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Acid-base sensitization of fluorescence by energy transfer in an ordered dye-lipid-electrolyte lamella
Volume
26. number
2
CHEMICAL
15 May 1974
PHYSICS LETTERS
ACID-BASE SENSITIZATION OF FLUORESCENCE BY ENERGY TRANSFER IN AN ORDERED DYE-LIPID-ELECTROLYTE LAMELLA Peter FROMHERZ Max-Planck-Institutjiir Biophysikalische Chemie (Karl-Friedrich-Bonhoeffer-Ittstittcrj,D-34Gijttingen. Germmz_v Received 21 December
Revised
manuscript
received
1973 25 February
1974
_ A pH sensitive coumarine dye is embedded in an electrically charged solid/electrolyte
interfmx. It sxves OSan en-
ergy donor for a cyanine
dye. The sensitizer, through proton transfer, loses this donor capacity. This trhnsformation affects the excitation of the acceptor. In the complete lamelIar assembly the agents affecting the sensitizer (UV light, acid-base, electrical charges and eiectrolytc) are coupled to the reaction of the exited acceptor (red fluorescence), which intrinskdly is not sensitive to these agents. The assembly is an esample how sn artiticial multi-molecular ~tructure may act as a new entity with chemical properties not present in the molecular constituents.
1. Introduction A dye S has the function of a donor in an energy transfer system. Through some agent it is transformable to a dye S’ which is not able to serve as a donor. Thus the agent affects the efficiency of the energy transfer and of all processes starting from the excited state of the acceptor A. Accordingly, by means of energy transfer, a property of a certain molecule (i.e., the agent sensitivity of S) may be transferred to another molecule (i.e.&, being intrinsically devoid of it. For example: a dye S- absorbs W and emits blue light. It is transformed by protons to a dye SH, which does not absorb W or/and does not emit blue photons. The fluorescence of a dye A, which is only capable of absorbing blue photons, becomes sensitive to photons if it is excited by S- via a photon transfer described by Fiirster’s formula of intermolecular energy transfer [l-3] (fig. la). This simple molecular device may be extended: the dyes SH/S-and A are located at a solid/electrolyte interface. The interfacial proton concentration is sensitive to the mterfacial electrical charge density and to the type tid concentration of the electrolyte [4]. Consequently, the transformation of S- to SH is sensitive to charges and electrolyte_ Finally the fluores.cence of A depends ou W,light, acid,.electrical charges and electrolyte, although intrinsically dye A is in-
sensitive to all these agents 151. The parts of this simple molecular circuit are illustrated in fig. 1b.
2. Method The molecular device shown in fig. 1 is realized in a monolayer assembly (fig. 2). A layer of cyanine dye molecules A (cf. fig. 3) is located at a distance of approximately 50 A from a layer of coumarine dye molecules SH/S- (cf. fig. 3) within methyl stearate monolayers. The chromophores of SH/S- are located in the plane of the head groups of electrically charged lipids (eicosyl trimethylammonium or eicosyl sulphate ions)_ This plane is in contact with a 10 mM sodiumchloride solution. When excited at 366 nm the dye S- emits a blue fluorescence (fig. 3), whereas dye SH exhibits no fluorescence .[6]. Dye A absorbs blue but no W photons and emits a red fluorescence [7] (fig. 3). Details of the experimental procedure may be found in ref. ISI-
3. Results Fig. 3 shows .the fluorescence spectrum of ihe rn.& noJayer assembly.ciepicted iu fig. 2 at a high pH (corn-,.
Volume
26, number
CHEMICAL PHYSICS LETTERS
2
15 May 1974
SH
UV
tight red
add/ base
fluorescence
b) electrical charges electrolyte Fig. 1. (a) Reaction graph of acid-base sensitizationof fluorescence. (b) Block diagram of acid-base sensitizationin an electrically charged interface.
plete dissociation of SH/S-),
with and without the layer of the acceptor A. The quenching of the S- fluorescence by A is apparent. At two different charge densities at the interface
the bulk pH is lowered step by step. The fluorescence spectra of A (obtained by subtracting the fluorescence component of S- from the measured spectra) at various pH values are depicted in fig. 4. The fluorescence of dye A is seen to be pH dependent and this pH sensitivity depends on the interfacial charge density.
1
Ik
biass
chromophore A ethylstearate chromophore SHISfixed charges diffuse double layer bulk
water
Fig. 2. Schematic drawingof the dye-lipid-electrolyte lamella e_tiiiting acid-basejensitized fluorescence. The lipid layer consists of three methyl stearate layers: the first (pure) one on the hydrophobic glasssupport, the second one mixed with the cyanine dye A(600 .&*/dyemolecule), the third one mixed with the coumarine dye SH(600 Ndye molecule) and additio.nal positivelyor negativelycharged lipid molecules (eicosyl trim~thylammonium.eicosyl sulphke). The hydrophilic groups .-of the third monolayer are in direct contact with a.10 mM NaCl~olution. :
In fig. 5 the sensitized titration of the A fluorescence is compared to the direct titration of the SH/Sfluorescence. The apparent pK values of SH/S- (pK (pox) = 6.2, pK (neg.) = 10) coincide precisely with the ‘pK’ values of the intrinsically pH insensitive dye A. (The fluorescence of A does not disappear completely at low pH due to little direct excitation of A by the exciting light (366 nm) bypassing the sensitizer s-.)
4. Discussion The ratio Id>+, of the S- fluorescence intensities at 450 nm with and without acceptor layer at the distance d is seen from fig. 3 fo ,be 0.4: By inserting this value and d = 52 A into the distance relation of ener-
Volume 26, number 2
CHE,MICAL PHYSICS LETPERS wavenumber
LSO
[cm -11
600
550
500
wavelength
15 May 1974
I nml
Fig. 3. Fluorescence and absorbance spectra of coumarine dye S- and cyanine dye A. 0 is the fluorescence spectrum of a lamellar system as shown in fig. 2 with: 600 AZ per dye S- and dye A molecule, respectively, 140 A2 per positive charge, 10 mM NaCl solution, pH 11, excitation at 366 nm. 0 is the fluorescence spectrum of a similar system without acceptor dye. 0 is the absorbance spectrum of a monolayer of cyardne dye A (600 AZ per dye molecule) measured in a dry monolayer assembly 171. The fluorescence spectra are corrected by the quinine-sulphate method [9] _cdl/d; = fluorescence intensity per wavenumber interval.)
wavenumber
[cm-r]
wavenumber
[cm-r]
wavelength [nml
wavelength [nml
al
b)
Fig. 4. Fhrorescence spectra of cyanine dye A (cf. fig. 3) (excitation at 366 nm) in the lamellar structure shown in f%. 2 at different pH values of the bulk aqueousphase (IO mM Nacl): (a) positively charged interface (140 R2 per charge), (b) negatively charged interface (200 A2 per charge). (dI/da = fluorescence intensity per wavenumber interval_) The numbers in the figures denote the bulk pH vahws.
L
5
6
7
0
9
10
11
12
bulk pH Fig. 5. Relative fluorescence intensity of cyanine dye A (cf. fig. 3) at 570 nm (circles) and of coumarine dye SH/S- (cf. fig. 3) at 450 ti (crosses) in the lam~llar system shown in tig. 2 (excitation at 366 rrm) versus the pH of the bulk ague-: ous phase (10 mM NaCl): @ positively charged interface (140 A2 per charge), @negatively
charged interface (200 AZ
per bharge). .. 223. :
.
Volume 26, number 2
CHEMICAL PHYSICS LE’ITERS
gy transfer for the geometry considered Id/IO = r1+(~&041-1 El01 an experimental characteristic distance do = 57 a is’obtained. A theoretical value of do is calculated by the formula ndo = CKC$‘~J”~ [IO]. Assuming an orientation factor Q = 0.1 I [lo] and a quantum yield of the S- fluorescence (I = 1) using the spectral data of fig. 3 to calculate J= JdP (P)-4f(P) A (P) (0 = wavenumber,f(G) = normalized fluorescence spectrum of S-,A@) = absorbance spectrum of A) and inserting the refractive index II = 1.52 [ 111 one obtaines a theoretical characteristic distance do = 63 A. The good agreement of the experimental and theoretical do values indicates that the interaction of Sand A, as indicated by the quenching of the S- fluorescence, is due to photon transfer as described by FBrster’s formula. The agreement of the titration characteristics of the fluorescence of A and S-(fig. 5) proves that A is excited by S-. The fluorescence of A observed with excitation by an external light source is not pH dependent under the conditions of the experiments described. The results prove that the acid-base sensitization of fluorescence, i.e., the molecular circuit depicted in fig. I is realized in the IameIIar structure depicted in fig. 2 *. The proton signal determines the position of the ‘switch’ SH/S- . The actual position is ‘read’ by the UV light. This information is transferred on the moiecuiar Ievel by the blue. photons to A, where it controls the red fluorescence. The threshold at which an external acid-base signal triggers the switch is adjustable by the charge density and the eIectrolyte. The
* For the syntheses of a function as shown in tip. lb it is not essential to synthesize a structure as shown in tig. 2 with molecular precision. Deviations from the ideal lamellar structure do not change the features of the function significantly as long as the essential molecular interactions, causing the non-additivity of the properties of the molecules involved, are maintained.
224
15 hlay 1974
integrated system possesses properties ( the coupling of the input UV light, acid-base, charge, electrolyte and the output red fluorescence), which are not inherent to the individual molecules involved. These properties are produced by the cooperation of different kinds of molecules in an ordered arrangement (cf. ref. [ 111). They are understood completely by the knowledge of the intrinsic properties of the molecules and their interactions in the ordered arrangement. The concept of acid-base sensitization by energy transfer may be generalized. Other kinds of chemical substances or physical agents (as temperature, light, electrical fieldsj are feasible as sensitizing agents. Besides fluorescence all kinds of photoreactions, e.g., photoprocesses in semiconductors, may be sensitized to some chemical or physical agent. Finally, the device of fig. lb may be extended: the acid may be produced in an ester hydrolysis by an interfacial enzyme. In this case the acid-base sensitization may act as a link between two stoichiometrically independent reactions, a hydrolysis and a photoreaction [5] _
References [l] Th. FGrster, Ann. Physik. 6 F., 2 (1948) 55. [2] L. Gomberoff and E.A. Power, Proc. Phys. Sot. (London) 88 (1966) 281. [3] P. Fromherz, Ber. Bunsenges. Physik. Chem. 77 (1973) 1019. [4] JJ. Davies and E.K. Rideal, Interfacial phenomena (Academic Press, New York, 1963) p_ 94. [5 J P. Fromherz, Chimia 27 (1973) 659. 16) A. Diener, C.V. Shank and A.M. Trozzolo, Appl. Phys. Letters 17 (1970) 189. [7] D. Miibius, 2. Naturforsch. 24a (1969) 251. [S] P. Fromherz, Biochim. Biophys. Acta 323 (1973) 326. [9] A. Schmiien and R. Legler, in: Landolt-BSrnstein, Gruppe II, Bd. 3, eds. K.H. Hellwege and A.M., Hellwege (Springer, Berlin, 1967) pp.228 ff. [ 10) H. Kuhn, J. Chem. Phys. 53 (1970) 101. [l l] H. Kuhn, D. Miibius and H. Biicher, in: Technique-s of chemistry, Vol. 1, part IIIB. eds. A. Weissberger and B.W. Rossiter Wiley, New York, 1972) pp. 577-702.
26. number
2
CHEMICAL
15 May 1974
PHYSICS LETTERS
ACID-BASE SENSITIZATION OF FLUORESCENCE BY ENERGY TRANSFER IN AN ORDERED DYE-LIPID-ELECTROLYTE LAMELLA Peter FROMHERZ Max-Planck-Institutjiir Biophysikalische Chemie (Karl-Friedrich-Bonhoeffer-Ittstittcrj,D-34Gijttingen. Germmz_v Received 21 December
Revised
manuscript
received
1973 25 February
1974
_ A pH sensitive coumarine dye is embedded in an electrically charged solid/electrolyte
interfmx. It sxves OSan en-
ergy donor for a cyanine
dye. The sensitizer, through proton transfer, loses this donor capacity. This trhnsformation affects the excitation of the acceptor. In the complete lamelIar assembly the agents affecting the sensitizer (UV light, acid-base, electrical charges and eiectrolytc) are coupled to the reaction of the exited acceptor (red fluorescence), which intrinskdly is not sensitive to these agents. The assembly is an esample how sn artiticial multi-molecular ~tructure may act as a new entity with chemical properties not present in the molecular constituents.
1. Introduction A dye S has the function of a donor in an energy transfer system. Through some agent it is transformable to a dye S’ which is not able to serve as a donor. Thus the agent affects the efficiency of the energy transfer and of all processes starting from the excited state of the acceptor A. Accordingly, by means of energy transfer, a property of a certain molecule (i.e., the agent sensitivity of S) may be transferred to another molecule (i.e.&, being intrinsically devoid of it. For example: a dye S- absorbs W and emits blue light. It is transformed by protons to a dye SH, which does not absorb W or/and does not emit blue photons. The fluorescence of a dye A, which is only capable of absorbing blue photons, becomes sensitive to photons if it is excited by S- via a photon transfer described by Fiirster’s formula of intermolecular energy transfer [l-3] (fig. la). This simple molecular device may be extended: the dyes SH/S-and A are located at a solid/electrolyte interface. The interfacial proton concentration is sensitive to the mterfacial electrical charge density and to the type tid concentration of the electrolyte [4]. Consequently, the transformation of S- to SH is sensitive to charges and electrolyte_ Finally the fluores.cence of A depends ou W,light, acid,.electrical charges and electrolyte, although intrinsically dye A is in-
sensitive to all these agents 151. The parts of this simple molecular circuit are illustrated in fig. 1b.
2. Method The molecular device shown in fig. 1 is realized in a monolayer assembly (fig. 2). A layer of cyanine dye molecules A (cf. fig. 3) is located at a distance of approximately 50 A from a layer of coumarine dye molecules SH/S- (cf. fig. 3) within methyl stearate monolayers. The chromophores of SH/S- are located in the plane of the head groups of electrically charged lipids (eicosyl trimethylammonium or eicosyl sulphate ions)_ This plane is in contact with a 10 mM sodiumchloride solution. When excited at 366 nm the dye S- emits a blue fluorescence (fig. 3), whereas dye SH exhibits no fluorescence .[6]. Dye A absorbs blue but no W photons and emits a red fluorescence [7] (fig. 3). Details of the experimental procedure may be found in ref. ISI-
3. Results Fig. 3 shows .the fluorescence spectrum of ihe rn.& noJayer assembly.ciepicted iu fig. 2 at a high pH (corn-,.
Volume
26, number
CHEMICAL PHYSICS LETTERS
2
15 May 1974
SH
UV
tight red
add/ base
fluorescence
b) electrical charges electrolyte Fig. 1. (a) Reaction graph of acid-base sensitizationof fluorescence. (b) Block diagram of acid-base sensitizationin an electrically charged interface.
plete dissociation of SH/S-),
with and without the layer of the acceptor A. The quenching of the S- fluorescence by A is apparent. At two different charge densities at the interface
the bulk pH is lowered step by step. The fluorescence spectra of A (obtained by subtracting the fluorescence component of S- from the measured spectra) at various pH values are depicted in fig. 4. The fluorescence of dye A is seen to be pH dependent and this pH sensitivity depends on the interfacial charge density.
1
Ik
biass
chromophore A ethylstearate chromophore SHISfixed charges diffuse double layer bulk
water
Fig. 2. Schematic drawingof the dye-lipid-electrolyte lamella e_tiiiting acid-basejensitized fluorescence. The lipid layer consists of three methyl stearate layers: the first (pure) one on the hydrophobic glasssupport, the second one mixed with the cyanine dye A(600 .&*/dyemolecule), the third one mixed with the coumarine dye SH(600 Ndye molecule) and additio.nal positivelyor negativelycharged lipid molecules (eicosyl trim~thylammonium.eicosyl sulphke). The hydrophilic groups .-of the third monolayer are in direct contact with a.10 mM NaCl~olution. :
In fig. 5 the sensitized titration of the A fluorescence is compared to the direct titration of the SH/Sfluorescence. The apparent pK values of SH/S- (pK (pox) = 6.2, pK (neg.) = 10) coincide precisely with the ‘pK’ values of the intrinsically pH insensitive dye A. (The fluorescence of A does not disappear completely at low pH due to little direct excitation of A by the exciting light (366 nm) bypassing the sensitizer s-.)
4. Discussion The ratio Id>+, of the S- fluorescence intensities at 450 nm with and without acceptor layer at the distance d is seen from fig. 3 fo ,be 0.4: By inserting this value and d = 52 A into the distance relation of ener-
Volume 26, number 2
CHE,MICAL PHYSICS LETPERS wavenumber
LSO
[cm -11
600
550
500
wavelength
15 May 1974
I nml
Fig. 3. Fluorescence and absorbance spectra of coumarine dye S- and cyanine dye A. 0 is the fluorescence spectrum of a lamellar system as shown in fig. 2 with: 600 AZ per dye S- and dye A molecule, respectively, 140 A2 per positive charge, 10 mM NaCl solution, pH 11, excitation at 366 nm. 0 is the fluorescence spectrum of a similar system without acceptor dye. 0 is the absorbance spectrum of a monolayer of cyardne dye A (600 AZ per dye molecule) measured in a dry monolayer assembly 171. The fluorescence spectra are corrected by the quinine-sulphate method [9] _cdl/d; = fluorescence intensity per wavenumber interval.)
wavenumber
[cm-r]
wavenumber
[cm-r]
wavelength [nml
wavelength [nml
al
b)
Fig. 4. Fhrorescence spectra of cyanine dye A (cf. fig. 3) (excitation at 366 nm) in the lamellar structure shown in f%. 2 at different pH values of the bulk aqueousphase (IO mM Nacl): (a) positively charged interface (140 R2 per charge), (b) negatively charged interface (200 A2 per charge). (dI/da = fluorescence intensity per wavenumber interval_) The numbers in the figures denote the bulk pH vahws.
L
5
6
7
0
9
10
11
12
bulk pH Fig. 5. Relative fluorescence intensity of cyanine dye A (cf. fig. 3) at 570 nm (circles) and of coumarine dye SH/S- (cf. fig. 3) at 450 ti (crosses) in the lam~llar system shown in tig. 2 (excitation at 366 rrm) versus the pH of the bulk ague-: ous phase (10 mM NaCl): @ positively charged interface (140 A2 per charge), @negatively
charged interface (200 AZ
per bharge). .. 223. :
.
Volume 26, number 2
CHEMICAL PHYSICS LE’ITERS
gy transfer for the geometry considered Id/IO = r1+(~&041-1 El01 an experimental characteristic distance do = 57 a is’obtained. A theoretical value of do is calculated by the formula ndo = CKC$‘~J”~ [IO]. Assuming an orientation factor Q = 0.1 I [lo] and a quantum yield of the S- fluorescence (I = 1) using the spectral data of fig. 3 to calculate J= JdP (P)-4f(P) A (P) (0 = wavenumber,f(G) = normalized fluorescence spectrum of S-,A@) = absorbance spectrum of A) and inserting the refractive index II = 1.52 [ 111 one obtaines a theoretical characteristic distance do = 63 A. The good agreement of the experimental and theoretical do values indicates that the interaction of Sand A, as indicated by the quenching of the S- fluorescence, is due to photon transfer as described by FBrster’s formula. The agreement of the titration characteristics of the fluorescence of A and S-(fig. 5) proves that A is excited by S-. The fluorescence of A observed with excitation by an external light source is not pH dependent under the conditions of the experiments described. The results prove that the acid-base sensitization of fluorescence, i.e., the molecular circuit depicted in fig. I is realized in the IameIIar structure depicted in fig. 2 *. The proton signal determines the position of the ‘switch’ SH/S- . The actual position is ‘read’ by the UV light. This information is transferred on the moiecuiar Ievel by the blue. photons to A, where it controls the red fluorescence. The threshold at which an external acid-base signal triggers the switch is adjustable by the charge density and the eIectrolyte. The
* For the syntheses of a function as shown in tip. lb it is not essential to synthesize a structure as shown in tig. 2 with molecular precision. Deviations from the ideal lamellar structure do not change the features of the function significantly as long as the essential molecular interactions, causing the non-additivity of the properties of the molecules involved, are maintained.
224
15 hlay 1974
integrated system possesses properties ( the coupling of the input UV light, acid-base, charge, electrolyte and the output red fluorescence), which are not inherent to the individual molecules involved. These properties are produced by the cooperation of different kinds of molecules in an ordered arrangement (cf. ref. [ 111). They are understood completely by the knowledge of the intrinsic properties of the molecules and their interactions in the ordered arrangement. The concept of acid-base sensitization by energy transfer may be generalized. Other kinds of chemical substances or physical agents (as temperature, light, electrical fieldsj are feasible as sensitizing agents. Besides fluorescence all kinds of photoreactions, e.g., photoprocesses in semiconductors, may be sensitized to some chemical or physical agent. Finally, the device of fig. lb may be extended: the acid may be produced in an ester hydrolysis by an interfacial enzyme. In this case the acid-base sensitization may act as a link between two stoichiometrically independent reactions, a hydrolysis and a photoreaction [5] _
References [l] Th. FGrster, Ann. Physik. 6 F., 2 (1948) 55. [2] L. Gomberoff and E.A. Power, Proc. Phys. Sot. (London) 88 (1966) 281. [3] P. Fromherz, Ber. Bunsenges. Physik. Chem. 77 (1973) 1019. [4] JJ. Davies and E.K. Rideal, Interfacial phenomena (Academic Press, New York, 1963) p_ 94. [5 J P. Fromherz, Chimia 27 (1973) 659. 16) A. Diener, C.V. Shank and A.M. Trozzolo, Appl. Phys. Letters 17 (1970) 189. [7] D. Miibius, 2. Naturforsch. 24a (1969) 251. [S] P. Fromherz, Biochim. Biophys. Acta 323 (1973) 326. [9] A. Schmiien and R. Legler, in: Landolt-BSrnstein, Gruppe II, Bd. 3, eds. K.H. Hellwege and A.M., Hellwege (Springer, Berlin, 1967) pp.228 ff. [ 10) H. Kuhn, J. Chem. Phys. 53 (1970) 101. [l l] H. Kuhn, D. Miibius and H. Biicher, in: Technique-s of chemistry, Vol. 1, part IIIB. eds. A. Weissberger and B.W. Rossiter Wiley, New York, 1972) pp. 577-702.