Talanta 96 (2012) 127–131 Contents lists available at SciVerse ScienceDirect Talanta j ourna l ho me page: www.elsevier.com/locate/talanta Serum alkaline phosphatase assay with paired emitter detector diode Kamil Strzelak, Robert Koncki, Lukasz Tymecki ∗ University of Warsaw, Department of Chemistry, Pasteura 1, 02-093 Warsaw, Poland a r t i c l e i n f o Article history: Available online 20 January 2012 Keywords: Alkaline phosphatase Serum Flow analysis Multicommutation Paired emitter detector diode a b s t r a c t A simple multicommutated flow system based on optoelectronic detector, three valves and peristaltic pump only has been developed for photometric determination of alkaline phosphatase activity in human serum. A miniaturized, compact flow-through detector dedicated to selective photometric detection of product formed in the course of the enzyme assay has been constructed using two paired light emitting diodes. The proposed analytical procedure based on kinetic methodology of enzyme activity detection and stopped-flow methodology of two-point measurements eliminates interferences caused by intense color of real samples and impurities present in commercial reagents. After optimization the system allows reproducible, mechanized analysis of human serum in relatively short time (8–9 samples per hour). Volume of serum required for single determination is 0.05 mL only. The system validated with real clinical samples is useful for determination of enzyme activity in human serum at physiological and pathological levels. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Although optical detection dominates in analytical chemistry, for many practical applications highly advanced and expensive spectrophotometers are not necessary. For many dedicated analyt- ical uses simple optical devices based on economic semiconductor components are sufficient. Light emitting diodes (LEDs), the cheap- est optoelectronic components, providing low-cost, high-intensity and quite monochromatic light sources with low-voltage power requirements. Various compositions of the semiconductor gate of diode lead to diversity of LEDs which can cover a broad spectral range from UV to NIR. Because of that, LEDs can be really useful in reducing the complexity of photometric devices by eliminating the need of wavelength selection, and offering simple measurement systems for determination of specific analytes that absorb at the wavelength emitted by selected LED. The utility of LEDs for opti- cal methods of analysis is well established and widely reported in the analytical literature [1]. Several applications of LEDs as light sources in flow analysis have been reviewed recently [2]. The electroluminescence process by which LED operates as a light source is reversible. Illuminated LED produces a small current proportional to the light. It means, that the internal photoelectric effect (opposite to electroluminescence phenomenon) allows the use of LED as light detector when it is applied in the reversed mode. LEDs will respond to fluctuations in light intensity of frequencies to ∗ Corresponding author. E-mail address: luktym@chem.uw.edu.pl (L. Tymecki). more than several hundred kHz. This makes them potentially use- ful as fast and reversible light detectors. Concluding, two LEDs can form a paired-emitter-detector-diode (PEDD) – a complete opto- electronic device for absorbance measurements. Unfortunately, the current generated by illuminated LED is very low and rather diffi- cult to precise measure without special instrumentation. To bypass this problem, an electromotive force generated by illuminated LED detector can be applied as an analytical signal of PEDD. In this approach, when PEDD is coupled with pH-meter [3] or voltmeter [4] the analytical signal (given in millivolts) is a linear function of absorbance. This linearity is easily explained by the compilation of Shockley equation for diodes and Lambert–Beer law for photome- try [3]. For practical uses, instead of relatively expensive pH-meter, a low-budget voltmeter can be applied [4]. In such case, due to low resistance of voltmeter, a partial discharging of LED detector is observed. Therefore, after the instrument replacement, the lin- earity in the wide range of concentration is lost but in the narrow range the sensitivity significantly increases. It is worth to notice, that this range of maximal sensitivity can be adjusted by selection of current supplying PEDD [4]. PEDDs operating according to the reported methodology of analytical signal transduction have just found first analytical appli- cations as extremely economic photometers for some cuvette enzyme assays [5], as hemoglobinmeters [6] as well as dedi- cated flow-through cells (additionally integrated with respective semiconductor light emitters and detectors) [3,4]. Recently, a flow-through sensor for redox species obtained by integration of sensing film with PEDD has been reported [7]. This sensor has been applied for glucose enzyme-based bioPEDD development [8]. In this 0039-9140/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2012.01.031