Abstract
Nowadays, studies of metabolic pathways and processes in living organisms cannot be easily done at the cellular level. That is why the development of a new analytical methods and approaches is needed, to allow detection of different biologically important species at very low concentrations levels and sample volumes, especially in individual cells. In the present work, we suggested a sensor to detect units of living cells by means determination of plant esterases (PE) based on fluorimetric detection of the products of the enzymatic hydrolysis of fluorescein diacetate in plant cell cultures (BY-2 tobacco cells and early somatic embryos of Norway spruce, clone 2/32). We standardized the sensor using a readily available esterase from pig liver. The detection limits were approximately 17 to 50 amol in 2 ml (8.5 to 25 femtomolar concentrations of esterases) of the enzyme contained in BY-2 tobacco cells and spruce early somatic embryos, respectively, after re-computation on the amounts of pig liver esterases. We assumed that the optimised sensor for the determination of PE in cell extracts accomplishes all requirements for a sensitive analysis which could be usable for single cell analysis. The detection limit was 1.5 in case of analysing BY-2 tobacco cells and 0.5 in early somatic embryos. Moreover, we were able to detect single protoplasts.
Highlights
Biotechnologies involving bioengineering, development of biosensors, genetic manipulation, single cell analysis and others has already become the pioneering focus in many scientific fields such as chemistry and molecular biology due to its potential for understanding the most important biochemical pathways and cycles occurring in living cells [1]
That is why we performed our fluorescein measurements in the presence of 250 mM phosphate buffer at pH 8.75
The plant esterases (PE) esterase activities measured according to the number of protoplasts are shown Fig. 5D
Summary
Biotechnologies involving bioengineering, development of biosensors, genetic manipulation, single cell analysis and others has already become the pioneering focus in many scientific fields such as chemistry and molecular biology due to its potential for understanding the most important biochemical pathways and cycles occurring in living cells [1]. Single-cell analysis gives great assistance to diagnosis and therapy of diseases. The state of diseases and the effects of treatments can be ascertained through analysing the change of components and contents, even DNA fragments in single-cells [2,3,4,5,6,7]. The amounts of components of the living cells such as peptides, proteins, enzymes, nucleic acids and others can be measured in a single living cell [11,12,13,14,15]. The use of sensitive and effective luminescent techniques in single cell analysis has increased [8,11,16,17,18,19]. A detection of fluorescence is possible to use for i) a direct determination of labelled compounds (labelled peptides, proteins etc.) [21,22,23,24] and/or ii) an indirect determination of compounds which is based on detection of the fluorescent product of the specific reaction proceeding between an analyte and a substrate [14,15,21,25]
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