Real time quantification of intracellular nickel using genetically encoded FRET-based nanosensor

Soleja, Neha; Mohsin, Mohd

doi:10.1016/j.ijbiomac.2019.07.115

Cited by 11 publications

(10 citation statements)

References 37 publications

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“…A binding partner of selenium, that is, SeBP, has been chosen to create a genetically encoded FRET-based sensor to monitor the selenium level within the living cells. For generating a FRET-based nanosensor, the fluorescent variants ECFP (donor) and Venus (acceptor) were attached with the SeBP at the N- and C-termini, respectively, to attain a recombinant protein. − The linear arrangement of the restriction sites and the construction of the selenium nanosensor are shown in Figure A. SeBP protein is a suitable biomolecule for FRET-based investigations due to its sandwiched selenium-binding cleft and conformational elasticity within its structure.…”

Section: Resultsmentioning

confidence: 99%

“…coli BL21-Codon Plus strain was transformed by nanosensor construct for expression. Cells were induced after growing at 37 °C with an OD 600 of between 0.4 and 0.6 to a final concentration of 1 mM IPTG and further grown in the dark for the next 16 h at 20 °C . The bacterial cell suspension (180 μL) was prepared by resuspending the cell pellet in 20 mM PBS buffer and mixing with 5 μM selenium (20 μL) and then dispensing in each of the 96-well microplates with triplicates.…”

Section: Methods and Methodologymentioning

confidence: 99%

“…Cells were induced after growing at 37 °C with an OD 600 of between 0.4 and 0.6 to a final concentration of 1 mM IPTG and further grown in the dark for the next 16 h at 20 °C. 14 The bacterial cell suspension (180 μL) was prepared by resuspending the cell pellet in 20 mM PBS buffer and mixing with 5 μM selenium (20 μL) and then dispensing in each of the 96-well microplates with triplicates. The FRET signal was recorded for 90 min at regular intervals of 5 min using a monochromator microplate reader.…”

Section: 4mentioning

confidence: 99%

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Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors

et al. 2023

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Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and antiinflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d ) of 0.198 × 10 −6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.

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Section: Resultsmentioning

confidence: 99%

Section: Methods and Methodologymentioning

confidence: 99%

Section: 4mentioning

confidence: 99%

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Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors

et al. 2023

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“…FRET occurs when the fluorescence emission spectrum of a donor group overlaps with the absorption spectrum of a receptor group in different fluorescence groups. When the distance between the two groups is appropriate (generally less than 10 nm), fluorescence energy transfer from the donor to the acceptor can be observed [19][20][21][22].…”

Section: Acute Myocardial Infarction (Ami) Is a Cardiovascular Diseasementioning

confidence: 99%

Development of a system for detecting cardiac troponin I by background fluorescence quenching based on internal filtration effect

Wang,

Zhang,

Song

et al. 2023

Nanotechnology

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The present study sought to develop a cardiac troponin I (cTnI) detection system based on background fluorescence quenching of internal filtration effect (IFE) and study the influence of IFE on the sensitivity of cTnI detection. Three nanogold materials were synthesized as fluorescence quenchers, and rhodamine 6 G (R6G) and Cy5 were used as fluorescence probes. Six experimental systems were established to detect cTnI in negative serum test solutions and clinical serum samples. The sensitivity of each system was compared to explore the contribution of IFE to the detection sensitivity of cTnI. When applied to negative serum test solutions, the R6G-nanogold material I system exhibited a superior detection effect for cTnI, with a limit of detection (LOD) of 0.15 ng ml−1. When applied to clinical serum samples, the Cy5-nanogold material Ⅲ system yielded a better detection effect for cTnI, with the lowest concentration of cTnI detected at 2 ng ml−1. The first and second internal filtering effects in the proposed system can be achieved simultaneously, effectively avoiding light absorption interference from clinical serum samples and enhancing the sensitivity of the background fluorescence quenching detection of cTnI.

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“…In previous research, various spectrometry techniques have been used that allowed for piloting of the biochemical properties of the living cell, but they have limited temporal and spatial resolution. The FRET (fluorescence resonance energy transfer) based genetically-encoded nanosensor has been proven to be a powerful tool that allows for the measurement of the metabolites at subcellular level in living cells and in a non-destructive manner [6][7][8][9]. The nanosensor consists of bacterial periplasmic binding proteins as a ligand sensing domain sandwiched between a FRET pair of fluorescent proteins [10].…”

Section: Introductionmentioning

confidence: 99%

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

Fatima

Okla

Mohsin

et al. 2020

IJMS

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Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N-and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.

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Real time quantification of intracellular nickel using genetically encoded FRET-based nanosensor

Cited by 11 publications

References 37 publications

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors

Development of a system for detecting cardiac troponin I by background fluorescence quenching based on internal filtration effect

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

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