the radical polymerization pro-
cess.22,25 Consistently we found that the 2,2,6,6-tetram-
ethyl-1-piperdinyloxyl (TEMPO)-based initiator gave no
polymer. However, the di-tert-butyl nitroxide derivative
initiator (3) worked well to produce polyNIPA. The di-
tert-butyl nitroxide initiator has higher reactivity than
the TEMPO initiator due to its steric hindrance which
weakens the C-O bond, thereby lowering the temper-
atures required for polymerization.26-28 Despite the use
of NMRP, GPC analysis of the block copolymer P -I
showed a bimodal elution profile with a broad molecular
weight distribution (Mn ) 51 604, Mw ) 233 100, PDI
) 4.52), which suggests that the resultant polymer
contains tri- and diblock copolymers. The observed
polydispersity is likely due to chain termination events.
Control experiments initiating NMRP of NIPA using
only initiator 3 with monomer-to-initiator ratios of 100
Cu(0), which reduces the Cu(II) spices that serve to
regenerate 2 and thereby drives the reaction to comple-
tion.
The water-soluble PPE 6 in Scheme 2 was synthesized
from monomers 4 and 519 and the end-capping/NMRP
initiator 3 by a Sonogashira cross-coupling using a Pd
catalyst and CuI cocatalyst in morpholine. This polymer
was purified by precipitation in ethyl acetate and
dialysis against water and was then isolated by lyo-
philization. NMR analysis provided the degree of po-
lymerization (DP) by integrating the signals from the
aromatic and amide groups in the PPE relative to those
from the tert-butyl moieties of the end-caps that are
well-resolved from other polymer signals. The DP of 6
was found to be 11.5. Gel permeation chromatography
Sch em e 2

718 Kuroda and Swager
Macromolecules, Vol. 37, No. 3, 2004
F igu r e 1. UV-vis absorption and fluorescence spectra of PPE
6 (solid lines with circles and with squares) and P -I (broken
lines with circles and with squares). The excitation wave-
lengths for the fluorescence spectra are 380 nm for 6 and 350
nm for P -I.
F igu r e 2. Fluorescence spectra of P -I in solution (solid line)
and solid on a filter paper (broken line). The excitation
wavelength is 350 nm.
1
gave a DP of 42 by H NMR and yields of 14%. (Mn,
in an ice bath. At these temperatures the polyNIPA
chains dissociate and are completely solvated by water.
Fluorescent assays are best performed at concentrations
lower than typically used in the precipitation of
polyNIPA, and in order to effect quantitative phase
separation, aqueous NaCl was added to the polymer
solutions.20 The addition of salt is well-known to reduce
the phase separation temperature.20,31 The polymer
solutions (polymer concentrations 0.25-0.025 mg/mL)
were heated at 50 °C, and the resultant precipitate was
collected by vacuum filtration with a small filter paper
(diameter 8 mm) on a Hirsch funnel and washed with
hot water to remove the salts. The filtrate had a slight
yellow color and blue fluorescence. It appears that only
trace amounts of the polymer passed though the filter
paper. After drying under vacuum at room temperature,
the filter paper was placed between a coverslip and a
glass microscope slide, and its fluorescence was mea-
sured. This procedure was reproduced many times, and
we found that the reproducibility of the emission
intensities was within 20%, which provides useful
analysis in many potential biosensory assays.
The fluorescence spectra of P -I on a filter paper
(Figure 2) reveal a slight red shift in the emission
relative to that in solution, and there are no new
emission signals. The fact that the emission band shape
is largely the same in the precipitate as in solution is
consistent with the absence of strong interchain π-as-
sociations (aggregation) that produce broad new low-
energy and undesired weakly emitting bands.32 Aggre-
gation of conjugated polymers is generally enhanced in
the solid state because of the potential of interchain π-π
stacking. Hence, the absence of such features indicates
that the polymer chains are largely prevented from
having strong interchain electronic interactions, per-
haps due to the dendritic side chains of P -I.
Mw, and PDI are 18 349, 25 745, and 1.4, respectively,
by GPC.) Precise polymerizations of NIPA are generally
difficult, and only Harth et al. have obtained polyNIPA
by the NMRP method with accurately controlled mo-
lecular weights and low polydispersities by using spe-
cially designed initiators.29 We investigated a related
ATRP method that has been successful for the formation
of short conjugated oligomer-polystyrene block copoly-
mers with low polydispersity.30 This method makes use
of oligo(p-phenylene-ethynylene)s end-capped with
ATRP initiators at their termini and subsequent po-
lymerization of styrene using a CuBr catalyst. We
attempted to end-cap PPE 6 with 2 as an ATRP initiator
by the same procedure in Scheme 2. However, we found
that 2 was not stable to the Sonogashira polymerization
conditions. Given that the observed polydispersity of P -I
does not present obvious limitations to the present study
and the resultant copolymer is highly water-soluble and
thermally precipitates upon heating, we have completed
the following studies with our high-PDI samples.
The UV-vis absorption spectra of P -I and the precur-
sor PPE 6 are shown in Figure 1. After copolymerization
by the NMRP method, a blue shift in P -I’s absorption
spectra was observed, whereas its fluorescence showed
no shift. A plausible explanation for the blue shift in
the absorption spectrum is that the polyNIPA block
disturbs the π-conjugation of the PPE segments and
creates a higher energy gap in their electronic structure.
The lack of change in the fluorescence is likely due to
energy migration from the terminal portions of the PPE
that are more affected by the polyNIPA to the center of
the PPE. In this way, the emission corresponding to the
lower energy gap in the center of the PPE dominates
the spectrum, and it appears to be the same as that of
6.
Th er m a lly In d u ced P r ecip ita tion . We are inter-
ested in developing sensor assays that make use of the
phase separation from aqueous solution upon heating.
To this end we examined a scheme whereby polymers
are collected as solid precipitates to remove interferants
and to facilitate analysis. To prepare completely soluble
polymer aqueous solutions, the polymers were stirred
The fluorescence spectra of the polymer (P -I) precipi-
tates were measured with variable polymer concentra-
tions (Figure 3), and the emission intensity from the
polymers collected on the filter papers increased linearly
with increasing polymer concentrations. These results
confirm that our collection method using filter paper
accurately reflects the polymer concentration in solu-

Macromolecules, Vol. 37, No. 3, 2004
Polymer Conjugates of Poly(N-isopropylacrylamide) 719
F igu r e 4. Normalized UV-vis absorption and fluorescence
spectra of P -I (A and B) and P -II (C and D) in water with
excitation at 350 nm.
F igu r e 3. Fluorescence spectra of P -I on filter papers (a) and
intensities at 470 nm (b). The intensity increases with increas-
ing the polymer concentration (0.015, 0.03, 0.075, and 0.15 mg/
mL). The excitation wavelength is 350 nm.
F igu r e 5. Absorption spectra of solution mixtures of P -I and
P -II. P -I concentration was constant at 0.12 mg/mL. P -II
concentrations are 0.05, 0.025, 0.0125, and 0.00625 mg/mL,
and the absorbance at 560 nm increased with increasing P -II
concentrations.
tion. As can be seen in Figure 3, the polymer emission
peak is slightly red-shifted with increasing polymer
concentrations. This is likely due to a spectral overlap
of strong background scattering from the filter paper
and the polymer emissions, which results in an apparent
red shift with the increase of the polymer emission.
Th er m a l P r ecip ita tion In d u ced En er gy Tr a n s-
fer . Through-space fluorescence resonance energy trans-
fer (FRET) is a distance-dependent dipolar interaction
of chromophores (a donor and acceptor) and is widely
used in sensory transduction schemes and assays for
the detection of biomolecules. In the FRET (Fo¨rster)
mechanism, the energy of the excited donor is efficiently
transferred to the acceptor without emission when they
are within the Fo¨rster radius (of 50-100 Å) of each
other.
transfer from the donor FSP which can capture a larger
percentage of the excitation photons due to its greater
optical cross-section.10,17 To test this approach, a dye-
polyNIPA conjugate P -II was prepared by a free radical
copolymerization of NIPA with a rhodamine B (RhB)
methacrylate monomer using AIBN in DMF (Mn
86 909, Mw ) 142 730, and PDI ) 1.64 by GPC).
)
Normalized UV-vis absorbance and fluorescence
spectra of P -I and P -II in water are shown in Figure 4.
The spectral overlap of the PPE (donor) emission from
P -I and the RhB (acceptor) absorption from P -II was
chosen to produce efficient FRET based on the Fo¨rster
mechanism and to also give an acceptor emission that
is well-resolved from that of P -I, thereby facilitating
analysis.
Our interest was to examine whether the thermally
induced phase separation of P -I can trigger FSP-
amplified FRET with dye molecules. The advantage of
this process is that the emission intensity from an
acceptor dye molecule can be enhanced by energy
The absorption and fluorescence spectra from mixed
solutions of P -I and P -II are shown in Figure 5 and
Figure 6, respectively. When the solution was excited

720 Kuroda and Swager
Macromolecules, Vol. 37, No. 3, 2004
F igu r e 6. Fluorescence spectra of the mixture of P -I and P -II in a solution (a) and on a filter paper (b). The spectra in (b) are
normalized relative to the peak intensities at 468 nm. P -I concentration in the solutions was 0.12 mg/mL. P -II concentrations
are 0.05, 0.025, 0.0125, and 0.00625 mg/mL in the solutions. P -I is the principal absorbing group with 350 nm excitation, and
P -II is selectively excited with excitation at 500 nm. Light scattering from the filter paper gives a peak at 540 nm in (b). Fluorescence
intensity at 580 nm increased with higher P -II concentrations. The solid lines in (c) and (d) show the dependence of the different
emissions as a function of P -II concentration.
at 350 nm, a major emission peak from the PPE
segments of P -I and a small one from the RhB bound
to P -II were observed at 470 and 580 nm, respectively.
With excitation at 500 nm, the RhB was selectively
excited, and its emission was observed at 580 nm. The
fluorescence intensity from RhB in solution at both
excitation wavelengths was found to be directly propor-
tional to the P -II concentration (Figure 6c). The ability
to directly excite the RhB thereby allows this signal to
be used as an internal reference to its concentration.
Thermally precipitated mixtures of P -I and P -II were
collected on a nonfluorescent filter paper using proce-
dures described earlier, and the normalized fluorescence
spectra from the filter paper are shown in Figure 6. We
find that the emission at 580 nm from the RhB is 5
times larger when we excited principally the PPE at 350
nm than when we selectively excited the RhB at 500
nm. This effect is not due to the differences of the
excitation intensities at the excitation wavelengths
because the photon fluxes with excitation at 350 and
500 nm are corrected to be the same by the spectrom-
eter. In this experiment the RhB was excited at 500 nm
rather than its absorption maximum to avoid an overlap
with strong light scattering from the filter paper. We
have further determined that the quantum yield of
precipitated P -I is independent of added polyNIPA.
Therefore, the increasing emission from RhB with
excitation at 350 nm is due to FRET induced by thermal
precipitation that localizes the RhB molecules to the
vicinity of the PPE segments. The RhB absorbance
maximum is at 560 nm, and on the basis of the
differences in excitation coefficient at 500 nm, we would
expect the emission to be 4 times larger than that
obtained with 500 nm excitation. As a result, in terms
of practical signal gains for sensory assays this system
only yields enhancements of about 20%. Nevertheless,
we emphasize that comparisons with the solution data
validate that thermal precipitation induces FRET. The
linear dependence on P -II concentration in the emission
intensity from the RhB with excitation at 500 nm in
the coprecipitates confirms, similar to P -I, that our
precipitation assay is quantitative and reflects the true
concentration of the polymer in solution (Figure 6d).
Interestingly, the RhB fluorescence intensity at 580 nm
from the filter papers (λex ) 350 nm) slightly levels off
with increasing the P -II concentrations (Figure 6d).
This is an additional signature of energy transfer and
is due to the fact that at high RhB concentrations there

Macromolecules, Vol. 37, No. 3, 2004
Polymer Conjugates of Poly(N-isopropylacrylamide) 721
Ch a r t 2
are multiple RhB acceptors within the Fo¨rster radius
and diffusion range of an exciton. In this way, the
amount of energy available to each RhB acceptor from
the PPE donors decreases, leading to leveling-off of the
RhB emission intensity.
Despite our successful demonstration of thermal
precipitation induced energy transfer from P -I to P -II,
there are a number of features that indicate that FRET
processes lack the efficiency that would be required for
a viable assay. First, we are only able to increase the
RhB emission about 20% beyond that which can be
obtained by the optimal direct excitation of this dye.
Second, the donor (PPE) emission does not decrease
significantly with increasing RhB concentrations, which
indicates that only a small amount of the donor’s energy
is being captured by the acceptor. We have considered
that the inefficiency in energy transfer from the PPE
segments can be attributed to one or more of the
following issues: (1) The polymers have different phase
separation temperatures, resulting in the polymers
precipitating separately into macrophase-separated do-
mains. (2) The RhB is imbedded in polyNIPA aggregates
and located too far from the PPE segments. (3) The
spectral overlap of the PPE emission and the RhB
absorption is not optimal (Figure 4). (4) The PPE
segment of P -I has a relatively low quantum yield,
which could be attributed to a weak aggregation of the
polymers.19 We can rule out the possibility of phase
separation by very rapidly heating the polymer mix-
tures, and the lack of any significant differences sug-
gests that this is not a consideration. The fact that the
PPE block polymer may microphase separate from P -II
was addressed by synthesizing a block copolymer with
polyNIPA blocks randomly containing a RhB comono-
mer (P -III, Chart 2). The incorporation of the dye into
the copolymer structure should work to better compati-
bilize the two materials. The absorption spectrum
(Figure 7) suggests that a small amount of RhB relative
to the PPE segment is incorporated into the polyNIPA
block of P -III, which is comparable to the mixtures of
P -I and P -II (Figure 5). The precipitation assay using
P -III was performed by the same procedure as P -I. As
can be seen in Figure 8, the intensity ratio of the RhB
emission excited at 350 nm to that excited at 500 nm is
about 5, which is almost same order of enhancement
as the mixture of P -I and P -II in Figure 6, indicating
still lack of highly efficient FRET.
F igu r e 7. Absorption spectrum of P -III in water.
Quade et al. reported highly efficient amplified emis-
sions from fluorescein by FRET from PPE thin films.17
In this case fluorescein has the advantage of a larger
spectral overlap with the PPE than RhB. However,
when the same experiments are carried out with a
fluorescein conjugate of polyNIPA, we observe a dra-
matic reduction in the emission from fluorescein with
precipitation. This loss of emission is likely the result
of the hydrophobic environment in the polyNIPA pre-
cipitates, which induces the conversion of this dye to
its colorless and nonfluorescent lactone. The low quan-
tum yields of these polymers are clearly a limitation,
and enhancing the emission efficiencies of our nonionic
water-soluble polymers is a challenging and ongoing
research goal.
Su m m a r y
New thermally responsive PPE conjugates of polyNIPA
have been synthesized, which precipitate from aqueous
solution upon heating, resulting in the isolation of PPE
for facile fluorescence measurements. A thermally in-
duced coprecipitation of these copolymers with a RhB-
polyNIPA conjugate induces FRET from PPE to RhB.
While this method presently lacks the necessary per-
formance to produce high amplification desired for
biological sensing, we have developed general proce-
To produce a higher spectral overlap, we also consid-
ered different fluorescent dyes as FRET partners. Mc-

722 Kuroda and Swager
Macromolecules, Vol. 37, No. 3, 2004
including antibody-antigen coupling and DNA base
pair matching. Once the target analytes (antigens or
DNAs) are captured by polyNIPA bioconjugates, ther-
mally induced coprecipitation with FSP-polyNIPA
conjugates will be performed, and the analytes can be
quantified by FSP-amplified fluorescent detection. This
thermal precipitation assay method with bioconjugates
of polyNIPA offers a new transduction scheme for FSP-
based biosensors.
Exp er im en ta l P r oced u r es
Gen er a l. Fluorescence spectra were measured on a SPEX
Fluorolog-τ2 spectrofluorometer. CuCl was purified by stirring
in acetic acid overnight. The remaining solid was filtered,
washed with EtOH, dried under vacuum, and stored under
Ar. NMR spectra were obtained on Varian Unity or Mercury
300 spectrometer. High-resolution mass spectra were obtained
at the MIT Department of Chemistry Instrumentation Facility
(DCIF) on a Bruker Daltronics APEX II 3 T FT-ICR-MS.
Polymer molecular weights were determined with a Agilent
1100 series HPLC equipped with a PL gel 5 µm guard column
and three columns (PL gel 5 µm, 105 Å, 104 Å, 103 Å) connected
in series using DMF containing 5 mM BuLi as an eluent. GPC
measurements were made relative to monodisperse polysty-
rene standards using a refractive index detector. The synthesis
of the monomers 4 and 5 was reported previously.19
N-{2-[2-(2-Am in oet h oxy)et h oxy]et h yl}-4-iod ob en za -
m id e (1). Compound 1 was prepared from 4-iodobenzoyl
chloride and 2,2′-(ethylenedioxy)bis(ethylamine). 4-Iodobenzoyl
chloride (5.00 g, 18.8 mmol) in CH2Cl2 (125 mL) was added
dropwise to 2,2′-(ethylenedioxy)bis(ethylamine) (22.2 g, 150
mmol) in CH2Cl2 (150 mL) and Et3N (5 mL). The reaction
mixture was stirred overnight and evaporated off. Water was
added to the residue, and the resultant precipitate was filtered.
The filtrate was extracted with EtOAc (400 mL). The organic
layer was dried over MgSO4 and evaporated off after filtration.
1
The oily residue was solidified under vacuum. H NMR (300
MHz, DMSO-d6): δ 8.621 (t, 1H, J ) 6.3 Hz), 7.82 (d, 2H, J )
8.4 Hz), 7.63 (d, 2H, J ) 8.7 Hz), 4.25-3.45 (m, 6H), 3.59-
3.33 (m, 4H), 2.64 (t, 2H, J ) 5.7 Hz), 2.34 (bs, 2H). 13C NMR
(75 MHz, DMSO-d6): 165.34, 136.94, 133.61, 129.01, 98.68,
72.90, 69.57, 69.47, 68.77, 41.31. HR-MS (ESI) calcd for C13H19
-
IN2O3: 379.0513 (M+ + H). Found: 379.0506 (M+ + H),
401.0356 (M+ + Na).
N-{2-[2-(2-Am in oet h oxy)et h oxy]et h yl}-4-iod ob en za -
m id e (2). 2-Chloropropinonyl chloride (0.71 g, 5.6 mmol) was
added to compound 3 (2.0 g, 5.3 mmol) in a mixture of CH2Cl2
(20 mL) and Et3N (0.74 mL, 5.3 mmol) in an ice bath. The
reaction mixture was allowed to warm room temperature and
stirred overnight. The solvent was evaporated off, and the
crude product was purified by a silica gel chromatography
F igu r e 8. Fluorescence spectra of P -III solution (a) and a
filter paper (b). The emission at 540 nm in the spectra
(excitation at 500 nm) (b) is light scattering from the filter
paper.
dures and synthetic methods that can be applied to
other optimized systems. Our ongoing efforts are in
progress, directing at solving the problems encountered,
which include efficient localization of dye molecules to
FSP segments by thermal precipitation, reductions in
self-quenching by aggregation of FSPs, increased spec-
tral overlaps of FSPs (donors) and dyes (acceptors), and
the design of new nonionic water-soluble polymers. It
is further anticipated that in some circumstances even
nonionic water-soluble polymers can exhibit nonspecific
weak binding of biomolecules and methods to prevent,
or mitigate the effects of, entrapment of these species
in thermally precipitation assays will need to be ad-
dressed.
In this report, we made use of a dye-polyNIPA
conjugate as a simple model analyte to examine the
basic assay principle. We also envision that improved
assays can make use of FSP-polyNIPA conjugates in
bioassay schemes with polyNIPA conjugates of antibod-
ies, enzymes, and DNAs.33-35 These polyNIPA biocon-
jugates will provide molecular recognition of analytes,
1
(CH2Cl2 (95%)/MeOH (5%)) (2.3 g, 91%). H NMR (300 MHz,
CDCl3): δ 7.88 (d, 2H, J ) 6.6 Hz), 7.53 (d, 2H, J ) 6.9 Hz),
6.98 (bs, 1H), 6.76 (bs, 1H), 4.44 (q, 1H, J ) 7.2 Hz), 4.25-
3.43 (m, 12H), 1.75 (d, 3H, J ) 6.9 Hz). 13C NMR (75 MHz,
DMSO-d6): 169.76, 166.88, 137.87, 133.99, 128.81, 98.71,
70.59, 70.54, 70.01, 69.71, 56.21, 40.18, 39.96, 23.07. HR-MS
(ESI) calcd for C16H22ClN2O4I: 491.0205 (M+ + H). Found:
469.0397 (M+ + H), 491.0197 (M+ + Na).
N-[2-(2-{2-[2-(N,N-Di-ter t-b u t yla m in ooxy)p r op ion yl-
a m in o]eth oxy}eth oxy)eth yl]-4-iod oben za m id e (3). Com-
pound 2 (300 mg, 0.640 mmol), Cu (61 mg, 0.96 mmmol), and
CuCl (25 mg, 0.26 mmol) were placed in a 10 mL Schlenk flask
and degassed by three quick cycles of vacuum and backfilling
with Ar. Dioxane was Ar-purged for 10 min. Tris[2-(dimethy-
lamino)ethyl]amine (Me6TREN)36 (59 mg, 0.26 mmol) in diox-
ane (3 mL) was poured into the Schenk flask, and di-tert-
butylnitroxide (125 mg, 0.867 mmol) was added under Ar. The
reaction mixture was stirred at 70 °C overnight. The reaction
mixture was evaporated, and the residue was chromatographi-
cally separated by a gradient silica gel column (ethyl acetate
(100%) to ethyl acetate (95%)/ethanol (5%)). The collected
product was dissolved in CH2Cl2 and washed with saturated

Macromolecules, Vol. 37, No. 3, 2004
Polymer Conjugates of Poly(N-isopropylacrylamide) 723
NH4Cl(aq) solution and saturated NaCl solution. The organic
layer was dried over MgSO4, and the solvent was evaporated
off after a filtration of MgSO4 to give an oily compound (190
pore size 8-12 µm, diameter 8 mm) on a Hirsch funnel and
washed with a hot water (50 °C, 3 mL) without a salt. The
filter paper was dried under vacuum overnight and placed
between a coverslip and a glass microscope slide. Fluorescence
from the collected precipitates on the filter paper was mea-
sured.
1
mg, 61%). H NMR (300 MHz, CDCl3): δ 7.76 (d, 2H, J ) 8.1
Hz), 7.53 (d, 2H, J ) 8.7 Hz), 6.89 (bs, 2H), 4.31 (q, 1H, J )
6.9 Hz), 3.64-3.41 (m, 12H), 1.45 (d, 3H, J ) 6.9 Hz), 1.21 (s,
18H). 13C NMR (75 MHz, DMSO-d6): 173.91, 166.77, 137.82,
134.03, 128.854, 98.65, 82.11, 70.55, 70.19, 70.05, 40.10, 38.90,
31.06, 30.61, 19.06. HR-MS (ESI) calcd for C24H40IN3O5:
600.1905 (M+ + Na). Found: 578.2081 (M+ + H), 600.1880
(M+ + Na), 1177.3847 (2M+ + Na).
Ack n ow led gm en t. We thank the National Science
Foundation through the Center for Materials Science
and Engineering at MIT and NASA for financial sup-
port.
Syn th esis of P P E 6. Monomers 4 (87.3 mg, 0.0779 mmol),
5 (40.8 mg, 0.0909 mmol), and alkoxyamine 3 (10 mg, 0.0173
mmol) were placed in a 25 mL Schlenk flask. Pd(PPh3)4 (5%)
and CuI (5%) were added to the flask in a glovebox. Morpholine
was passed through an alumina column and degassed by three
quick cycles of vacuum and backfilling with Ar before use. The
solvent was cannulated into the flask containing the monomers
(∼0.5 mL). The reaction mixture was degassed again and
stirred at 60 °C overnight and poured into EtOAc. The
precipitate was collected by centrifuge, washed with EtOAc
and diethyl ether, and dried under vacuum. The compound
was dissolved in water and dialyzed against water (Pierce,
Snake skin, MWCO 3500) for 2 days (500 mL water × 4 times
in a day). The polymer solution was then lyophilized (86 mg).
1H NMR (300 MHz, DMSO-d6): δ 7.75 (bs, 2H), 7.451 (bs, 2H),
7.074 (bs, 4H), 4.951 (bs, 16H), 4.596 (s, 8H), 4.098 (bs, 4H),
3.988 (bs, 4H), 3.560 (bs, 40H), 1.201 (s, 1.83H), 1.144 (s,
1.83H). DP ) 11.5 by 1H NMR. Mn ) 23 556, Mw ) 34 411,
and PDI ) 1.46 based on GPC analysis.
Refer en ces a n d Notes
(1) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev.
2000, 100, 2537-2574.
(2) (a) Yang, J . S.; Swager, T. M. J . Am. Chem. Soc. 1998, 120,
5321-5322. (b) Yang, J . S.; Swager, T. M. J . Am. Chem. Soc.
1998, 120, 11864-11873.
(3) Cumming, C. J .; Aker, C.; Fisher, M.; Fox, M.; la Grone, M.
J .; Reust, D.; Rockley, M. G.; Swager, T. M.; Towers, E.;
Williams, V. IEEE Trans. Geosci. Remote Sensing 2001, 39,
1119-1128.
(4) Liu, Y.; Mills, R. C.; Boncella, J . M.; Schanze, K. S. Langmuir
2001, 17, 7452-7455.
(5) Marsella, M. J .; Swager, T. M. J . Am. Chem. Soc. 1993, 115,
12214-12215.
(6) Wang, B.; Wasielewski, M. R. J . Am. Chem. Soc. 1997, 119,
12-21.
(7) Kim, J .; McQuade, D. T.; McHugh, S. K.; Swager, T. M.
Syn th esis of P P E-P olyNIP A Con ju ga te (P -I). PPE 6
(10 mg) and NIPA (500 mg) were placed in a 10 mL Schlenk
flask and degassed by three cycles of vacuum and backfilling
with Ar. Ar-purged DMF or DMSO (0.5 mL) was added to the
flask by a syringe. The reaction mixture was stirred at 120 °C
overnight. The polymer was precipitated in ethyl ether and
collected by a centrifuge. The dried polymer was dissolved in
water and purified by a dialysis against water. The polymer
solution was then lyophilized after filtration to give a yellow
powder (22 mg). Mn ) 51 604, Mw ) 233 100, and PDI ) 4.52
based on GPC analysis.
Syn th esis of Rh B-P olyNIP A Con ju ga te (P -II). NIPA
(0.50 g, 4.4 mmol), methacyloxyethyl thiocarbamoyl rhodamine
B (7.4 mg, 0.011 mmol, Polysciences, Inc.), and AIBN (3.6 mg,
0.022 mmol) were placed in a 25 mL Schlenk tube and
degassed by three vacuum-Ar filling cycles. Ar-purged DMF
(1.5 mL) was added to the tube by a syringe. The reaction
mixture was stirred at 60 °C overnight. The polymer was
precipitated in ethyl ether and collected by a centrifuge and
dried under vacuum. Mn ) 86 909, Mw ) 142 730, and PDI )
1.64 based on GPC analysis.
Syn th esis of P P E-Rh B-P olyNIP A Con ju ga te (P -III).
PPE 6 (5 mg), NIPA (0.5 g), and methacyloxyethyl thiocar-
bamoyl rhodamine B (0.4 mg, 0.6 mmol, Polysciences, Inc.)
were placed in a 25 mL Schlenk tube and degassed by three
cycles of vacuum and backfilling with Ar. Ar-purged DMSO
(0.5 mL) was added to the tube by a syringe. The reaction
mixture was stirred at 120 °C overnight. The polymer was
precipitated in ethyl ether and collected by centrifuge. The
dried polymer was dissolved in water and purified by a dialysis
against water (Pierce, Snake skin, MWCO 3500). The polymer
solution was then lyophilized after filtration.
Angew. Chem., Int. Ed. 2000, 39, 3868-3872.
(8) Crawford, K. B.; Goldfinger, M. B.; Swager, T. M. J . Am.
Chem. Soc. 1998, 120, 5187-5192.
(9) Gaylord, B. S.; Heeger, A. J .; Bazan, G. C. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 10954-10957.
(10) Gaylord, B. S.; Heeger, A. J .; Bazan, G. C. J . Am. Chem. Soc.
2003, 125, 896-900.
(11) Fan, C.; Plaxco, K. W.; Heeger, A. J . J . Am. Chem. Soc. 2002,
124, 5642-5643.
(12) Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl,
F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
12287-12292.
(13) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore´,
K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002,
41, 1548-1551.
(14) Swager, T. M. Acc. Chem. Res. 1998, 31, 201-207.
(15) Wosnick, J . H.; Swager, T. M. Curr. Opin. Chem. Biol. 2000,
4, 715-720.
(16) Zhou, Q.; Swager, T. M. J . Am. Chem. Soc. 1995, 117, 7017-
7018.
(17) McQuade, D. T.; Hegedus, A. H.; Swager, T. M. J . Am. Chem.
Soc. 2000, 122, 12389-12390.
(18) Kimura, M.; Horai, T.; Hanabusa, K.; Shirai, H. Adv. Mater.
1998, 10, 459-462.
(19) Kuroda, K.; Swager, T. M. Chem. Commun. 2003, 26.
(20) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163-249.
(21) Matyjaszewski, K.; Woodworth, B. E.; Zhang, X.; Gaynor, S.
G.; Matzner, Z. Macromolecules 1998, 31, 5955-5957.
(22) Hawker, C. J .; Bosman, A. W.; Harth, E. Chem. Rev. 2001,
101, 3661-3688.
(23) Stalmach, U.; de Boer, B.; Post, A. D.; van Hutten, P. F.;
Hadziioannou, G. Angew. Chem., Int. Ed. 2001, 40, 428-430.
Gen er a l P r otocol for Th er m a lly In d u ced P r ecip ita -
tion . Polymer stock solutions (20 mL) were prepared by
stirring the polymers in deionized water and cooling in an ice
bath. The polymer concentrations were 0.15 mg/mL for P -I
and 0.25 mg/mL for P -II to give about 0.5 and 0.4 at the
maximum in UV-vis absorbance, respectively. These polymer
stock solutions were diluted to the desired concentration, and
they were mixed to give polymer sample solutions. The sample
solution (1 mL) was transferred to a 25 mL sample tube, and
a saturated NaCl aqueous solution (0.1 mL) was added into
the tube. The solution was heated at 50 °C in a water bath
without stirring for 3 min. The resultant precipitate was
vacuum-filtered with a filter paper (S&S filter paper, medium,
(24) Stalmach, U.; de Boer, B.; Videlot, C.; van Hutten, P. F.;
Hadziioannou, G. J . Am. Chem. Soc. 2000, 122, 5464-5472.
(25) Benoit, D.; Chaplinski, V.; Braslau, R.; Hawker, C. J . J . Am.
Chem. Soc. 1999, 121, 3904-3920.
(26) Ohno, K.; Tsujii, Y.; Miyamoto, T.; Fukuda, T.; Goto, M.;
Kobayashi, K.; Akaike, T. Macromolecules 1998, 31, 1064-
1069.
(27) Ohno, K.; Izu, Y.; Yamamoto, S.; Miyamoto, T.; Fukuda, T.
Macromol. Chem. Phys. 1999, 200, 1619-1625.
(28) J ousset, S.; Hammouch, S. O.; Catala, J .-M. Macromolecules
1997, 30, 6685-6687.
(29) Harth, E.; Bosman, A. W.; Benoit, D.; Helms, B.; Fre´chet, J .
M. J .; Hawker, C. J . Macromol. Symp. 2001, 174, 85.

724 Kuroda and Swager
Macromolecules, Vol. 37, No. 3, 2004
(30) Tsolakis, P. K.; Kallitsis, J . K.; Godt, A. Macromolecules 2002,
(34) Ding, Z.; Chen, G.; Hoffman, A. S. Bioconjugate Chem. 1996,
7, 121.
35, 5785.
(31) Freitag, R.; Garret-Flaudy, F. Langmuir 2002, 18, 3434-
(35) Umeno, D.; Mori, T.; Maeda, M. Chem. Commun. 1998,
1433-1434.
(36) Ciampolini, M.; Nardi, N. Inorg. Chem. 1966, 5, 41-44.
3400.
(32) Kim, J .; Swager, T. M. Nature (London) 2001, 411, 1030-
1034.
(33) Monji, N.; Hoffman, A. S. Appl. Biochem. Biotechnol. 1987,
14, 107-120.
MA035373+