fluorescent ultrahigh-molecular-weight polyacrylamide probes for dynamic flow systems: synthesis,...

Fluorescent Ultrahigh-Molecular-WeightPolyacrylamide Probes for Dynamic Flow Systems:Synthesis, Conformational Behavior and Imaging

Professor Abraham Warshawsky, a main contributor to this work, died suddenly on November 12, 2001, in Paris. This article isdedicated to his memory

Ying Wang,*1 a Abraham Warshawsky,1 Chengyong Wang,1 Nava Kahana,1 Corinne Chevallard,2 Victor Steinberg2

1Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel2Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, 76100, Israel

Keywords: conformational analysis; fluorescence; imaging; polyacrylamide; rheology

Introduction

Polyacrylamides and chemicallymodified polyacrylamides

undergo remarkable conformational changes or phase tran-

sitions in response to external stimuli such as surfactant,[1]

pH,[2] and salts.[1a,c] Polymer chain elongation, micro-

environmental effects and molecular behavior of linked

macromolecules are studied by various methods, such as

single-molecule force spectroscopy,[3] light scattering,[4]

fluorescence,[1b,5] viscosity,[1b,6] electron spin resonance in

conjunction with transmission electron microscopy,[7] and

microcalorimetry.[8] Hydrosoluble polymers show promise

in biological,[9] medical, and agricultural,[10] material[11]

Full Paper: The synthesis and characterization of ultrahigh-molecular-weight fluorescent partially hydrolyzed polyacry-lamide (HPAm) of the type P[Am*]x[Am]85[AA]15�x, whereAm is an acrylamide unit, Am* is a fluorescent-labeled Amunit, and AA is an acrylic acid unit, are reported. The dansylprobeN-(8-aminooctanyl)-5-dimethylamino-1-naphthalene-sulfonamide [DNSNH(CH2)8NH2], sensitive to conforma-tional changes of the polymer, was covalently bound to theHPAm. The fluorescence of the dansyl-labeled polymer andthe influence of various external stimuli, such as pH, NaCland metal ions, are described. The dansyl probe acts as ahydrophobic pendant on the chain of HPAm, and shows ablue-shifted emission in comparisonwith the unattached dye,suggesting that the dansyl probe may cause a coiling effecttoward a more compact structure of the polymer. Fluores-cence emission studies reveal significant compaction of thepolymer chain for pH in the range 4.7–9.8 and extension forpH below 3.7 or above 11.0. Increased fluorescence intensity,with respect to the fluorescence signal of the free dansylprobe in NaCl, is emitted by the dansyl-labeled polymer.Metal ions, such as Cd(II), Cu(II) and Co(II), modulate thefluorescence emission. Viscosity measurements yield furtherevidence of structural changes of HPAm, following theattachment of the dansyl probe. Fluorescence imaging ofP[Am*]x[Am]85[AA]15�x allows the observation of the dyna-mic behavior and the kinetics of conformational changes ofthe labeled polymer. Single polymer visualization is routinely

achieved on DNA molecules, however, to our knowledge,nobody has so far extended this technique to synthetic poly-mers. It is shown by fluorescencemicroscopy that the dansyl-labeled polymer is stretched by a circular shear flow.

Fluorescence spectra of the free dansyl probe with those of adansyl-labeled HPAm sample at pH 7, excitation at 345 nm.

Macromol. Chem. Phys. 2002, 203, 1833–1843 1833

Macromol. Chem. Phys. 2002, 203, No. 12 � WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2002 1022-1352/2002/1208–1833$17.50þ.50/0

a Current address: Department of Chemistry, Wayne StateUniversity, 5101 Cass Avenue, Detroit, MI, 48202-3489, USAFax: 313-5778822; E-mail: [email protected]

and environmental[12] applications. Some key reasons for

these interests are the low cost of acrylamidemonomers and

polyacrylamides, the relative ease of copolymerization of

acrylamide with other functional acrylamide monomers,

the versatility in applications and, above all else, a good

biocompatibility with DNA in vitro.

Among the different available polyacrylamides, partially

hydrolyzed polyacrylamide (HPAm) is gaining consider-

able importance and interest in fundamental and applied

research. Fundamental research conducted on the rheo-

logical behavior of HPAm solutions underlines the sen-

sitivity of these solutions to surfactant,[13] salinity,[14]

temperature,[14] and monovalent and divalent metal

cations.[14c,15] The light scattering,[16] and fluorescent

methods[17] were applied to the study of chain aggregation

and interpolymer complex formation. Besides, and very

importantly, the use of HPAm is well-known to enhance oil

recovery.[18] The carboxyl group (–COOH) complexing

properties are very effective in removing metal ions[19] and

phosphates[20] from dilute solutions and wastewater. As a

polyelectrolyte, HPAm is also applied as a coagulant in

treatment of solid-containing water,[21] and zinc and

chromium-containing wastewater.[22]

The aim of the present study was to synthesize a

fluorescent-labeled copolymer P[Am*]x[Am]85[AA]15�x

(where Am is an acrylamide unit; Am* is a fluorescent-

labeled Am* unit, and AA is an acrylic acid unit), directly

from HPAm in order to avoid the partial hydrolysis step,

usually required to label polyacrylamides, which results in

the polymer degradation by chain scission. Another aim

was to visualize and record the behavior pattern of the

polymer chain at a molecular level by fluorescence micro-

scopy. Such a visualization tool would certainly improve

the understanding of the dilute polymer solution hydro-

dynamics in complex flows, especially as regards dynamic

behavior and kinetics of conformational changes. To per-

form such a study, it is imperative that the fluorescent poly-

mer exhibits the same physical properties as the unlabeled

polymer HPAm, in particular the same flexibility. Another

condition is to obtain fluorescent ultrahigh-molecular-

weight P[Am*]x[Am]85[AA]15�x directly from high mole-

cular weight HPAm (Mw1.5� 107). The issuewas therefore

to synthesize a high molecular weight fluorescent poly-

acrylamidewithout changing significantly either themolec-

ular weight or other important physical properties of the

polymer, with the additional requisite to be able to tune the

fluorescence intensity.

The molecular weight of HPAm used in most of the

alternative synthetic methods reported in the literature lies

in the range104 to 106 Daltons.[23] The ultrahigh molecular

weight HPAm does not dissolve in most common organic

solvents. Therefore, the reaction of the fluorescent probe

with HPAm must take place in an aqueous or mixed

aqueous–organic solvent medium, and under mild condi-

tions to avoid the breakage of the polymer chain.Moreover,

to get clear indication that the fluorescent probe has been

successfully attached, one might take advantage of a probe

that is sensitive to polarity or microenvironmental changes

around the fluorophore.

To date, two probes, pyrene[24] and dansyl,[25] have been

used to study interactions in polyacrylamide gels,[25c,d]

that is, to evaluate chain–solvent interactions in network

polymers,[25e] and to probe conformational changes[25b] in

response to surfactant,[24a] pH[24b] or solvent variations.[25a]

Information on the pyrene pendant microenvironment can

be derived from the ratio of different vibronic band inten-

sities of the pyrene emissions, pyrene excimer emission, or

energy transfer from pyrene to naphthalene. Yet, amino-

pyrene is very sluggish in reacting with the –COOH group

of HPAm, in aqueous or in mixed aqueous solvent, due to

strong hydrophobic interactions. Therefore, dansyl amine

was chosen to perform the labeling of HPAm.

In this paper, we present the controlled synthesis of a

dansyl-labeled HPAm, which keeps an adjustable and

controllable number of CO2H groups (see Scheme 1). As a

result of the increasing numbers of dansyl probes on the

chain of the polymer, the maximal emission of the cova-

lently linked dansyl probe is blue-shifted with regard to

the free [DNSNH(CH2)8NH2], or to a mixture of free

[DNSNH(CH2)8NH2] and HPAm, indicating that the label

Scheme 1.

1834 Y. Wang, A. Warshawsky, C. Wang, N. Kahana, C. Chevallard, V. Steinberg

was successfully attached to the chain. Viscosity measure-

ments give further support to this contention.Moreover, the

dansyl-labeled P[Am*]x[Am]85[AA]15�x polymer shows

very interesting sensitivities to pH, NaCl and metal ion

variations. The polymer can be stretched under flow and

observed by fluorescence imaging.

Experimental Procedures

Materials

Dansyl chloride (98%), 1,8-diaminooctane (98%), Caproicacid, N,N 0-dicyclohexylcarbodiimide (DCC) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(EDC) were obtained from Aldrich. HPAm (15%, Mw

1.5� 107) was provided by Dr. M. Lerner.[26] Stock solutionsof 1 000 ppm HPAm were prepared by dissolving 1 g ofpolymer into 1 L of aqueous solution containing (a) 5% NaCland 250 ppmNaN3, and (b) 250 ppmNaN3. The stock solutionswere then left for several days to ensure complete dissolutionprior to reaction with DNSNH(CH2)8NH2. Other compoundsand solvents were analytical grade and were used withoutfurther purification. A solution of 10�2 mol �L�1 DNSNH-(CH2)8NH2 in ethanol was freshly prepared. Phosphate buffer(pH 5.6) of 66� 10�3

M was made from potassium dihydrogenphosphate and anhydrous disodium hydrogen phosphate.

Proton NMR spectra were measured on a Bruker DPX-250NMR spectrometer. All chemical shifts are reported in unitsdownfield from tetramethylsilane (TMS) as an internalstandard. The following abbreviations are used: s-singlet,d-doublet, t-triplet, q-quartet, and m-multiplet. UV-vis spectrawere measured on a Hewlett-Packard 8450 diode arrayspectrophotometer. Solution fluorescence was detected on aSLM 8100 spectrofluorometer. Thin layer chromatography(TLC) was carried out on E. Merck Kieselgel 60 F254 plates.The structure of all synthesized dyes was confirmed by NMRand their purity was checked by TLC.

Viscosity measurements of polymer samples were per-formed on a commercial rheometer (Rheolyst AR 1000-Nfrom TA Instruments). The labeled polymeric chains wereeventually visualized under flow in a homemade fluorescencemicroscope equipped with an oil-immersion objective (ZeissPlan-Apochromat 63x) and an intensified CCD camera(Videoscope ICCD-350F). See later discussion for the setup.

Synthesis

N-(8-aminooctanyl)-5-dimethylamino-1-naphthalenesulfonamide (DNSNH(CH2)8NH2)(Compound 1)

The spacer amino dansyl derivative DNSNH(CH2)8NH2 wassynthesized by a close adaptation of the literature proce-dure.[27] Dansyl chloride (2.757 g, 10 mmol) was partiallydissolved into 150 ml of CHCl3, then the turbid, yellowsolution was transferred into a 250 ml funnel. 1,8-diamino-octane (2.288 g, 20 mmol), triethylamine (1.4 ml, 10 mmol)andCHCl3 (70ml) were placed in a 500-ml three-necked flask,equipped with a magnetic stirrer, and an HCl gas absorption

inlet. The solution of dansyl chloride was dropped into themixture at such a rate that the reaction mixture always showeda light green fluorescent color for about 4 h, and was then leftovernight. The precipitate formed during reaction was isolatedby filtration and washed with chloroform. The chloroform,now containing the dye, and the remaining reacted solutionwere combined and then washed with saturated NaHCO3

(2� 150 ml), water (2� 150 ml), and dried overnight overNa2SO4. The solvent was evaporated using a rotavaporator,then under vacuum (1 mm Hg) to give a yellow, oily material.The crude product was further purified by flash chromato-graphy (Firstly using hexane/EtOAc (3:2), Secondly usingCHCl3:CH3OH:NH3 (25% aq.) (5:1:0.1)) to yield a yellow,oily product 1 (1.108 g, yield 29%) (Scheme 2).

TLC: Rf¼ 0.30 (CHCl3:CH3OH:NH3 (25% aq.)¼ 5:1:0.1),staining with ninhydrine.

1H NMR(CDCl3, TMS): d¼ 1.17 (br, m, 8H, –SO2NHCH2-CH2CH2CH2CH2CH2CH2–), 1.39 (m, 4H, –SO2NHCH2CH2-CH2CH2CH2CH2CH2CH2NH2), 2.2–2.4 (br, 2H, –CH2NH2),2.67 (t, 2H, –SO2NHCH2–), 2.96 (m, 8H, –N(CH3)2,–CH2NH2), 7.17 (d, 1H, arom, H6), 7.56 (q, 2H, arom, H7,H3), 8.25 (q, 2H, arom, H2, H8), 8.55 (d, 1H, arom, H4).

Model Reaction: Synthesis of N-(8-pentamido-n-octanyl)-5-dimethylamino-1-naphthalenesulfonamide(DNSNH(CH2)8NHCO(CH2)4CH3) (Compound 2)

DNSNH(CH2)8NH2 (80mg, 0.21mmol) and dichloromethane(5 ml, dried over CaCl2) were placed in a 25-ml round-bottomed flask, fitted with a magnetic stirrer and plunged intoan ice bath. Caproic acid (27 ml, 0.21 mmol) was added to therapidly stirred and cooled mixture. A solution of DCC (54 mg,0.26 mmol) in dichloromethane (4 ml) was dropped into themixture. After five minutes, the ice bath was removed. Thereaction was followed by TLC [CH3Cl:CH3OH:NH3 (25%aq.)¼ 5:1:0.1] until DNSNH(CH2)8NH2 disappeared. Theprecipitate formed was filtered out, and then washed withdichloromethane (2 ml) three times. The combined filtrateswere washed with 4% NaHCO3 solution (2� 5 ml), then oncewith saturated NaCl. The organic phase was dried overnightover Na2SO4. The solvent was removed under high vacuum(1 mm Hg). Further purification by flash chromatography wasneeded (hexane/EtOAc¼ 1:2.5) to afford 75 mg of product(Scheme 2).

Rf¼ 0.35 (hexane: EtOAc¼ 1:2.5).1H NMR (CDCl3, TMS): d¼ 0.91 (t, 3H, –CH3), 1.20–1.40

(br, m, 18H, –SO2NHCH2CH2CH2CH2CH2CH2CH2CH2-NHCOCH2CH2CH2CH2CH3), 2.15 (t, 2H, –NHCOCH2–),2.89 (m, 8H, –N(CH3)2, SO2NHCH2), 3.18 (m, 2H, –CH2-NHCO–), 4.54 (t, 1H, –NHCO–), 5.37 (br, s, –SO2NH–), 7.20

Scheme 2.

Fluorescent Ultrahigh-Molecular-Weight Polyacrylamide . . . 1835

(d, 1H, arom, H6), 7.53 (q, 2H, arom, H7, H3), 8.26 (q, 2H,arom, H2, H8), 8.55 (d, 1H, arom, H4).To simulate the reaction of DNSNH(CH2)8NH2 with HPAm

in dimethyl sulfoxide (DMSO)/water solution, a modifiedversion of a procedure routinely used in bioconjugatedprotein chemistry[28] was applied. The model compoundDNSNH(CH2)8NHCO(CH2)4CH3 was prepared by reactionof DNSNH(CH2)8NH2 with caproic acid in the presence ofEDC. DNSNH(CH2)8NH2 (24.6 mg, 0.065 mmol) wasdissolved into 0.5 ml of DMSO and rotated very slowly on arotavaporator. The mixture of EDC (74.5 mg, 0.38 mmol) andcaproic acid (6.3 ml, 0.05 mmol) in 5.0 ml of pH 5.6 phosphatebuffer was added to the mixture, and then left for 5 h underrotation in the dark. After removal of the water under highvacuum, a yellow, oily material was obtained. The residue wasdissolved in CHCl3 (5 ml). The white precipitate was filteredout and the colored product in CHCl3 was washed with 5%NaHCO3 (3� 2 ml), then water (2� 2 ml). The organic phasewas dried over Na2SO4 to yield 26 mg of the crude product.The pure compound 2 (10 mg) was received by flashchromatography [hexane/EtOAc¼ 1:2.5]. The structure wasconfirmed by NMR.

Synthesis of P[Am*]x[Am]85[AA]15�x

Aseries of products of P[Am*]x[Am]85[AA]15�xwith differentnumbers of dansyl probes were prepared. The products aredescribed in Table 1. The typical procedure is given below: Thedesired amount of EDC (10 times in excess with respect to themolar quantity of CO2H group) was added to a solution of1 000 ppmHPAm (20 ml) containing 250 ppm NaN3, and thenslowly rotated (0.5 rpm; slow rotation is needed to preventchain degradation of HPAm) for half an hour. Later on, theDNSNH(CH2)8NH2-containing DMSO solution was added tothe polymer solution by aliquots of 3 ml. The dye concentra-tion in the DMSO solution ranges from 2� 10�4 to 3.58�10�3 mol � l�1. After each addition, the sample was kept underrotation for 20 minutes. This two-step procedure was repeateduntil the total 20 ml of the solution has been added. Aftercompletion of the addition, themixturewas allowed to rotate inthe dark for one week.

Purification of Dansyl-Labeled P[Am*]x[Am]85[AA]15�x

Size-exclusion chromatography (SEC) was used to test theneed for purification. At first, we tested the elution of thepolymer itself (HPAm) by use of Sephadex150 in a 15 mm�

1.5 mm column. The polymer HPAm was eluted by water andfractions of 1 ml were collected. The 25 fractions wereanalyzed by UV spectrophotometry. The results showed thatHPAm comes out in the first fractions, as indicated by the UVspectrum that contains two peaks at 231 and 322 nm. Next, theseparation of the polymer HPAm/dye mixture was carried outin the same mode. That is, HPAm (2 ml, 231 ppm), togetherwith 1� 10�5 mol � l�1 DNSNH(CH2)8NH2, was separated bythe procedure presented above. The pure polymer HPAm cameout after one or two fractions, as detected by UV measure-ments, with a total lack of fluorescence for these fractions.After 10 fractions, the free DNSNH(CH2)8NH2 was found toshow an emission maximum around 554 nm. These resultsshow that SEC is the way to separate HPAm from the free orunreacted DNSNH(CH2)8NH2.

Prior to the isolation of the labeled product P[Am*]x-[Am]85[AA]15�x by SEC, dialysis against pH 4 is essential toremove DMSO, EDC and possible unreacted dye. A 2-mlpolymer solution (Table 1, sample 4) was loaded directly on aSephadex150 in a 15� 1.5 mm column. Dansyl-labeledP[Am*]x[Am]85[AA]15�x polymer was eluted by water in thefirst fractions, and characterized by fluorescence measure-ments (maximum emission: 520 nm) and TLC using CH3Cl/CH3OH (9:1). No unreacted DNSNH(CH2)8NH2 was detectedon the TLC plate. Applying the same tests (fluorescence andTLC) to the remaining fractions, no free dye was found at all.The achievement of a test experiment aiming to separate amixture of P[Am*]x[Am]85[AA]15�x and 1� 10�5 mol � l�1

free DNSNH(CH2)8NH2 further confirmed that unreactedDNSNH(CH2)8NH2 can be separated from P[Am*]x[Am]85-[AA]15�x. This leads to the conclusion that the dansyl-labeledP[Am*]x[Am]85[AA]15�x sample does not contain any freedye after dialysis. To prevent bacterial growth, 250 ppm NaN3

was added to the dansyl-labeled P[Am*]x[Am]85[AA]15�x

solution along with 5% NaCl.

Analysis of the Product of P[Am*]x[Am]85[AA]15�x

After dialysis, the actual concentration of the polymerP[Am*]x[Am]85[AA]15�x was determined by accurate weigh-ing after lyophilization. More precisely, 10 ml of the dansyl-labeled polymer stock solution was dried for 4d by lyophi-lization. The white residue was then accurately weighed. Theconcentration of polymer in the original solution was calcu-lated and expressed in ppm. The number of dansyl groupsattached to the polymer chain was later on determined fol-lowing a method known from the literature.[29] Standard

Table 1. Optimization the synthesis of P[Am*]x[Am]85[AA]15�x in cosolvent.

Sample no. Mole ratio of –COOH/dansylgroup in the reaction mixture

Polymer concentrationafter dialysis ppm

Number of dansylprobes per chain

Solution status

1 1:0.095 61.4 777 clear2a) 1:0.20 45.1 17 394 clear3 1:0.44 88.5 18 881 clear4 1:0.66 66.4 19 182 clear5 1:1 turbid6 1:2 turbid

a) The reaction time was 4 d.


solutions of DNSNH(CH2)8NH2 and dansyl-labeled polymersolutions were prepared in pH 5.6 buffer. Standard curvesgiving concentration versus absorption at 320 nm for DNSNH-(CH2)8NH2 and at 340 nm for dansyl-labeled P[Am*]x[Am]85-[AA]15�x were plotted. The ratio of the slopes from the twocurves was used to calculate the number of dansyl groupsattached to the polymer. Such a method is valid as long asthe concentration of the dansyl-labeled polymer used to get thestandard curves is lower than 35 ppm. Actually, although theabsorption band of the dansyl probe and HPAm partiallyoverlap, the absorption of a HPAm solution at 340 nm remainsnegligible up to 35 ppm. The absorption intensity at 340 nm istherefore assumed to originate only from the unattached dansylprobe. The calculated results are summarized in Table 1.

Results and Discussion

Optimization of the Synthesis of Dansyl-LabeledHPAm (P[Am*]x[Am]85[AA]15�x) in MixedAqueous Solvent

Initial attempts were devoted to exploring the reactivity of

an alkyl carboxylic acid (caproic acid) as an analogue of

HPAm. DNSNH(CH2)8NH2 was allowed to react with

caproic acid using DCC or EDC as a coupling agent to yield

the compound 2 (Scheme 2). 1H NMR of compound 2

showed that the reaction proceeds cleanly and that the same

synthetic strategy can be applied to couple HPAm to

DNSNH(CH2)8NH2. However, it is crucial to find the right

conditions to perform the coupling reaction between the

organic soluble DNSNH(CH2)8NH2 with the large, water

soluble, conformationally sensitiveHPAmmacromolecule.

Since DNSNH(CH2)8NH2 is incompatible with aqueous

systems of pH higher than 3, a water-miscible cosolvent,

which is able to dissolve DNSNH(CH2)8NH2, without

inducing the precipitation of HPAm upon the addition of

dye into the polymer solution, must be employed. Common

solvents, such as dimethylformamide, DMSO and tetra-

hydrofuran cannot solubilize HPAm at all, but a DMSO and

water mixture is an excellent solvent for dansyl-labeled

P[Am*]x[Am]85[AA]15�x. Optimizing the DMSO/water

ratio is essential to prevent precipitation during the addition

of the DNSNH(CH2)8NH2 into the polymer solution.

Several attempts with different DMSO/H2O ratios even-

tually showed that a 1:1 ratio allows the execution of a

panel of experiments in which varying amounts of

DNSNH(CH2)8NH2 are blended with a fixed amount of

HPAm. In the labeled product P[Am*]x[Am]85[AA]15�x,

multiple covalently bound dansyl groups, distributed along

the polymer, can cause interpolymeric entanglements. To

avoid turbid solutions, it is important to optimize the

number of dansyl molecules on the HPAm chain. Table 1

summarizes the experimental conditions and results. The

following ratio valueswere chosen for themolar quantity of

carboxylic groups, present on polymers backbone, to the

molar quantity of dye molecules in solution, 1:0.2, 1:0.44,

thus specifying the concentration in DNSNH(CH2)8NH2

for each solution. Themolar ratio of 1:0.66 is clearly a limit

above which serious interpolymer entanglement may be

encountered.

Photophysical Properties of P[Am*]x[Am]85[AA]15�x

Free DNSNH(CH2)8NH2 Behaviorin Various Solvents

The maximum emission of the dansyl probe and its deri-

vatives has been amatter of dispute for years.[30] In Table 2,

we summarize the lowest energy absorption, maximum

fluorescence emission and relative fluorescence intensity

of 1� 10�5 mol � l�1 DNSNH(CH2)8NH2 in 12 solvents.

The lowest absorption band is red-shifted for all solvents in

comparison with DNSNH(CH2)8NH2 in water, but the ab-

solute value remains constant among these solvents. On the

contrary, the maximum of emission varies with the solvent.

Results shown in Table 2 agree with reports which claim

that the dansyl probe shows an increased emission intensity

and a maximum emission blue shift when going from

water to less polar media.[30] Such spectral variations are

employed in studies on conformational transitions in

protein[31] and synthetic polymers.[25] We can therefore

anticipate that DNSNH(CH2)8NH2 could be used as a

sensitive probe to detect the existence of interpolymer and/

or intrapolymer interactions in aqueous solutions, since the

relative fluorescence intensity would be an indication of the

extent of interpolymer interaction in an aqueous medium.

Dansyl Probe DNSNH(CH2)8NH2 in Free andAttached States

To provide some comparison, the behavior of the dansyl

probes DNSNH(CH2)8NH2 was studied in the free and

Table 2. Selected photophysical properties ofDNSNH(CH2)8NH2 in a series of solvents.

Solvent labsa) lmax

b) RFIc)

nm nm

water 329 549 7 171dimethyl sulfoxide 339 555 41 498methanol 339 503 44 444dimethylformamide 340 503 61 424ethanol 338 500 43 448acetone 345 500 10 411dichloromethane 346 500 25 651tetrahydrofuran 339 493 93 597dioxane 333 483 116 884benzene 341 481 68 916diethyl ether 338 471 121 398hexane – 455 7 127

a) labs¼The lowest energy absorption band.b) lmax¼The maximum fluorescence emission.c) RFI¼Relative fluorescence intensity.


attached modes by fluorescence spectroscopy. As shown in

Figure 1, free DNSNH(CH2)8NH2 shows a maximum

emissionat554nm,while labeledP[Am*]x[Am]85[AA]15�x

emission is blue-shifted. This blue shift is proportional to

the extent of dansylation of HPAm. With up to several

hundreds of dansyl probes attached to the polymer chain,

the fluorescence peak is shifted from554 to 536 nm. Further

increase in the number of dansyl probesmakes the emission

peak shift up to 526 nm. This band originates from the

twisted intramolecular charge transfer state,[30] and is

highly sensitive to the polarity of the medium. Therefore,

this shift is the direct expression of a change in the dansyl

probe microenvironment. It is possible that increasing the

number of dansyl groups along the polymer chain enhances

hydrophobic interactions between the dansyl probes and the

polymer chain, and/or the interaction among dansyl probes,

bringing about a shift to shorter wavelengths.

The Influence of pH on Photophysical Propertiesof P[Am*]x[Am]85[AA]15�x

First,we review the results obtained forDNSNH(CH2)8NH2

at different pH values. The emission wavelength, lmax, and

the corresponding fluorescence intensity are almost con-

stant, irrespective of the pH (see Figure 2). In contrast to

that, the lmax of dansyl-labeled HPAm depends on the pH.

Following the decrease of pH from 3.7 to 1.9, lmax shifts to

higher wavelengths, indicating an increase in the polarity of

the medium due to the protonation of the (CH3)2N– group

of the dansyl probe. Over the pH range 3.7–9.8, lmax shifts

to a shorter and constant wavelength and a slight increase

in the fluorescence intensity is noticed (Figure 2b). This

suggests that the dansyl probe stands in a hydrophobic

microenvironment since it is known that, for the dansyl

probe, such an environment results in a maximum of

emission at shorter wavelength and in an increase of the

fluorescence intensity compared to a hydrophilic environ-

ment. Above pH 9.8, lmax shifts to higher wavelength and

the fluorescence intensity decreases gradually. This can be

attributed to the increase of the polymer charge (–COO�),

which causes chain expansion due to electrostatic repulsion.

The Effect of NaCl on Photophysical Propertiesof P[Am*]x[Am]85[AA]15�x

The addition of salt, such as NaCl, to dilute HPAm solu-

tions, is known to significantly change their behavior under

flow.Consequently,we decided to study the influence of 5%

NaCl on the fluorescence of the dansyl probe and dansyl-

labeled HPAm (Figure 3). The DNSNH(CH2)8NH2 emis-

sion is quenched by an aqueous NaCl solution whereas an

increased fluorescence is emitted by the dansyl-labeled

HPAm. This phenomenonmay be explained by considering

Figure 1. Fluorescence spectra of the free dansyl probe withthose of a dansyl-labeled HPAm sample at pH 7.

Figure 2. (a) pH dependence of the fluorescence peak wave-length for 5� 10�5 mol �L�1 free dansyl probe and 33.2 ppmdansyl-labeled HPAm (19 182 dansyl probes per chain). (b) pHdependence of the relative intensity at the maximum emission. Alloriginal samples are in pH 7.


the influence of the ionic strength on the polymer con-

formation. Polyacrylamides can dissociate in solution to

form polyvalent macroions that are able to produce a strong

electric field to attract the counterions.We therefore assume

that the dansyl-labeled HPAm will hold the same proper-

ties. This means that the interaction of macroion with

counterion will lead to significant effect on the properties of

polyelectrolytes.

The viscosity data for P[Am*]x[Am]85[AA]15�x shows

a significant decrease upon the addition of NaCl (see

Figure 6a). This addition effectively reduces the repulsive

forces between similar charges on the polymer chain,

causing a shielding effect of the counterions around the

anionic sites of the P[Am*]x[Am]85[AA]15�x chain, and

consequently reducing the size of the polymer coil. The

sample viscosity is therefore lowered. The reduced size of

the polymer coil brings the dansyl pendants closer to each

other so that the dansyl probe locates in amore hydrophobic

microenvironment. This in turn leads to an increase of the

quantum yield. Such an increase of the fluorescence

intensity in the presence of NaCl is essential to achieve

fluorescence imaging on polymer molecules in flow.

Effect of Metal Ions on Fluorescence ofDansyl-Labeled HPAm

The use of ‘‘polychelatogens’’, water-soluble polymers, in

separation science is an active area of interest.[12] The

–COOH and –CONH2 functional groups in HPAm have

complexing propertieswithmetal ions.[16] Thus, the dansyl-

labeled HPAm will hold both photoluminescence and

metal-complexing properties. The fluorescence of P[Am*]x-

[Am]85[AA]15�x couldbe sensitive tometal-inducedfluores-

cence changes. Figure 4 shows that the addition of cobalt

and copper quenches the fluorescence, while cadmium

causes an increase in fluorescence. The mechanism of

quenchingmay consist of a dansyl-to-metal energy-transfer

or a metal-to-dansyl electron-transfer mechanism due to

complexation, as claimed in the literature.[32] A dynamic

quenching is also possible. The increase of dansyl fluores-

cence by cadmiumhas been explained in the literature.[32] It

is claimed that the deprotonation of sulfamide complexes

induces a higher electronic density on the naphthalene

ring of the dansyl groups. Since the concentration of

P[Am*]x[Am]85[AA]15�x is quite low (less than 100 ppm)

after dialysis, quantitative fluorescence analysis of metal

ions by P[Am*]x[Am]85[AA]15�x has not yet been achiev-

ed. However, we believe that our results open an oppor-

tunity to use the material as a metal collector and an

indicator in environmental science.

Viscosity Measurements

Rheological measurements provide an effective means

of probing changes in polymer physical properties. In

Figure 3. (a) Influence of 5%NaCl on the fluorescence of dansylprobe; (b) influence of 5% NaCl on the fluorescence of 45.1 ppmdansyl-labeled HPAm sample (17 394 dansyl probes per chain).

Figure 4. Perturbation of the fluorescence emission of 22.2 ppmdansyl-labeled HPAm (19 182 dansyl probes per chain) by6.60� 10�5 mol � l�1 metal ions.


particular, if the fluorescent labeling does alter the

conformation of the polymeric chain, one expects a

concomitant change in the sample viscosity. The viscosity

of a 45.1 ppm HPAm solution was therefore compared to

the one of a dansyl-labeled HPAm solution at the same

concentration. The measurements were performed using a

cone-and-plate geometry. The rotation of the upper cone

induces a circular shear-flow in the fluid that fills the space

between the two parts of the geometry. For a small angle of

the cone (a¼ 18), this geometry ensures a constant shear-

rate throughout the sample. The steady-shear viscosity,

Z of the sample is then simply defined as the coefficient

that relates the shear stress s to the shear rate dg/dt in the

steady-shear flow:[33] s¼ Z(dg/dt) dg/dt. Results are shownin Figure 5.

The difference between the two curves s(dg/dt) clearlyindicates a change in the rheological behavior of the

polymeric solution following the labeling procedure. The

unlabeled sample shows a constant viscosity ZUL¼1.22 mPa � s over the range 2 to 100 s�1 of shear rates.

After labeling of the HPAm solution, the viscosity becomes

shear-rate dependent. It strongly decreases as the shear rate

increases, before it reaches a plateau at high shear rate

values. The viscosity at the plateau is ZL¼ 1.46 mPa � s,which is about 20% higher than ZUL.The enhancement of the low-shear viscosity and the in-

crease in the degree of shear thinning in concentrated poly-

mer solutions by adding hydrophobes has been reported.[34]

These studies were conducted with associative emulsion

polymers and with several types of hydrophobes. It was

found that the more complex the structure of the hydro-

phobes used, the larger degree of shear thinning was

observed. It was pointed out that the physical chemistry,

which provides a larger degree of shear thinning in

associative polymer solutions is still unknown, but at least

two different mechanisms have been identified: the rupture

of the network junctions under shear, and the non-affine

deformation of the network. The enhancement of the low

shear viscosity that was indeed observed in our experiments

on the dilute polymer solution of HPAm, is quite surprising

since one expects a more compact structure of the labeled

polymer with respect to the unlabeled one owing to the

hydrophobic interactions of the attached dansyl probes, and

therefore a smaller viscosity. Plotting the shear stress versus

the shear rate highlights this change in rheological behavior.

Moreover, after labeling, a finite stress (yield stress) is

needed tomake the sample flow. This characterizes a plastic

behavior, which can be described by the Herschel–Bulkley

model (see the insert in Figure 5). The latter feature was not

observed in the concentrated polymer solutions studied

previously.[34]

This change in behavior probably results from the for-

mation of complex polymeric superstructures due to the

strong hydrophobic interactions between the dyemolecules

present on the polymer chains. One expects this effect to

become weaker as the number of dansyl probes per chain

decreases, which is actually confirmed by the curve pre-

sented in Figure 6b. A modification of the pH, with the

related change in the fluorophore microenvironment, leads

to a small but visible decrease in viscosity (see Figure 6c).

As explained previously, the protonation of the (CH3)2N–

group of the dansyl probe is achieved at pH 2, and the strong

hydrophobic interactions between dyes are possibly com-

pensated by the electrostatic repulsion between charged dye

molecules. To check the existence of macromolecule com-

plexes,we stirred the unlabeled and dansyl-labeled polymer

solutions for 3 h with the aim of destroying the complexes.

The applied mechanical stirring was not sufficiently strong

to modify the unlabeled sample viscosity (see Figure 6d),

indicating therefore that chain breakage did not happen

during mechanical stirring. However, the labeled sample,

once mechanically stirred, exhibits a rheological behavior

that is closer to the unlabeled sample behavior than before

mechanical stirring. This certainly means that the mechan-

ical stirring was able to destroy, at least partially, the

associations of macromolecules responsible for a high

viscosity at low shear rates.

In conclusion, the chemical labeling of the HPAm chain

with dansyl significantlymodifies the physical properties of

the polymer. The conformation, as well as the susceptibility

under flow of the polymer is altered. Thus, the labeling not

only induces a quantitative change in viscosity but also

more deeply affects the rheological behavior of the sample.

Nevertheless, the concentration of dansyl-labeled HPAm

solution needed to study single-molecule dynamics by

fluorescence microscopy is very low, less than 2 ppm,

Figure 5. Viscosity measurements for a 45.1 ppm HPAmsolution with 5% NaCl, and 257 ppm NaN3: * before labeling;& after labeling. Insert: shear stress with respect to shear rate.Curves are fitted with the Herschel–Bulkley model s¼ syþK(dg/dt)n: * before labeling sy(yield stress) ¼ 3.6 � 10�7 Pa,K¼ 1.44 � 10�3(Pa � s)n, n¼ 0.9615; & after labeling sy¼1.19� 10�2 Pa, K¼ 2.82� 10�3(Pa � s)n, n¼ 0.8335.


compared with those used for viscosity measurements. One

can therefore expect that the influence of the hydrophobic

interactions between the bound dye molecules become

negligible at this concentration, and that the physical

properties of the labeled polymer can be identified to those

of the unlabeled polymer. The hydrophobic interactions can

also be weakened by a reduction of the number of dansyl

probes per chain. Lastly, we have seen that it is possible to

partially balance the hydrophobic interactions by electro-

static interactions invery acid conditions, or by using higher

concentrations of NaCl, and that mechanical stirring can

help to break the polymeric complexes. These considera-

tions give hope to further experiments on polymer dyna-

mics in complex flows.

Microscopy Observations

Direct observation of the labeled chains by fluorescence

microscopy is probably the most convincing way to check

the efficiency of the labeling protocol. We intend to use this

technique later on to carry out investigations on polymer

dynamics in complex flows. Single-polymer visualization

is routinely achieved on the DNA molecule,[35] but, to our

knowledge, nobody has so far extended this technique to

synthetic polymers.Weobtained images using a homemade

fluorescence microscope equipped with an intensified CCD

camera. The pictures were first recorded on a VCR and

then digitized on a computer. A schematic representation

of the set-up is given Figure 7. Figure 8 shows labeled

P[Am*]x[Am]85[AA]15�x polymers stretched by a circular

shear flow. The polymer sample is placed between two

parallel horizontal disks. At rest, the polymer chains adopt a

coiled state.[33] Using the microscope, one can then see

bright points on a black background (not shown). The

rotation of the upper plate generates a circular shear-flow in

the fluid. For a high enough speed of rotation, the velocity

gradients developed within the fluid are able to stretch the

polymer molecules. This occurs when the hydrodynamic

Figure 6. (a) Influence of 5% NaCl on polymer sample viscosity in the presence of 257 ppm NaN3. Samples 1 and 2: 45.1 ppm HPAmwith and without NaCl, respectively. Samples 3 and 4: 46.7 ppm dansyl-labeled HPAm (17 394 dansyl probes per chain) with and withoutNaCl, respectively. (b) Influence of the number of dansyl probes per chain on the 45.1 ppm sample rheological behavior in the presence of257 ppm NaN3 and 5% NaCl. Sample 1: HPAm. Sample 2: dansyl-labeled HPAm, 17 394 dansyl probes per chain. Samples 3 and 4:dansyl-labeled HPAm, after 1 day and 2 days reaction, respectively (the molar ratio of COOH/dansyl group in the reaction mixture is1:0.11). (c) Influence of the pHon the 45.1 ppm sample rheological behavior in the presence of 257 ppmNaN3 and5%NaCl. Samples 1 and2: HPAm at pH 7 and pH 2, respectively. Samples 3 and 4: dansyl-labeled HPAm, after 1 day reaction, at pH 7 and pH 2, respectively (themolar ratio of COOH/dansyl group in the reaction mixture is 1:0.11). (d) Influence of mechanical stirring on the sample rheologicalbehavior in the presence of 5% NaCl and 257 ppm NaN3. Samples 1 and 3: 46.7 ppm HPAm without stirring and with 3-h stirring,respectively. Samples 2 and 4: 46.7 ppm dansyl-labeled HPAm (17 394 dansyl probes on polymer chain) without stirring and with 3-hstirring, respectively.


force exerted on the molecule overcomes the entropic force

that tends to keep the polymer in a coiled state. The molec-

ular extension on Figure 8 is around 15 mm. The quality of

these images is actually limited by the use of a metal-halide

lamp, whose emission spectrum is rather poor in the UV

range, and could therefore be greatly improved, by the use

of a UV lamp. However, these images demonstrate the

efficiency of the chemical labeling and pave theway for the

realization of experiments on single-molecule dynamics

with synthetic polymers.

Conclusion

The dansyl probe, which is sensitive to environmental pro-

perties such as pH or solvent polarity, was linked to HPAm.

The synthetic method described in this work could be

transposed to any attachment procedure of a fluorescent

probe to a high molecular weight, water-soluble poly-

acrylamide. Spectroscopic behavior of the dansyl-labeled

polymer provides clues to the P[Am*]x[Am]85[AA]15�x

conformational state. Lastly, first observations of the poly-

mer under flow by fluorescence imaging were achieved,

thus paving the way to further study on the dynamical

behavior and the kinetics of conformational changes of the

polymer in dilute solutions.

Acknowledgement: We are grateful for financial support by theMinerva Center for Nonlinear Physics of Complex Systems, by aresearch grant from the Henry Gutwirth Fund and by an IsraelScience foundation grant. We acknowledge Dr. Zvi Ludmer for hisconstructive role in the formation of this project. We are alsograteful to Dr. M. Lerner who kindly provided us with an HPAmsample and helped in choosing the proper polymer. V. S. alsothanks Dr.M. Lerner for discussions of the polymer properties andmethods of their characterization.

Received: September 9, 2001Revised: January 2, 2002

Accepted: February 11, 2002

Figure 7. Experimental setup used for the visualization ofpolymer molecules under flow. The fluorescence microscope isan optical microscope to which fluorescence filters have beenadded in order to excite the fluorescent dye (emitter) and to isolatethe fluorescence signal (exciter). A sample of labeled HPAmsolution is introduced into a disk-disk system. The rotation of theupper disk induces a circular shear flow in the fluid.

Figure 8. Experimental photographs (processed) of HPAmmolecules labeled with the dansyl probe in a random flow at thesame rotation speed. The extension of the molecules evident in allthree images is due to the velocity gradients present in the circularshear-flow.


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