Excitation energy transfer between acriflavine and rhodamine 6G as a pH sensor

Sensors and Actuators B 63 Ž2000. 18–23 www.elsevier.nlrlocatersensorb

Excitation energy transfer between acriflavine and rhodamine 6G as a pH sensor Vinita Misra, H. Mishra ) , H.C. Joshi 1, T.C. Pant Photophysics Laboratory, Department of Physics, D.S.B. Campus, Kumaun UniÕersity, Nainital-263 002, India Received 15 May 1999; received in revised form 1 November 1999; accepted 18 November 1999

Abstract Excitation energy transfer between acriflavine Ždonor. to rhodamine 6G Žacceptor. molecules in water with varying pH has been studied by using steady state measurements. From absorption and fluorescence spectra of donor and acceptor, overlap integrals for donor fluorescence and donor absorption Ž V DD . as well as donor fluorescence and acceptor absorption Ž V DA . have been calculated. The corresponding critical transfer distances for dipole–dipole Forster mechanism of excitation energy transfer between donor–donor Ž R 0D . and donor–acceptor Ž R 0A ., reduced concentration and efficiency of energy transfer have also been calculated. It has been found that the V DA change with pH whereas V DD remain unchanged, resulting in change in energy transfer from donor to acceptor with pH, but no change in energy migration. The efficiency of excitation energy transfer is maximum for a highly basic solution and it decreases with a decrease in pH. The energy transfer efficiency, reduced concentration, and overlap integrals show a linear dependence on pH. It has been proposed that the system may be used as a wide range Ž1.4 to 12. pH sensor with an accuracy of 0.01. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Energy transfer; pH sensor; Acriflavine; Rhodamine 6G

1. Introduction Excitation energy transfer process is one type of the photophysical processes which arises due to the change in the physical state of a molecule, while leaving the chemical properties unchanged. In this process, transfer of electronic excitation energy takes place from excited molecule of one species Ždonor. to unexcited molecule of another Žacceptor.. Similar transfer among molecules of the same species is known as energy migration. Several theories have been given to explain energy transfer phenomenon. Forster w1x was the first to develop a theory of energy transfer, taking dipole–dipole interaction into consideration between molecules. Later on, Dexter w2x and Inokuti and Hirayama w3x generalized Forster’s theory and applied it to the case of multiple and exchange interactions. In these theories, molecule is assumed to be stationary during its excited state. However, mobility of interacting molecules also affects rate of energy transfer. Yokota and )

Corresponding author. Fax: q91-5942-35576. E-mail address: [email protected] ŽH. Mishra.. 1 Present address: Spectroscopy Group, Institute for Plasma Research, Gandhinagar-382 428, India.

Tanimoto w4x theoretically explained diffusion effect on energy transfer process and Burshtein w5x gave a theory to explain effect of rapid energy migration on energy transfer process. Huber w6x theoretically explained the effect of energy migration in the case of comparable donor–donor and donor–acceptor interactions. Loring et al. w7x have given one of the most general theory of transport and trapping of excitation energy. Jang et al. w8x have recently developed a new theory for the effect of migration and diffusion on energy transfer process, which eventually agreed with the already known experimental finding of Pandey w9x. Joshi et al. w10x have also found from the time resolved study in a dye pair that the observed diffusion coefficient is greater than the sum of translational diffusion coefficients and diffusion coefficients for energy migration among donors, which has been attributed to increase the migration due to translational diffusion. In recent years, a great deal of attention has been paid towards the development of optical sensors based on fluorescence optrode w11x. An optrode consists of a bifurcated optical fiber to carry the excitation radiation to, and emitted radiation from, the matrix containing the fluorescence molecule held at the end of the fiber. Non-radiative excitation energy transfer has been used as pH sensor by Jordan

0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 Ž 0 0 . 0 0 2 9 6 - 3

V. Misra et al.r Sensors and Actuators B 63 (2000) 18–23

et al. w12x using such an optrode. They have found that non-radiative energy transfer increases with increasing pH in eosin Ždonor. to phenol red Žacceptor. dye system. Oxygen and sulfur dioxide sensors based on energy transfer have also been investigated by Sharma and Wolfbeis w13,14x. The distance dependence of energy transfer between two fluorophors fluorescein Ždonor. and rhodamine Žacceptor. has been used as glucose biosensor w15x. The distance variation with ionic strength has also been used in energy transfer based sensors for hydrogen sulfide, nitrous oxide, pH, and alkali metal ions w16x. The advantage of energy transfer sensor is that all absorbance-based indicators can be used in this method. Lifetime-based energy transfer pH sensor has also been projected where pH modulates the lifetime w17x. Fluorescence lifetime-based measurements have a number of advantages over intensity based measurements — it is independent of intensity fluctuation, photodetector response, photobleaching, etc. Optical measurement of pH using phase modulation fluoremetry for decay time has been found to result in increased precision by Szmacinski and Lakowicz w18x. The aim of this work is to investigate the effect of pH in excited state energy transfer and also discuss its application for fabrication of pH sensor. Acriflavine and rhodamine 6G dye pair in water solution, having varying pH, has been taken for this purpose. Excitation of an aromatic molecule possessing acidic or basic functional groups alters the distribution of electronic charge in the molecule, as a result of which the electron density in the region of the acidic or basic functional groups may be increased or decreased. Functional groups are either electron acceptor or electron donor types. Molecules containing electron acceptor type functional groups are stronger bases in lowest excited state than in the ground state, whereas molecules containing electron donor type functional groups are generally stronger acids in the excited state than in the ground state w19x. Shifting of the absorption and fluorescence band to shorter wavelength upon dissociation indicates increase in basicity upon excitation from ground to lowest excited singlet state. Similarly, shifting of the absorption and fluorescence bands to longer wavelengths upon excitation indicates decrease in basicity upon excitation from the ground to excited singlet state. Acriflavine molecules contain electron donor type functional group while rhodamine 6G molecules contain electron acceptor type functional group. Hence such acid–base properties of ground and excited state of molecule shall affect the strength of energy transfer process between these molecules in solution of different pH.

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further purification. Solution of different pH were prepared by mixing sulfuric acid and distilled water, as well as sodium hydroxide and distilled water, in different proportion and dissolving appropriate amount of dyes in these solutions. The concentration of acriflavine Ž10y6 M. and rhodamine 6G Ž10y5 M. was kept constant in all the samples. Samples were also prepared in distilled water with the same dye concentration. The study has been done via steady state measurements. Absorption and fluorescence spectra were recorded using Jasco V-550 spectrophotometer and Jasco FP-777 spectrofluorometer, respectively. pH was measured by pH meter HANNA instruments ŽPortugal. with accuracy of "0.2 and graphs have been plotted by sigma plot and Microsoft excel software. Absorption and fluorescence spectra of donor and acceptor have been taken separately under varying conditions of pH. The analysis has been done by overlapping fluorescence spectra of donor with absorption spectra of donor, as well as fluorescence spectra of donor with absorption spectra of acceptor. Critical transfer distance, overlap integral, reduced concentration, and efficiency of energy transfer have been calculated for these spectra.

3. Theory The value of critical transfer distance Ž R 0A . between donor and acceptor molecules is calculated spectroscopically by using Forster’s energy transfer relation w20,21x: R 60 A s

Ž 5.86 = 10y2 5 . V DA f D Ž n.

4

where V DA is overlap integral for overlapping fluorescence spectra of donor with absorption spectra of acceptor,

2. Experimental Acriflavine ŽAldrich Chemical, USA., rhodamine 6G ŽE. Merck Darmstadt, made in Germany., sulfuric acid and sodium hydroxide ŽE. Merck, India. were used without

Fig. 1. Overlap between Acriflavine Ždonor. emission and absorption and absorption of Rhodamine 6G Žacceptor. in acidic medium ŽpH s1.4.; Ž1. donor emission, Ž2. donor absorption, Ž3. acceptor absorption.

V. Misra et al.r Sensors and Actuators B 63 (2000) 18–23

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Energy transfer efficiency Žh T . has been calculated by using the relation w9,21x 2

h T s g DA 'p exp Ž g DA . 1 y erf Ž g DA . where erfŽg DA . is error function of reduced concentration g DA . Similarly, critical transfer distance Ž R 0D ., overlap integral Ž V DD ., and reduced concentration Žg DD . for selfoverlap have been calculated as: R 60 D s

Ž 5.86 = 10y2 5 . V DD fD Ž n.

4

`

V DD s

H0

f d Ž n . ´ d Ž n . d nr Ž n .

4

and g DD s

`

H0

Fig. 2. Overlap between Acriflavine Ždonor. emission and absorption and absorption of Rhodamine 6G Žacceptor. in basic medium ŽpH s12.; Ž1. donor emission, Ž2. donor absorption, Ž3. acceptor absorption.

fd Ž n . dn

CD C0 D

.

4. Results and discussion

f D is quantum yield of donor, n is refractive index of medium. V DA has been calculated by using the formula w20x: `

V DA s

H0

f d Ž n . ´ a Ž n . d nrn 4 `

H0

fd Ž n . dn

where H0` f d Ž n . ´ aŽ n .d n is area of overlapping region, H0` f d Ž n .d n s area of donor fluorescence and n is average wave number of the overlapping region. Reduced concentration Žg DA . for donor–acceptor overlapping is calculated by using w1,20x

g DA s

CA C0 A

where CA is acceptor concentration and C0A is critical acceptor concentration. Critical acceptor concentration can be calculated by the relation w22x 3000

C0 A s

3

2p 2 N Ž R 0 A .

3

where N is Avagadro’s number.

The absorption spectra of acriflavine Ždonor. and rhodamine 6G Žacceptor. along with fluorescence spectra of donor have been shown for different pH solutions in Figs. 1 and 2. It is found that for the same donor, acceptor concentration Ž10y6 and 10y5 M. and excitation wavelength Ž430 nm., there is considerable change in overlap of the donor fluorescence and acceptor absorption spectra Ž V DA . for acidic and basic solutions, whereas overlap of donor fluorescence and donor absorption Ž V DD . is not much affected by change in pH. It is interesting to note that there is no change in the shape of fluorescence of acriflavine and absorption spectra of rhodamine 6G. The absorption spectrum of rhodamine 6G remains unchanged while the fluorescence maximum of acriflavine shifts considerably with pH, which results in change of overlap only. The value of overlap integral Ž V DA . increases with increasing pH. For highly acidic solution ŽpH 1.4., its value is 15 = 10y1 4 cm6 My1 and for highly basic solution ŽpH 12., its value is 48 = 10y1 4 cm6 My1 and corresponding ˚ assuming critical transfer distances are 52 and 64 A, fluorescence quantum yield of acriflavine as 0.9 w22x. Calculated value of overlap integral and critical transfer distances for intermediate pH solutions have been given in Table 1. In case of self-overlapping, the value of V DD for

Table 1 Calculated value of different spectroscopic parameters for energy transfer in different pH solution Solution no.

pH

Donor–donor overlap y1 5

V DD Ž10 1 2 3 4 5

1.4 3.6 6.9 10.0 12.0

54 52 57 50 46

6

y1 .

cm M

Donor–acceptor overlap

˚. R 0D ŽA

g DD Ž10

V DA Ž10y14 cm6 My1 .

˚. R 0A ŽA

g DA Ž10y2 .

44.0 43.8 42.5 43.5 43.0

0.190 0.187 0.171 0.184 0.177

15 21 31 41 48

52 54 57 62 64

0.314 0.392 0.434 0.533 0.586

y3 .

V. Misra et al.r Sensors and Actuators B 63 (2000) 18–23

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Table 2 Energy transfer efficiency Žh T ., reduced concentration g DA in different pH solution Solution no.

pH

g DA Ž10y2 .

Erf g DA

h T Ž10y3 .

1 2 3 4 5

1.4 3.6 6.9 10.0 12.0

0.314 0.392 0.434 0.533 0.586

0.0033 0.0040 0.0045 0.0056 0.0067

5.5 6.5 8.0 9.3 10.2

highly acidic solution is 54 = 10y1 5 cm6 My1 and for highly basic solution, its value is 46 = 10y1 5 cm6 My1 ; their corresponding critical transfer distances are 44 and 43 ˚ indicating no change in energy migration with pH. The A, energy transfer efficiency and reduced concentration in different pH solutions has been given in Table 2. The reduced concentration and efficiency of energy transfer are maximum for highly basic solution and minimum for acidic solution. Wavelengths for absorption and fluorescence maxima have been given in Table 3. Donor and acceptor absorption spectra for varying pH solutions show that peak of absorption maxima is nearly at the same wavelength for all solutions Ži.e., for acriflavine, l max is 456 nm for highly acidic solution and 453 nm for highly basic solution, while for rhodamine 6G, lmax is 527 nm for highly acidic solution and 522 nm for highly basic solution.. However, a large wavelength shift occurs in the donor fluorescence peaks in acidic and basic media Ž lmax s 534 nm for acidic media and 507 nm for basic media.. These results can be explained on the basis of excited state acidrbase properties of the molecules. Since acriflavine contains electron donor type functional group, it will be more acidic in excited state than in the ground state and it becomes even more acidic in excited state when mixed with an acidic solution. Therefore, large wavelength shift in fluorescence occurs towards longer wavelength. However, there is negligible change in absorption spectra of rhodamine 6G due to change in pH. Thus, the overlap decreases and therefore, overlap integral and critical transfer distance decrease with increasing acidity Žor decreasing pH.. Similarly, acidity of excited state of acriflavine goes on decreasing when mixed with more and more basic solution. Hence, fluorescence peak shifts towards shorter wavelength, as a result of which there is an

Fig. 3. Graph between Ža. overlap integral vs. pH and Žb. reduced concentration vs. pH.

increase in overlap, and therefore the overlap integral and critical transfer distance increase. Thus, the energy transfer is most efficient in basic medium for this pair of dyes. The graph between the overlap integral Ž V DA . vs. pH and reduced concentration Žg DA . vs. pH are linear ŽFig. 3.. Therefore, pH dependence of energy transfer between these pairs of dye makes the system attractive for sensing pH. Any of these parameters can be used to sense pH after suitable calibration of measured data. The accuracy of these measurements is 0.01 pH units. It is also possible to measure pH by using pH dependence of acriflavin decay time due to change in nonradiative energy transfer observed here. With the currently available instruments in this time domain, decay time can be measured with an accuracy of "10y2 ns. The observed value of the reduced concentration Žg DA . from decay time analysis under Forster mechanism of energy transfer can be measured with an accuracy of 0.001, which corresponds to an accuracy of 0.01 in pH measurements. Interference in these measurements may occur due to excitation energy transfer to dissolved metal ions like Cuqq w23x, Coqq w24x, etc., which have their absorption spectra overlapping with the emission spectra of acriflavin. In an earlier work, Jordan et al. w12x also employed energy transfer for pH measurements with an accuracy of 0.01 pH unit over a limited physiological pH range. Egami et al. w25x have introduced an evanescent wave spectroscopic fiber optic pH sensor, using polymer doped with

Table 3 Wavelength of maximum absorption and fluorescence for donor and acceptor in solution of different pH Solution no.

pH

Donor

Acceptor

lma x Žabs., nm

lmax Žem., nm

lmax Žabs., nm

lmax Žem., nm

1 2 3 4 5

1.4 3.6 6.9 10.0 12.0

456 453 452 453 453

534 531 509 507 507

527 527 523 522 522

556 556 553 551 551

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V. Misra et al.r Sensors and Actuators B 63 (2000) 18–23

either cango red ŽpH range 3 to 5. or methyl red ŽpH range from 5 to 7.. Peterson et al. w26x have developed a pH sensor based on measurement of absorption of phenol red having a physiological range of 7–7.4 pH and accuracy of 0.01. Thus, our system has a wide range Ž1.4 to 12 pH. of measurement capability with an accuracy of 0.01. Another important aspect of the present system is that unlike the work of Jordan et al., it does not suffer from the draw back due to change in acceptor concentration. In our system, overlap is changed simply due to the scanning of acceptor absorption spectrum by the shifting of the donor emission spectrum with change in pH. Therefore, the present system appears to be very useful as it covers a wide pH range Ž1.4 to 12. with good accuracy of 0.01 in pH. Further investigations are being carried out in Nafion film in order to search for a probe matrix and the results appear to be encouraging w27x.

5. Conclusion The excitation energy transfer from acriflavine to rhodamine 6G shows a pH dependence due to change in overlap of acriflavine emission and rhodamine 6G absorption spectrum. This change is mainly due to the shifting of the donor emission with pH, which scans through the absorption spectrum of the acceptor. Linear dependence of overlap integral, reduced concentration, and efficiency of excitation energy transfer with pH makes the system attractive as pH sensor from 1.4 to 12 in this dye pair. An indicator-mediated pH sensor, based on this energy transfer in a suitable matrix that will have an accuracy of 0.01, has been suggested.

Acknowledgements The authors thank Prof. H.B. Tripathi for the useful discussions. One of the authors ŽVinita Misra. is thankful to UGC ŽDSArCOSIST., New Delhi, India for financial assistance and ŽHM. is thankful to CSIR ŽIndia. for senior research fellowship.

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w6x D.L. Huber, Donor fluorescence in high trap concentration, Phys. Rev. B 20 Ž1979. 2307–2314. w7x R.F. Loring, H.C. Anderson, M.D. Fayer, Electronic excited state transport in solution, J. Chem. Phys. 76 Ž1982. 2015–2077. w8x S. Jang, K.J. Shin, S. Lee, Effect of excitation migration and transnational diffusion in the luminescence quenching dynamics, J. Chem. Phys. 102 Ž1995. 815–827. w9x K.K. Pandey, Electronic excitation transport, diffusion and trapping, Chem. Phys. 165 Ž1992. 123. w10x H.C. Joshi, H. Mishra, H.B. Tripathi, T.C. Pant, Role of diffusion in energy transfer: a time resolved study, J. Lumin. Ž2000. Žin press.. w11x O.S. Wolfbeis ŽEd.., Fiber Optic Chemical Sensors and Biosensors Vols. 1 and 2 CRC Press, Boca Raton, FL, 1991. w12x D.M. Jordan, D.R. Walt, F.P. Milanovich, Physiological pH fiberoptical sensor based on energy transfer, Anal. Chem. 59 Ž1987. 437–439. w13x A. Sharma, O.S. Wolfbeis, Fiber optic oxygen sensor based on fluorescence quenching and energy transfer, Appl. Spectrosc. 42 Ž1988. 1009. w14x A. Sharma, O.S. Wolfbeis, Fiber optic fluorosensor for sulfurdioxide based on energy transfer and exciplex quenching, Proc. SPIE 990 Ž1989. 116. w15x L. Tolose, H. Malak, G. Raob, J.R. Lakowicz, Optical assay for glucose based on luminescence decay time of the long wavelength dye Cy5e, Sens. Actrators, B 45 Ž1997. 93–99. w16x L.M. Christian, W.R. Seitz, Optical ionic strength sensor based on polyelectrolyte association and fluorescence energy transfer, Talanta 35 Ž1988. 119. w17x O.S. Wolfbeis ŽEd.., Fiber Optic Chemical Sensors and Biosensors Vol. 1 CRC Press, Boca Raton, 1991, p. 380. w18x H. Szmacinski, J.R. Lakowicz, Optical measurements of pH using fluorescence lifetimes and phase modulation fluoremetry, Anal. Chem. 65 Ž1993. 1668–1674. w19x E.L. Wehry ŽEd.., Modern Fluorescence Spectroscopy Vol. 2 Plenum, New York, 1976, p. 245. w20x K.K. Pandey, T.C. Pant, Migration modulated donor acceptor energy transfer in PMMA, J. Lumin. 47 Ž1991. 319–325. w21x J.B. Birks, Photophysics of Aromatic Molecules, Wiley-Interscience, New York, 1970. w22x K.K. Pandey, T.C. Pant, Diffusion modulated energy transfer, Chem. Phys. Lett. 170 Ž1990. 244–252. w23x O.J. Rolinski, D.J.S. Birch, A fluorescence lifetime sensor for CuŽI. ions, Meas. Sci. Technol. 10 Ž1999. 127–136. w24x A.S. Holmes, D.J.S. Birch, K. Suhling, R.E. Imhof, T. Salthammer, H. Dreeskamp, Evidence of donor–donor energy transfer in lipid bilayers: perylene fluorescence quenching by Coqq ions, Chem. Phys. Lett. 186 Ž1991. 189–194. w25x C. Egami, Y. Suzuki, O. Sugihora, H. Fujimura, N. Okamoto, Wide range pH fiber sensor with cango red and methyl red doped poly Žmethyl methacrylate. cladding, Jpn. J. Appl. Phys. 36 Ž1997. 2902–2905. w26x J.I. Peterson, S.R. Goldstein, R.V. Fitzgerald, D.K. Buckhold, Fiber optic pH optical probe for physiological use, Anal. Chem. 52 Ž1980. 864. w27x V. Misra, H. Mishra, H.C. Joshi, T.C. Pant, An optical pH sensor based on excitation energy transfer in nafion film ŽManuscript under preparation..

Biographies Vinita Misra did her MSc ŽPhysics. in 1994, from Almora Campus, Kumaun University, Nainital. She is working as a research fellow in Photophysics Laboratory, Kumaun University, Nainital, since 1996. Her field of research is Excitation energy transfer and its application in pholuminescent system.

V. Misra et al.r Sensors and Actuators B 63 (2000) 18–23 H. Mishra passed his MSc ŽPhysics. with specialization in Electronics Ž1993. and in Laser and Molecular Spectroscopy Ž1996., from Kumaun University, Nainital. Presently, he is working as a Senior Research Fellow of CSIR New Delhi ŽIndia. in Photophysics laboratory, Kumaun University, Nainital. His current interest is in the field of excited state ultrafast processes in photo luminescence and its application. Dr. H.C. Joshi obtained his PhD degree in 1990 from Kumaon University Nainital ŽIndia.. He was a postdoctoral fellow at laser laboratory, I.I.T. Kanpur Ž1992–1993. and later on EPSRC fellow at University of Strathclyde ŽGlasgow, UK. from 1994–1995. He is in a permanent position at Institute of Plasma Research, Gandhi nagar ŽIndia.. His research interest is energy transfer, plasma physics, ultrafast spectroscopy, LSC and optical sensor.

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Dr. T.C. Pant received his PhD in ‘‘Ion–Phonon Interaction and Excitation Energy Transfer’’ from Kumaun University, Nainital, India in 1979. After about two and half years as Junior Research Fellow of CSIR, New Delhi ŽIndia., he became Lecturer in Physics Dept. DSB Govt. P.G. College, Nainital. In 1996, he became Professor of Physics at DSB Campus, Kumaun University, Nainital ŽIndia.. From 1990 to 1994, he was a Senior Lecturer in Egerton University, Kenya ŽEA.. His field of research has been Excitation Energy Transfer and its application to material aspect of Luminescence Solar Collectors ŽLSC. and recently Optical Sensors.