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Optical pH sensor based on a dual life time referencing (DLR) methodRka VARGA1, Aleksandar SZCHENYI 2, Barna KOVCS1,2

1 University of Pcs, Faculty of Natural Sciences, Department of General and Physical Chemistry H-7624 PCS, Ifjusg str. 6 2 South Trans Danubian Cooperative Research Centre H-7632 PCS, Mra F. Utca 72/a

Introduction: Optical parameters that can be exploited for development of sensors are: absorbance, reflectance and fluorescence. In the case of the fluorescence the most commonly used parameter is fluorescence intensity, nevertheless it also has some drawbacks. It requires additional calibration, it is influenced by dye leaching, photo bleaching, the sample turbidity can abrogate the measured values. Several methods have been developed to overcome these problems, like: intensity ratio measurements, or Dual Lifetime Referencing (DLR) method [1]. It is a principle to reference fluorescence intensities via fluorescence decay times. DLR method uses two fluorescent dyes with overlapping spectroscopic properties, one pH-sensitive, short-lived indicator and a pH-insensitive reference dye with a decay time in the s or ms range. In the present work use N-allyl-4-piperazinyl-1,8-naphthalimide (APN) as a pH sensitive dye [2], and tris(diphenylphenanthroline)ruthenium(II) complex as reference fluorophore [3]. The dyes has been co immobilized in the sol-gel matrix and has been tested with respect to their spectral characteristics, reversibility and response time.

DLR Method Aref cos * ref Aind 1 A ! cot * ref ind Aref sin * ref sin * ref Aref

cot * m !

Spectral properties of APN

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Fig 4. APN absorbance on different pH

Experimental: The synthesis of APN was similar to that reported by Niu et al. [5]. The whole synthesis route is shown in Fig. 1. The pH sensing principle of APN is shown in Fig 2. The Ru-complex was encapsulated in a sol- gel matrix. 1,81 mg Ru(dyph) was dissolved in a solution comprising 0,2 ml of ethanol, 0,1 ml of distilled water and 0,247 ml tetraethoxysilane (TEOS). The sol gel process was then initiated by the addition of 0,02 ml of 0,1 M HCl to the solution. Condensation was allowed to proceed for 10 min, at which point the solvents were rapidly removed under vacuum at a temperature of 100 C. The resulting gel was powdered in mortar and tempered in the argon atmosphere at 200 C overnight. The glassy powder prepared this way is impermeable for oxygen [2]. The pH sensing layers was prepared as follows: 0,19 mg APN was dissolved in 0,05 ml ethanol and 0,1 ml vinyltriethoxysilane (VTES). The solution was sonificated for 5 minutes and irradiated with UV light (385 nm) for 30 minutes, 0,1 ml tetraethoxysilane (TEOS) and 1,9 mg of Ru-complex containing micro particles was added. The sol gel process was then initiated by the addition of 0,02 ml of 0,1 M HCl to the solution. The solution was sonicated for 5 minutes and stirred for 20 minutes. The sensing layers have been spin casted on the activated microscope glass cover, dried at room temperature for 1 hour, and then gelated in the oven at 80 C for 3 hours. The sensing membrane was conditioned in diluted ammonia solution for 1 hour and washed truly with deionized water before measurements. For pH calibration Britton-Robinson buffer was used Apparatus: The phase shift measurements were performed with SR830 DSP Lock in Amplifier. Blue LED 405 nm was used for excitation, modulated light was coupled into a bifurcated fiber bundle. The light was reflected from the sensor that was placed in a flow cell. For the detection of the fluorescent light and determination of phase shift a photomultiplier was used with sample rate of 1 Hz (Fig 3.). Results: Absorbance (Fig 4.) and fluorescence (Fig. 5.) spectra of APN was taken with Avantes 2048 fiber optic spectrophotometer and Xe light source. In the order with absorbance spectra an appropriate light source was chosen. Proton strongly enhance the fluorescence intensity of APN, which shows no fluorescence above pH 12. It has been found that APN fluorescence intensities have a liner correlation with pH in the pH range 3-6. The phase shift of the reference material and the overall sensor layer as a function of the modulation frequency is shown in the Fig 6. It has been found that the difference in the phase shift have a maximal value at the 3,5 kHz modulation frequency. Fig. 7. shows the calibration curve of the sensor layer Conclusion: A pH sensor has been prepared with covalently bonded pH sensitive dye and co immobilized reference powder. The sensor calibration have a liner correlation in the pH range 3-11,5. The examined combination of the materials shows promising results for the further development of pH sensor.

Fig 1. The synthesis route of APN

Fig 2. Fluorescence enhancement mechanism

LED

LOCK IN

PM

BROAD FILTER 400 nm BROAD FILTER 500 nm

BIFURCATED FIBER

FLOW CELL SENSING LAYER

Fig. 5. APN fluorescence on different pH

Fig. 3. Apparatus

Phase shift measurements

Calibration

Fig. 6. Phase shift as a functiom of modulation frequency

Fig. 7. Calibration of sensing layer

[1] I. Klimant, C. Huber, G. Liebsch, G. Neurauter, A. Stangelmayer, O. S. Wolfbeis, New Trends in Fluorescence Spectroscopy, Springer Series on Fluorescence, 2001, 257-274. [2] Zhi-Zhang Li, Cheng-Gang Niu, Guang-Ming Zeng, Yun-Guo Liu , Pan-Feng Gao, Guo-He Huang, You-An Mao, Sensors and Actuators B 114 (2006) 308 315. [3] Torsten Mayr, Ingo Klimant, Otto S. Wolfbeis, Tobias Werner , Analytica Chimica Acta 462 (2002) 1 10 [4] C.G. Niu, G.M. Zeng, L.X. Chen, G.L. Shen and R.Q. Yu, Analyst 129 (2004), pp. 20 24.

Acknowledgement: To Peter cs for the work on the sythesis of the sensing materials