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A Fluorimetric Sensor for Detection of One Living Cell

View Article: PubMed Central

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.

No MeSH data available.


Reaction scheme of fluorescein diacetate hydrolysis catalyzed by plant esterases (PE).
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f1-sensors-07-00222: Reaction scheme of fluorescein diacetate hydrolysis catalyzed by plant esterases (PE).

Mentions: Recently, it has been published that abundant proteins in plant cells, esterases, could be utilized as a marker of growth and viability [26-29]. When a cell were dying or has been damaging, the activity of these enzymes were decreasing. Based on this phenomenon, we concluded that a suggesting of a sensor to determine plant esterases could be used to detect living cells. Thus we focused on the development of an effective and sensitive analytical sensor to determine one living plant cell. Based on our previously published papers, fluoresceine diacetate (FDA) has been selected as a suitable substrate to determine plant esterases. The basic scheme of FDA hydrolysis catalysed by plant esterases is shown in Fig. 1.


A Fluorimetric Sensor for Detection of One Living Cell
Reaction scheme of fluorescein diacetate hydrolysis catalyzed by plant esterases (PE).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756718&req=5

f1-sensors-07-00222: Reaction scheme of fluorescein diacetate hydrolysis catalyzed by plant esterases (PE).
Mentions: Recently, it has been published that abundant proteins in plant cells, esterases, could be utilized as a marker of growth and viability [26-29]. When a cell were dying or has been damaging, the activity of these enzymes were decreasing. Based on this phenomenon, we concluded that a suggesting of a sensor to determine plant esterases could be used to detect living cells. Thus we focused on the development of an effective and sensitive analytical sensor to determine one living plant cell. Based on our previously published papers, fluoresceine diacetate (FDA) has been selected as a suitable substrate to determine plant esterases. The basic scheme of FDA hydrolysis catalysed by plant esterases is shown in Fig. 1.

View Article: PubMed Central

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.

No MeSH data available.