Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells
- 156 Downloads
Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10−7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.
KeywordsMercury Fluorescent proteins Genetically encoded FRET Nanosensors
The first author (NS) is thankful to University Grants Commission for Senior Research Fellowship. Financial Assistant in the form of research grant under nanobiotechnology scheme (No. BT/PR22248/NNT/28/1272/2017) from Department of Biotechnology, Govt. of India for conducting this research work is gratefully acknowledged.
NS and MM designed the study and prepared the original manuscript. NS and MM conducted all in vitro and cellular experiments and analyzed the data. NS and MM did the live cell imaging of yeast and HEK cells and analyzed the data. AQ performed the cytotoxicity test. NS, MM and MAJ revised the manuscript. All authors were engaged in commenting on the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 9.WHO (2005) Mercury in Drinking-water. (Vol. WHO/SDE/WSH/05.08/10): World Health OrganizationGoogle Scholar
- 10.Gray JE, Theodorakos PM, Fey DL, Krabbenhoft DP (2015) Mercury concentrations and distribution in soil, water, mine waste leachates, and air in and around mercury mines in the Big Bend region, Texas, USA. Environ Geochem Health 37:35–48. https://doi.org/10.1007/s10653-014-9628-1 CrossRefPubMedGoogle Scholar
- 12.Zeyaullah M, Islam B, Ali A (2010) Isolation, identification and PCR amplification of merA gene from highly mercury polluted Yamuna river. Afr J Biotechnol 9(24):3510–3514Google Scholar
- 15.Kobal AB, Horvat M, Prezelj M, Briski AS, Krsnik M, Dizdarevic T, Mazej D, Falnoga I, Stibilj V, Arneric N, Kobal D, Osredkar J (2004) The impact of long-term past exposure to elemental mercury on antioxidative capacity and lipid peroxidation in mercury miners. J Trace Elem Med Biol 17(4):261–274. https://doi.org/10.1016/S0946-672X(04)80028-2 CrossRefPubMedGoogle Scholar
- 16.Srikanth K, Ahmad I, Rao JV, Trindade T, Duarte AC, Pereira E (2014) Modulation of glutathione and its dependent enzymes in gill cells of Anguilla anguilla exposed to silica coated iron oxide nanoparticles with or without mercury co-exposure under in vitro condition. Comp Biochem Phys C 162:7–14. https://doi.org/10.1016/j.cbpc.2014.02.007 CrossRefGoogle Scholar
- 17.Linšak Ž, Linšak DT, Špirić Z, Srebočan E, Glad M, Milin Č (2013) Effects of mercury on glutathione and glutathione-dependent enzymes in hares (Lepus europaeus Pallas). J Environ Sci Health A Tox Hazard Subst Environ Eng 48(11):1325–1332. https://doi.org/10.1080/10934529.2013.781869 CrossRefPubMedGoogle Scholar
- 20.Corbisier P, van der Lelie D, Borremans B, Provoost A, de Lorenzo V, Brown NL, Lloyd JR, Hobman JL, Csöregi E, Johansson G, Mattiasson B (1999) Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal Chim Acta 387(3):235–244. https://doi.org/10.1016/S0003-2670(98)00725-9 CrossRefGoogle Scholar
- 21.Bagheri H, Afkhami A, Khoshsafar H, Rezaei M, Shirzadmehr A (2013) Simultaneous electrochemical determination of heavy metals using a triphenylphosphine/MWCNTs composite carbon ionic liquid electrode. Sens Actuators B Chem 186:451–460. https://doi.org/10.1016/j.snb.2013.06.051 CrossRefGoogle Scholar
- 24.Nevado JJ, Martin-Doimeadios RC, Bernardo FJ, Moreno MJ (2005) Determination of mercury species in fish reference materials by gas chromatography-atomic fluorescence detection after closed-vessel microwave-assisted extraction. J Chromatogr A 1093(1–2):21–28. https://doi.org/10.1016/j.chroma.2005.07.054 CrossRefPubMedGoogle Scholar
- 34.Gupta S, Sarkar S, Katranidis A, Bhattacharya J (2019) Development of a cell-free optical biosensor for detection of a broad range of mercury contaminants in water: a plasmid DNA-based approach. ACS Omega 4:9480–9487. https://doi.org/10.1021/acsomega.9b00205 CrossRefPubMedPubMedCentralGoogle Scholar
- 43.Khan P, Rahman S, Queen A, Manzoor S, Naz F, Hasan GM, Luqman S, Kim J, Islam A, Ahmad F, Hassan MI (2017) Elucidation of dietary polyphenolics as potential inhibitor of microtubule affinity regulating kinase 4: in silico and in vitro studies. Sci Rep 7(1):9470. https://doi.org/10.1038/s41598-017-09941-4 CrossRefPubMedPubMedCentralGoogle Scholar
- 54.Orij R, Postmus J, Ter Beek A, Brul S, Smits GJ (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. Microbiology 155(1):268–278. https://doi.org/10.1099/mic.0.022038-0 CrossRefPubMedGoogle Scholar
- 57.Kiyono M, Omura T, Fujimori H, Pan-Hou H (1995) Lack of involvement of merT and merP in methylmercury transport in mercury resistant Pseudomonas K-62. FEMS Microbiol Lett 128(3):301–306. https://doi.org/10.1111/j.1574-6968.1995.tb07540.x CrossRefPubMedGoogle Scholar