t-DLR Imaging of Sensors Integrated in Microtiterplates

The DLR sensor presented were also exploited for time-resolved luminescence imaging.

Therefore the DLR scheme is transferred from the frequency domain into the time domain [21]. In this case, the luminescence can be detected by a CCD-camera because no sinusoidal modulation is necessary. The camera maps an area of 20 wells of a microtiterplate with integrated sensor spots of type M3. The wells were filled with copper(II) solution of various concentrations. The uniform illumination of the areas is crucial and for this reason a source for inhomogeneities. However, this effect can be eliminated to a large extent by forming the ratio of the excitation window and the emission windows representing (a) the luminescence of both the indicator and the reference and (b) the solely emission of the reference standard.

Figure 5.9 shows the grey-scale image of the wells and the surface plot of the image. R reflects the copper(II) concentration of the solution in each well. The effects of nonhomogeneous excitation (through a nonhomogeneous lightfield) or nonhomogeneous dye distribution are referenced out, which is displayed by the uniform grey distribution of the sensor spots. Furthermore, the pixel data of each well were averaged and plotted vs. the copper(II) concentration (see Figure 5.10). In addition, Figure 5.10 displays the calibration plot of a conventional intensity measurement at two wavelengths. Apart from the scale, the calibration plots show identical shape. The homogeneous intensity distribution of the sensor spots demonstrates successful referencing.

500 µM 50 µM 25 µM 5 µM 0 µM

Fig. 5.9. Referenced grey-scale picture (left) and surface plot (right) of the mapped area of the microtiterplates with sensor spots integrated into the wells. X and y-axes represent the pixels of the image, R values (eq. 2) are plotted in direction of the z-axes.

10 100 1.6

1.8 2.0 2.2 2.4

[Cu²⁺] /µM

R

0.8 1.0 1.2 1.4 1.6 1.8 2.0

Imaging

Intensity

F530 /F620

Fig. 5.10. Calibration plots of (a) the imaging measurement and (b) the ratiometric intensity measurement. R is the fraction of the intensity of the reference and the indicator in the excitation window and the intensity of the reference in the emission window. F530 is the intensity measured at 530 nm and F620 at 620 nm, respectively, when excited at 420 nm.

4.4. Conclusion

A powerful sensor scheme for the determination of copper(II) with internal referencing of luminescence intensities is presented. The scheme is based on combining a fluorescent indicator with a phosphorescent reference standard. The method provides the conversion of a copper(II)-dependent fluorescence intensity into a phase shift that can be detected by non-sophisticated instrumentation. The sensing membrane is capable of measuring copper(II) with an outstandingly high selectivity over a wide range. The dynamic range is between 5 and 1000 µM which matches the guide line set by the WHO and the EC. The advantages of the referencing method over intensity-based measurements was demonstrated by the measurement of turbid solutions. The intensity signal changes strongly, while the phase shift remains at a constant level. This simplifies determination of copper(II) of real samples where turbidity is likely to occur.

In addition, the sensing scheme enables measurements both in the frequency domain and in the time domain. This was demonstrated by performing 2-dimensional measurements by time-resolved imaging of sensors integrated into microtiterplates. The images obtained show a homogenoeus intensity distribution due to a efficient intrinsic referenciation of the heterogeneous light-field and of dye distribution. The method also demonstrates its high potential over sequential methods in view of a data acquisition in less than 1 s and the collection of the data in one image which allows further interpretation.

4.5. References

[1] I. Klimant, Ch. Huber, G. Liebsch, G. Neurauter, A. Stangelmayer, O. S. Wolfbeis, in New Trends in Fluorescence Spectroscopy, B. Valeur, J. C. Brochon (eds.), Springer Verlag, Berlin, (2001).

[2] I. Klimant, (inv.) German Patent Application, DE 198.29.657, Aug. 1 (1997).

[3] C. Huber, I. Klimant, C. Krause, O. S. Wolfbeis, Dual lifetime referencing as applied to a chloride optical sensor, Anal. Chem., 73, 2097 (2001).

[4] I. Oehme, O. S. Wolfbeis, Fundamental Review – Optical Sensors for Determination of Heavy Metal Ions, Microchim. Acta, 126, 177 (1997).

[5] C. Sanchez-Pedeno, J. A. Ortuno, M. I. Albero, M. S. Garcia, A new procedure for the construction of flow-through optodes. Application to determination of Copper, Fresenius J. Anal. Chem., 366, 811 (2000).

[6] N. Malcik, P. Caglar, R. Narayanaswamy, Investigations into optical sensing of cupric ions using several immobilized reagents, Quím. Anal., 19, 94 (2000).

[7] L.A. Saari, W.R. Seitz, Immobilized Calcein for metal ion preconcentration, Anal.

Chem., 56, 810 (1984).

[8] F. V. Bright, G. E. Poirer, G. M. Hieftje, A new ion sensor based on fiber optics, Talanta, 113 (1988).

[9] J. W. Parker, O. Laksin, C. Yu, M. L. Lau, S. Klima, R. Fischer, I. Scott, B. W.

Atwater, Fiber-Optic Sensor for pH and Carbon Dioxide Using a Self-Reerencing Dye, Anal. Chem., 65 2329 (1993).

[10] D. J. S. Birch, O. J. Rolinski, D. Hatrick, Fluorsecence lifetime sensor of copper ions in water, Rev. Sci. Instrum., 67(8), 2732 (1996).

(1999).

[12] J. R. Lakowicz, F. N. Castellano, J. D. Dattelbaum, L. Tolosa, G. Rao, I. Gryczynski, Low-Frequency Modulation Sensors Using Nanosecond Fluorophores, Anal. Chem., 70, 5115 (1998).

[13] G. Liebsch, I. Klimant, C. Krause, O. S. Wolfbeis, Fluorescent Imaging of pH with Optical Sensors Using Time Domain Dual Lifetime Referencing, Anal. Chem., 73, 4354 (2001).

[14] C. Huber, I. Klimant, C. Krause, T. Werner, T. Mayr, O. S. Wolfbeis, Optical Sensor for Seawater Salinity, Fresenius J. Anal. Chem., 368, 196 (2000).

[15] W. W. Stewart, Synthesis of 3,6-Disulfonated 4-aminonaphthalimides, J. Am. Chem.

Soc., 103, 7615 (1981).

[16] C. T. Lin, W. Boettcher, W. Chou, C. Creutz, N. Sutin, J. Am. Chem. Soc., Mechanism of the Quenching of the Emission of Substituted Polypyridineruthenium(II)

Complexes by Iron(III), Chromium(III) and Europium(III) Ions, 98, 6536 (1976).

[17] I. Klimant, O. S. Wolfbeis, Oxygen-Sensitive Materials Based on Silicon-Soluble Ruthenium Complexes, Anal. Chem., 67, 3160 (1995).

[18] A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser, A. von Zelewsky, Ru(II) Polypyridine Complexes: Photophysics, Photochemistry and Chemiluminescence, Cood. Chem. Rev., 84, 85 (1988).

[19] G. Liebsch, I. Klimant, O. S. Wolfbeis, Luminescence lifetime temperature sensing based on sol-gels and poly(acrylonitrile)s dyed with ruthenium metal-ligand complexes, Adv. Mater. 11 1296 (1999).

Chapter 5

Multi-Ion Imaging Using Selective Fluorescent Sensors in a Microtiterplate Array Format

A novel type of sensor array destined for water analysis is described. The sensor array delivers simple on/off patterns of complex ion mixtures. Fluorescent indicators for calcium(II), sodium(I), magnesium(II), sulfate, chloride and mercury(II) were dispersed in thin films of water-soluble polymer on the bottom of the wells of microtiterplates. Indicator and polymer dissolve after adding the aqueous sample and the interaction of analyte and dissolved indicator result in a fluorescence signal that can be quantified. The fluorescence intensity of the indicators was transferred into a time-dependent parameter applying a scheme called dual lifetime referencing (DLR). In this method, the fluorescence decay profile of the indicator is referenced against the phosphorescence of an inert reference dye added to the system. The intrinsically referenced measurements were performed using blue LEDs as light sources and a fast gateable CCD camera.

5.1. Introduction

The arrangement of several chemical sensors in an array format enables the simultaneous determination and assessment of multiple chemical information. This is essential in the design of, for example, artificial noses and tongues. Existing sensing schemes employ a variety of chemical interaction strategies. These include the use of conductive polymers [1], metal oxide field effect transistors [2], surface acoustic wave devices [3], catalytic (tin oxide) [4,5], electrochemical [6,7] or optical sensors [8-14]. By making use of certain sensors out of the multitude of existing sensors for environmental contaminants, almost any species may be sensed, but this is associated with a considerable instrumental effort, since practically all sensors are different in terms of sensing scheme and hence instrumentation. Ideally, however,

technology (e.g. optical), can sense all parameters simultaneously, yet is compatible with standard methods for sample preparation. We have tackled this challenge by (a) using a uniform analytical protocol, (b) using fluorescent indicators with very similar excitation and emission wavelengths, (c) integrating the format into microtiterplate technology (in order to form disposable sensor arrays), (d) making use of chemical imaging (which allows fast reading of all parameters simultaneously), and (e) using comparative and inexpensive semiconductor and optical components. This combination paves the way for analyzing aqueous solutions for a large number of parameters using a single opto-electronic system, in a short time.

The sensing array introduced here incorporates sensor spots yielding signals at almost identical excitation and emission wavelengths and within the same range of decay times. It parallels a recent report (using different materials) to ‘see’ a variety of odors by making use of a set of sensors spots whose coloration is affected by vapors of certain odorants [8]. In contrast to previous work by McDevitt and co-workers who have described arrays based on micro-beads contained in micromachined cavities [9]. The scheme presented here does not require any highly sophisticated steps in peparation and signal processing, but rather makes use of the widely accepted microtiterplate technique.