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FEATURE ARTICLER.VargheseandH.-A.WagenknechtDNAasasupramolecularframeworkforthehelicalarrangementsofchromophores:towardsphotoactiveDNA-basednanomaterials

ISSN1359-7345COMMUNICATIONAndreasHirsch et al.FractioningHiPcoandCoMoCATSWCNTsviadensitygradientultracentrifugationbytheaidofanovelperylenebisimidederivativesurfactant 1359-7345(2009)19;1-Y

www.rsc.org/chemcomm Number19|21May2009|Pages2593–2772

Chemical Communications

As featured in:

See Seiichi Uchiyama and Yumi Makino, Chem. Commun., 2009, 2646.

www.rsc.org/chemcommRegistered Charity Number 207890

Designed copolymers sharply change their fluorescence properties in a narrow pH range. That is, only one pH unit is enough for their fluorescence off–on switching. This unusual digital-type response is expected to be useful for a sensitive pH monitoring and a molecular computing which operates efficiently with a small variation in input.

Title: Digital fluorescent pH sensors

Showcasing research from Seiichi Uchiyama and Yumi Makino’s laboratory, based at The University of Tokyo, Japan.

FEATURE ARTICLER. Varghese and H.-A. WagenknechtDNA as a supramolecular frameworkfor the helical arrangements ofchromophores: towards photoactiveDNA-based nanomaterials

ISSN 1359-7345

COMMUNICATIONAndreas Hirsch et al.Fractioning HiPco and CoMoCATSWCNTs via density gradientultracentrifugation 1359-7345(2009)19;1-Y

www.rsc.org/chemcomm Number 19 | 21 May 2009 | Pages 2593–2768

Chemical Communications

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Digital fluorescent pH sensorsw

Seiichi Uchiyama* and Yumi Makino

Received (in Cambridge, UK) 15th January 2009, Accepted 24th February 2009

First published as an Advance Article on the web 10th March 2009

DOI: 10.1039/b900889f

We designed polymeric sensors that created a digital-type

fluorescence response to pH variation in an aqueous solution.

An important challenge in developing new chemical ion

sensors is to design them with the ability to generate a

digital-type output signal1 that can be used in binary switching

systems as highly sensitive ion indicators2 and in molecular

devices3 such as data storage tools.4 Until now, an inner filter

effect2 and a dynamic multichromophore array5 have been

utilized to sharply respond to ion concentration variation;

however, the inflexible requirements of these methods limit

their widespread use. In this communication, we demonstrate

digital-type fluorescent pH sensors based on the incorporation

of a water-sensitive fluorophore into a pH-responsive polymer.6,7

The fluorescence output of these polymeric pH sensors is

switched in a significantly narrow pH range (i.e., within almost

a unit). In addition, their functions, such as operating pH

range and switching direction (i.e., on–off and off–on actions),

can be easily tuned by structural modification of a proton

receptor. It is noteworthy that many digital signal transductions

occur in living organisms, in which the digital processors are

biological versions of macromolecules.8

Fig. 1a depicts the chemical structure of a digital fluorescent

pH sensor, 1 (Mn ¼ 44 600, Mw/Mn ¼ 2.31), which was

prepared by random copolymerization ofN-isopropylacrylamide

(NIPAM), N,N-dimethylaminopropylacrylamide (DMAPAM)

bearing an amino group as a proton receptor, and fluorescent

N-{2-[(7-N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazol-4-yl]-

(methyl)amino}ethyl-N-methylacrylamide (DBD-AA) (see ESIwfor experimental details). The use of a similar copolymer

as a pH sensor has already been reported by Onoda et al.9

Nevertheless, the present communication is the first to discuss

the digital sensory behavior of these copolymers. Fig. 1b and c

indicate the relationship between the fluorescence intensity of

1 in an aqueous solution and medium pH. As depicted in

Fig. 1b (filled circles), 1 in aqueous solution emitted 6.6-fold

stronger fluorescence under basic conditions (pH 4 9) than

under acidic conditions (pH o 8) at 50 1C. Under the latter

conditions, 1 assumes a hydrated open form due to the high

hydrophilicity of the protonated DMAPAM units, and solvent

water molecules approach the DBD-AA units7e to quench

their fluorescence. On the other hand, 1 exists in a dehydrated

globular form in a basic solution because of the hydrophobic

interactions between the NIPAM and unprotonated DMAPAM

units, where the DBD-AA units fluoresce at a location far

from the water molecules. It should be noted that the

fluorescence response of 1 disappeared when the solution

temperature was decreased to 20 1C (see open circles in

Fig. 1b). This temperature-specific function is a typical feature

of polymeric pH sensors.9,10 At the lower temperatures,

hydration by the solvent is a dominant interaction, even under

basic conditions, such that 1 never assumes the dehydrated

globular form.11

In comparison to the conventional 8-hydroxypyrene-1,3,6-

trisulfonate (HPTS) pH sensor12 (Fig. 2), the fluorescence

switching of 1 was completed in a narrower pH range (within

about one unit from pH 8 to 9). In order to quantify the

similarity of the fluorescent pH sensor output to a perfect

digital signal, the fluorescence signals were fitted to the

following proposed equation by assuming that the fluorescence

intensity of 1 was linear to the proportion of the protonated

DMAPAM units as the fluorescence intensity of HPTS is

linear to the proportion of the phenolate form:

�log[(FImax � FI)/(FI � FImin)] ¼ a(pH � pKa) (1)

where FI is the observed fluorescence intensity at a fixed

wavelength, FImax and FImin are the corresponding maximum

Fig. 1 (a) Chemical structure of 1. (b) Fluorescence responses of 1

(0.01 w/v%) to pH variation at 50 1C (K) and 20 1C (J) in a

Britton–Robinson buffer. Excitation: 450 nm. The fluorescence

quantum yield was 0.035 at 50 1C and pH 3. (c) Fluorescence spectra

of 1 at 50 1C. Conditions are the same as in (b).

Graduate School of Pharmaceutical Sciences, The University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.E-mail: seiichi@mol.f.u-tokyo.ac.jp; Fax: þ81 3 5841 4768;Tel: þ81 3 5841 4768w Electronic supplementary information (ESI) available: Experimentaldetails. See DOI: 10.1039/b900889f

2646 | Chem. Commun., 2009, 2646–2648 This journal is �c The Royal Society of Chemistry 2009

COMMUNICATION www.rsc.org/chemcomm | ChemComm

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and minimum, respectively, and a is the slope (equal to 1 for

common pH sensors according to the Henderson–Hasselbalch

equation13). By analyzing the experimental data, we observed

that compound 1 gave a ¼ 3.84 � 0.25 (pKa ¼ 8.68 � 0.09),

whereas HPTS gave a ¼ 1.01 � 0.02 (pKa ¼ 7.42 � 0.02)

(av � SD). This high a value for 1 clearly shows that it has a

digital-type output signal with a sharp edge. One of the

advantages of digital fluorescent ion sensors is that their

responses are sensitive to a relatively small variation in ion

concentration. In fact, 1 required only a pH variation of

0.7 units (5-fold change in proton concentration) for the

fluorescence output to change from 10 to 90% of the entire

response (cf., HPTS required 1.9 pH units, a 79-fold change in

[H1]). Such an efficient switching of a chemical sensor is

valuable, especially if the device is to be used for molecular

computation.1,3,14

The mechanism of this sharp response is related to a

decrease in the Lewis basicity of the receptor amine in the

DMAPAM units with a conformational change occurring in

the molecule of 1 from the open form at a low pH to the

globular form at a high pH. In general, the pKa value of the

protonated amine receptors decreases by more than one unit in

a hydrophobic environment due to the destabilization of the

protonated receptors by dielectric effects.15 In the case of 1, a

pKa value for the protonated DMAPAM units at the lower pH

(o8) should be nearly identical to that in water (pKa ¼ 9.316),

whereas the pKa value at the higher pH (49) can be estimated

to be less than 8.3. In the intermediate pH range between 8 and

9, the pKa value for the protonated DMAPAM units gradually

decreases from 9.3 to less than 8.3 as a function of increasing

pH because the structural change of 1 to the globular form

makes the local environments near the DMAPAM units

hydrophobic (cf., apparent pKa for 1 obtained from eqn (1)

was an intermediate value of 8.68, as discussed in the

former paragraph). Thus, the response curve of 1 in the

fluorescence intensity–pH diagram becomes steeper than that

of conventional pH sensors working with a pKa value that

remains unchanged as the pH changes.17

In order to confirm that the fluorescence response of 1

originated from the combination of the NIPAM, DMAPAM,

and DBD-AA units, the fluorescence properties of control

compounds (Fig. 3) were studied in a broad temperature range

between 20 and 70 1C. Neither DBD-IA7e (a model fluorophore

of the DBD-AA unit) nor the copolymer 2, consisting of only

DMAPAM and DBD-AA units (i.e., without NIPAM units)

(Mn ¼ 30 700, Mw/Mn ¼ 1.92), was sensitive to pH

variation (data not shown). Indeed, the insensitivity of 2

clearly demonstrates that the NIPAM units were required

for 1 to give the digital-type output signal. Most likely, the

NIPAM units of 1 maintained a hydrophobic–hydrophilic

balance that allowed for a conformational change between

the open and globular forms with pH variation. In other

words, the excess DMAPAM units in 2 made the copolymer

too hydrophilic to assume a globular form, even under basic

conditions. In this sense, replacement of the NIPAM units

by more hydrophobic units could decrease the functional

temperature of the copolymer from 50 1C.9

The functional pH range of our digital fluorescent pH

sensor can easily be tuned by modifying the chemical structure

of the ionizable units. For instance, when N-(3-morpholin-4-

ylpropyl)acrylamide (MPAM) units with a weaker proton

receptor (pKa for the conjugate acid of MPAM is 7.0 in

water16) were incorporated into the copolymer instead of the

DMAPAM units, the functional pH range of the resultant

copolymer 3 (Fig. 4, Mn ¼ 34 000, Mw/Mn ¼ 3.04) was shifted

to the acidic region (apparent pKa ¼ 6.16 � 0.03), and the

output signal remained digital (a for eqn (1) ¼ 2.54 � 0.05), as

indicated in Fig. 5a. In contrast, the use of a stronger receptor

unit, N-[3-(diethylamino)propyl]acrylamide (DEAPAM)

(pKa for the conjugate acid of DEAPAM is 10.3 in water16),

as in copolymer 4 (Fig. 4, Mn ¼ 33 800, Mw/Mn ¼ 2.25),

moved the functional pH to the basic region (apparent

pKa ¼ 9.12 � 0.05 and a ¼ 4.75 � 0.44), as displayed in

Fig. 5b. In addition, an acrylic acid (AA) unit (pKa of

isobutyric acid is 4.9 in water18) can also be used as an

ionizable unit. Then, the fluorescence response of copolymer

5 (Fig. 4,Mn ¼ 38 100,Mw/Mn ¼ 3.17), composed of NIPAM,

AA, and DBD-AA units, was reversed from that of 1; that is,

it emitted a stronger fluorescence under acidic conditions

(Fig. 5c, apparent pKa ¼ 5.17 � 0.13 and |a| ¼ 4.57 � 0.59).

This is because 5 assumes a globular form at a low pH wherein

a hydrophobic interaction is induced between NIPAM and the

unionized AA units.

In summary, we have developed digital fluorescent pH

sensors based on the copolymers of NIPAM, an ionizable

acrylic acid derivative (DMAPAM, MPAM, DEAPAM, or

AA), and DBD-AA. These pH sensors switch their output

signals in a narrow pH range (within almost one unit). In

addition to changing the functional pH ranges, the switching

direction can be changed by adopting different ionizable units.

Fig. 2 (a) Chemical structure of HPTS. (b) Fluorescence responses of

HPTS (1 mM) to pH variation in a Britton–Robinson buffer at 25 1C.

Excitation: 454 nm.

Fig. 3 Chemical structures of DBD-IA (N,2-dimethyl-N-(2-{methyl-

[7-(dimethylsulfamoyl)-2,1,3-benzoxadiazol-4-yl]amino}ethyl)propan-

amide) and 2.

This journal is �c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 2646–2648 | 2647

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The polymeric design of our new digital sensors also offers

advantages in modifying the fluorescence color7a,7f and attaching

its function to bulk materials.19 More examples and a further

study on the functional mechanism are needed to develop

these digital fluorescent pH sensors further.

We thank Ms. C. Gota and Ms. M. Onoda for valuable

comments and discussions. S.U. thanks the Foundation for

Promotion of Material Science and Technology of Japan

(MST Foundation) for financial support.

Notes and references

1 K. Szaci"owski, Chem. Rev., 2008, 108, 3481–3548.2 G. Gabor and D. R. Walt, Anal. Chem., 1991, 63, 793–796.3 V. Balzani, A. Credi and M. Venturi, Molecular Devicesand Machines, Wiley-VCH, Weinheim, 2nd edn, 2008.

4 M. Irie, T. Fukaminato, T. Sasaki, N. Tamai and T. Kawai,Nature, 2002, 420, 759–760.

5 J. A. Riddle, X. Jiang, J. Huffman and D. Lee, Angew. Chem., Int.Ed., 2007, 46, 7019–7022.

6 Some pH-responsive gels encounter a digital-type phase transitionwith changing environmental pH. See: (a) T. Tanaka, D. Fillmore,S.-T. Sun, I. Nishio, G. Swislow and A. Shah, Phys. Rev. Lett.,1980, 45, 1636–1639; (b) R. A. Siegel and B. A. Firestone, Macro-molecules, 1988, 21, 3254–3259.

7 The relevant combination of a water-sensitive fluorophore and athermo-responsive polymer has been applied to fluorescentthermometers showing digital-type output signals. See:(a) S. Uchiyama, Y. Matsumura, A. P. de Silva and K. Iwai, Anal.Chem., 2003, 75, 5926–5935; (b) S. Uchiyama, Y. Matsumura,A. P. de Silva and K. Iwai, Anal. Chem., 2004, 76, 1793–1798;(c) C. Gota, S. Uchiyama and T. Ohwada, Analyst, 2007, 132,121–126; (d) Y. Shiraishi, R. Miyamoto, X. Zhang and T. Hirai,Org. Lett., 2007, 9, 3921–3924; (e) C. Gota, S. Uchiyama,T. Yoshihara, S. Tobita and T. Ohwada, J. Phys. Chem. B, 2008,112, 2829–2836; (f) Y. Shiraishi, R. Miyamoto and T. Hirai,Langmuir, 2008, 24, 4273–4279.

8 T. Tian, A. Harding, K. Inder, S. Plowman, R. G. Parton andJ. F. Hancock, Nat. Cell Biol., 2007, 9, 905–914.

9 M. Onoda, S. Uchiyama and T. Ohwada, Macromolecules, 2007,40, 9651–9657.

10 (a) S. Beltran, J. P. Baker, H. H. Hooper, H. W. Blanch andJ. M. Prausnitz, Macromolecules, 1991, 24, 549–551; (b) G. Chenand A. S. Hoffman, Nature, 1995, 373, 49–52; (c) S. Uchiyama,N. Kawai, A. P. de Silva and K. Iwai, J. Am. Chem. Soc., 2004,126, 3032–3033.

11 X. Yin, A. S. Hoffman and P. S. Stayton, Biomacromolecules, 2006,7, 1381–1385.

12 O. S. Wolfbeis, E. Furlinger, H. Kroneis and H. Marsoner,Fresenius0 Z. Anal. Chem., 1983, 314, 119–124.

13 (a) L. J. Henderson, Am. J. Physiol., 1908, 21, 173–179;(b) K. A. Hasselbalch, Biochem. Z., 1917, 78, 112–144;(c) H. N. Po and N. M. Senozan, J. Chem. Educ., 2001, 78,1499–1503.

14 (a) F. M. Raymo, Adv. Mater., 2002, 14, 401–414; (b) U. Pischel,Angew. Chem., Int. Ed., 2007, 46, 4026–4040; (c) A. P. de Silva andS. Uchiyama, Nat. Nanotechnol., 2007, 2, 399–410.

15 (a) M. S. Fernandez and P. Fromherz, J. Phys. Chem., 1977, 81,1755–1761; (b) C. J. Drummond, F. Grieser and T. W. Healy,J. Chem. Soc., Faraday Trans. 1, 1989, 85, 521–535;(c) S. Uchiyama, K. Iwai and A. P. de Silva, Angew. Chem., Int.Ed., 2008, 47, 4667–4669.

16 P. G. Righetti, E. Gianazza, C. Gelfi, M. Chiari and P. K. Sinha,Anal. Chem., 1989, 61, 1602–1612.

17 A related pKa shift has been discussed on a drastic ionization ofnitrated poly(4-hydroxystyrene) with increasing pH. See:D. Westover, W. R. Seitz and B. K. Lavine, Microchem. J.,2003, 74, 121–129.

18 K. Kashiwagi, R. Sugise, T. Shimakawa, T. Matuura andM. Shirai, J. Mol. Catal. A: Chem., 2007, 264, 9–16.

19 A. P. Herrera, M. Rodrıguez, M. Torres-Lugo and C. Rinaldi,J. Mater. Chem., 2008, 18, 855–858.

Fig. 4 Chemical structures of 3–5.

Fig. 5 Fluorescence responses of (a) 3 at 50 1C, (b) 4 at 50 1C, and

(c) 5 at 45 1C to pH variation in a Britton–Robinson buffer.

Concentration: 0.01 w/v%. Excitation: 450 nm.

2648 | Chem. Commun., 2009, 2646–2648 This journal is �c The Royal Society of Chemistry 2009

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