Fluorometric paired emitter detector diode (FPEDD)

Abstract

Integration of two light emitting diodes allows construction of a fluorometric paired emitter detector diode (FPEDD)—a compact optoelectronic device useful as a complete flow-through fluorescence detector.

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According to the concept of PEDD-based photometry developed by the Diamond group,^1,2 a complete absorbance detector can be constructed using only two light emitting diodes (LEDs). In such a device one of them (LED-emitter) is a source of nearly monochromatic light compatible with the absorption spectrum of analytes, whereas the second one (LED-detector) plays the role of detector of this light. Integration of such compatible LEDs leads to construction of compact PEDD-based devices useful as dedicated complete absorbance detectors compatible with advanced analytical systems like FIA and chromatography. Examples of such flow systems developed for photometric determination of selected dyes, metal ions and anions are widely reported in the analytical literature.^3–8 Our group has developed a simple method for transduction of electric signal generated by absorbance-PEDD based on the recording of electromotive force generated by illuminated LED-detector.⁹ Further simplification of this approach allows the use of ordinary, low-budget multimeter as instrument reading the voltage signal generated by PEDD.^10,11 Such simplification offers an additional increase in sensitivity.¹⁰ Exploring the reported instrumental approach, some bioanalytical uses of absorbance-PEDDs for photometric detection of enzyme activity (namely urease and alkaline phosphatase) as well as for respective enzyme substrates have been demonstrated.^11–14 Another current trend in the development of the PEDD concept is its implementation to the sensor format. Only recently Diamond's group has published two papers devoted to PEDDs integrated with optically pH-sensitive films. These gas sensing devices were applied for photometric detection of acid vapors¹⁵ and for sweat monitoring.¹⁶

Fluorimetry is another important field of modern analytical chemistry where LEDs are intensively investigated, since it was found that LED induced fluorescence (LED-IF) can be useful for analytical purposes. LED-IF based systems are widely reported in the analytical literature,^17–22 however the main attention is put into the improvement of fluorescence excitation efficiency, for instance by changing LED-emitter geometry. For the fluorescence detection rather expensive photomultiplier tubes (PMTs) or photodiodes are applied. Surprisingly, until now the PEDD concept, intensively explored in the field of photometric devices and methods of measurements, has been not adapted for fluorometric methods of analysis.

The main goal of this study is the development of FPEDD—the optical device where as a fluorescence exciting light source and as a fluorescence detector two appropriate LEDs are applied. The final goal is the development of integrated flow-through FPEDD. There are two main practical advantages expected for such an approach: (i) extremely low cost of FPEDD because LEDs are one of the cheapest optoelectronic components and (ii) simplicity of integration and miniaturization of such a device to the format of compact flow-through detector (especially when compared with detectors based on PMTs).

For effective induction of fluorescence the emission spectrum of LED emitter should be compatible with the absorption spectrum of analyte. As LEDs are sensitive for light of higher energy than the light emitted by them,^9,12 LED-based fluorometric detectors should generate light of lower energy that measured fluorescence. Fluorescein has been chosen as a model fluorogenic analyte. The maximum of its absorption spectrum is at 490 nm, thus for the induction of its fluorescence, blue LEDs (470 nm) have been applied. The maximum of analyte fluorescence is observed at 521 nm, thus for its detection, red LEDs (630 nm) have been used. Both LEDs (diameter: 5 mm; with transparent lens), were obtained from Optosupply (Hong Kong). The excitation/emission spectra for calcein, very similar to those for fluorescein, also are compatible with the selected pair of LEDs.

For reference measurements of LED induced fluorescence a fiber optic spectrofluorometer (model USB2000FLG from Ocean Optic Inc., USA) was applied. Measurement setup for preliminary experiments is shown in Fig. 1. Both LEDs and optical fibers from the fluorometer have been mounted in a 1× 1 × 4 cm cuvette holder made of LEGO bricks. LED-emitter was supplied using a home-made electronic circuit. For measurements of voltage generated by the LED detector ordinary multimeter (Axiomet model AX-18B, China) connected with data storage PC viaUSB interface was applied.

Fig. 1 Measurement setup for primary experiments under stationary conditions and LED-IF of fluorescence of fluorescein detected using LED-detector (black lines) and spectrofluorometer (grey lines). Currents supplying blue LED emitter are given in the Figure.

Stationary measurements with the use of FPEDD prototype shown in Fig. 1 clearly confirm utility of LEDs for fluorescence detection. The graphs for calibrations performed in the ppm range of fluorescein using red LED-based detectors and conventional fiber optic fluorometers are fully comparable. Moreover, in both cases the same effect of LED-IF intensity on the sensitivity of detection was observed. The detection limits for 5, 20 and 80 mA current driven LEDs emitters were 30, 13 and 9 ppb, respectively. What is more, further increases in sensitivity can be caused using an array of LEDs inducing fluorescence. For instance, the mounting of the second LED-emitter vis-a-vis the first one (see Fig. 1) resulted in nearly twice increase in the sensitivity of detection with LED-detector. The same effect was observed when a fluorometer was applied as the detector. For further experiments the blue LED emitter was supplied with an 80 mA current.

Calcein and its calcium complex have nearly the same excitation and emission spectra as those for fluorescein, thus the reported sensing system is also useful for their detection. Fig. 2 presents dependency of fluorescence of these species (and fluorescein) on pH. In the case of calcein solutions a small amount of EDTA was added to mask effects from Ca traces. In the case of measurement of the complex an excess of calcium ions was applied. As before, for fluorometric detections both, the fiber optic fluorometer and red LEDs, have been applied and fully comparable results have been obtained. The results collected in Fig. 2 confirm that optimal the pH for fluorescein and calcein detection ranges from pH of 7 to 9. Moreover, it is obvious that a pH of 11–13 is optimal for indirect fluorometric detection of calcium using calcein as a fluorogenic ligand, because at this pH the fluorescence of the complex is significantly higher than for free ligand.

Fig. 2 pH dependence of LED-IF for fluorescein (A), calcein (B) and Ca-calcein complex (C) detected using LED-detector (left). For comparison, the results of reference fluorescence measurements using spectrofluorometer are shown (right).

As reported previously in papers devoted to photometric analysis,^3–14 LED-based light detectors are rapid, so they are useful for flow analysis applications, where non-stationary signals are measured. FIAgram shown in Fig. 3 (top) for FPEDD coupled with conventional flow-through cell (depicted in the Fig. 3) confirms this statement also for fluorometric measurements. Integration of LEDs allows fabrication of compact flow-through FPEDD. To achieve this goal, the LEDs were mechanically prepared with a micromilling machine for obtaining the flat surface with the angle of 45 degrees to the symmetry axis of the diode. Then, two diodes of desired shape were glued with 0.5 mm thick black plastic shutter in between. Finally, the channel was drilled through the shutter for the obtained space for thin wall PTFE tubing (0.8 mm OD). The resulting device plays a triple role: a source of light inducing fluorescence, a fluorescence detector and a flow-through cell for fluorescence measurements. Construction of such integrated FPEDD as well as the corresponding FIAgram are shown in the bottom of Fig. 3. The developed flow-through FPEDDs were examined in a simple single channel FIA system with manual sample injection valve and Minipuls-3 peristaltic pump from Gilson (France). As can be seen from recordings shown in Fig. 3 the peaks were well reproducible in both cases. The obtained detection limits for conventional-type and integrated-type flow-through detectors were 0.24 ppm and 0.04 ppm respectively.

Fig. 3 Construction of flow-through FPEDDs and their application to fluorescein detection under FIA conditions. Data for LEDs coupled with conventional flow-through cell (top) and for compact flow-through FPEDD (bottom).

Taking into account that both FIAgrams shown in Fig. 3 have been obtained under the same experimental conditions and using the same pair of LEDs it could be concluded that the integration causes some increase in detection sensitivity. We can only speculate, that this improvement is caused by changes in the geometry of flow-through cell (mainly smaller distance between chips of applied LEDs and thinner layers of plastic LED bulbs causing higher efficiency of fluorescence induction as well as detection). Fig. 4 presents the effect of LED-IF intensity on fluorescein calibration graphs obtained under the same FIA conditions. The results are fully compatible with those shown in Fig. 1, obtained under stationary conditions. In the case of calcein detection, similar results have been obtained. An increase in current causes both increase of sensitivity and increase of baseline signal. The shift of baseline signal (potential measured in the absence of fluorescein) is the evidence that a part of the light emitted by blue LED is measured by the red LED detector. Despite this, determination of fluorescein in ppm range of concentration using integrated FPEDD is still possible, also under non-stationary conditions.

Fig. 4 Effect of current supplying LED emitter on fluorescein detection under FIA conditions using compact flow-through FPEDD. Corresponding calibration graphs are given in the inset.

The developed integrated FPEDD can be applied for calcium detection. For such a purpose this device was implemented into double channel FIA system in which calcium standards were injected into the water stream. By the second channel carrier containing 0.25 g L⁻¹calcein ligand in 0.02M KOH was delivered. As shown in Fig. 2, under such conditions the Ca-calcein complex exhibits fluorescence, whereas the fluorescence from free ligand is negligible. The recording of measurements for Ca standards and the corresponding calibration graph shown in Fig. 5 clearly demonstrates that the system offers fast and reproducible detection of calcium in the ppm range of concentrations. The last nine peaks of the FIAgram have been obtained for real undiluted samples of low calcium mineral waters. As shown in Table 1, the results of such fluorometric analysis are comparable with declared values as well as with the results obtained in the course of complexometric titration of samples with EDTA. These results clearly indicate the utility of developed devices for “real scenario” applications.

Fig. 5 Calibration of compact FPEDD-based double channel FIA system dedicated for calcium determination and corresponding calibration graph. Last nine peaks are obtained for low-calcium mineral water samples S1–S3 (see Table 1).

Table 1 Results of calcium determination in low mineralized drink waters using developed flow-through FPEDD and reference method

Sample	calcium content [ppm]
	Declared	Reference	Found
Żywiec (S1)	42.6	41.7 ± 0.1	43.3 ± 0.2
Górska Natura (S2)	25.3	26.1 ± 0.1	28.7 ± 0.0
Primavera (S3)	48.1	47.3 ± 0.2	48.8 ± 0.1

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To the best of our knowledge this is the first report on complete fluorometric devices containing a fluorescence inductor and detector in the integrated form of flow-through cell made of only two ordinary LEDs. In conclusion, the PEDD-based fluorometry reported in this paper has two important advantages. Firstly, it enables optical measurements using only two extremely low-cost LEDs and ordinary voltmeter. The total cost of FPEDD reported in this work does not exceed €0.5. Secondly, the flow-through FPEDD is easily miniaturized and integrated with advanced analytical systems like FIA, chromatographyetc. In our opinion, the FPEDD detector concept reported in this paper can be applied for many other dedicated analytical applications.

Acknowledgements

This work was supported by the Polish Ministry of Scientific Research and Information Technology (Project no. MNISW-N-N-204-128538 - PhD student grant for M.P.). Aga & Stefa assistance in the course of the manuscript preparation is kindly acknowledged.

Notes and references

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Footnote

† This contribution has been awarded in the course of VIII Polish Conference on Analytical Chemistry (4–9 July 2010, Kraków, Poland).

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