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Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

ABSTRACT: In vivo physiological measurement is a major challenge in modern science and technology, as is environment conservation at the global scale. Proper toxicological testing of widely produced mixtures of chemicals is a necessary step in the development of new products, allowing us to minimize the human impact on aquatic ecosystems. However, currently available bioassay-based techniques utilizing small aquatic organisms such as fish embryos for toxicity testing do not allow assessing in time the changes in physiological parameters in the same individual. In this study, we introduce microencapsulated fluorescent probes as a promising tool for in vivo monitoring of internal pH variation in zebrafish embryos. The pH alteration identified under stress conditions demonstrates the applicability of the microencapsulated fluorescent probes for the repeated analysis of the embryo's physiological state. The proposed approach has strong potential to simultaneously measure a range of physiological characteristics using a set of specific fluorescent probes and to finally bring toxicological bioassays and related research fields to a new level of effectiveness and sensitivity.

SUBMITTER: Gurkov A

PROVIDER: S-EPMC5646854 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

Gurkov Anton A Sadovoy Anton A Shchapova Ekaterina E Teh Cathleen C Meglinski Igor I Timofeyev Maxim M

PloS one 20171018 10

In vivo physiological measurement is a major challenge in modern science and technology, as is environment conservation at the global scale. Proper toxicological testing of widely produced mixtures of chemicals is a necessary step in the development of new products, allowing us to minimize the human impact on aquatic ecosystems. However, currently available bioassay-based techniques utilizing small aquatic organisms such as fish embryos for toxicity testing do not allow assessing in time the cha ...[more]

PMID: 29045437

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Project description:The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (?pHenv), whether the concentration of ionophores used can effectively abolish the ?pHenv is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ?pHenv can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6)-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma), BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r-square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ?pHenv can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore nigericin required to collapse the ?pHenv was then studied. The establishment of a non-destructive method of monitoring the stromal pH will be valuable for studying the roles of the ?pHenv in chloroplast physiology.

Dataset Information

Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

Publications

Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

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