FRET-based Nanosensor Research Articles

Cells stably expressing FRET-based nanosensor AMPfret: A versatile tool to study cell energetics

Cells stably expressing FRET-based nanosensor AMPfret: A versatile tool to study cell energetics

Genetically Encoded FRET-Based Nanosensor for Real-Time Monitoring of A549 Exosomes: Early Diagnosis of Cancer.

Exosomes contain a plethora of unique disease biomarkers involving cellular homeostasis, infection dissemination, cancer development, and cardiac diseases. Exosomes originating from cancer cells have promising biomarkers for the early detection and assessment of the therapeutic response to cancer. The exosomal epidermal growth factor receptor (EGFR) is a potential biomarker which is overexpressed in cancer; thus, the level of EGFR expression is investigated by so many methods in a liquid and solid biopsy. The optimal method for isolating pure exosomal EGFRs has not been well understood so far. Current approaches are complicated and time-consuming, therefore hampering their clinical applications. Here, we demonstrate the creation of an innovative fluorescence resonance energy transfer (FRET) sensor, named ExoSen (exosome sensor), which can be implemented to determine the concentration of exosomal EGFRs at in vitro as well as in vivo levels. In this study, a sensing element for A549 exosomes, mitogen-inducible gene 6 (MIG6), has been employed between the FRET pair ECFP and Venus. MIG6 binding to ExoSen induced a conformational change that can be monitored by a variation in the FRET ratio. Moreover, the developed sensor, expressed in bacterial, yeast, and HEK-293T cells, demonstrates an increased FRET ratio with the addition of A549 exosomes, which can quantify the A549 exosomes noninvasively. The ExoSen enables rapid detection of A549 exosomes with great sensitivity at a concentration of 3.5 × 109 particles/mL. ExoSen is stable to pH fluctuations and provides a highly accurate, real-time optical readout in cell-based experiments by using confocal microscopy.

Designing of nanosensor and its applications for spatiotemporal visualization and real time quantification of biotin in biological samples

Biotin is a 244 Da water-soluble vitamin involved in vital metabolic processes by acting as an essential cofactor for several enzymes. When biotin levels are low, critical enzymes such as pyruvate carboxylase, 3-methylcrotonyl-CoA, and propionyl-CoA carboxylase become dysfunctional leading to fatal human disease. Traditional methods for measuring biotin in biological fluids yield valuable results for particular applications, but the majority of them are intrusive, expensive, and lack sensitivity and specificity. A highly effective genetically encoded FRET-based nanosensor OSenB (Optical Sensor for Biotin) is developed using FRET pair enhanced cyan fluorescent protein (ECFP) and Venus attached to the N- and C-termini of the avidin module, allowing real-time monitoring and quantification of biotin at a trace level. OSenB exhibits discernible FRET ratio shifts, measures biotin with excellent specificity and selectivity, provides a rapid detection response, is resilient to pH fluctuations, and produces a very precise FRET signal with a detection range of 12 pM to 165 nM biotin quantification. Developed mutant sensor OSenB-44 with 0.717 nM apparent affinity (Kd), was found to be the most successful nanosensor. Confocal images show that OSenB-44 was successfully expressed in vivo and dynamically measured biotin in Escherichia coli, yeast, and human embryonic kidney (HEK)− 293T in real-time.

FRET-based nanosensor AMPfret distinguishes physiological from toxic stress.

Cells can deal with different kinds of stress by responding with compensatory or detoxifying mechanisms. Only when such systems become saturated or overstrained, the stress may emerge as toxic. In energy metabolism, the AMP-activated protein kinase (AMPK) has evolved to detect physiologically relevant energy stress in form of decreased ATP/AMP and ATP/ADP ratios. Once activated, AMPK induces compensatory measures that increase ATP generation and reduce ATP consumption. We have exploited this naturally evolved energy sensing mechanism by engineering the genetically-encoded, FRET-based nanosensor AMPfret. This sensor allows for direct, real-time monitoring of cellular energy state as the cell perceives it. We further developed clonal cell lines with site-specific integration of the AMPfret genes, showing AMPfret expression at suitable levels (Abi Nahed et al., 2023, Meth Mol Biol). With the example of HEK293T cells treated with nanoparticles or prospective anti-cancer drugs, we show that the early time course of the AMPfret signal is predictive for physiological versus toxic stress. While the former leads to only mild or transient increase in the AMPfret signal, the latter triggers a possibly slow but persistent and strong increase in AMPfret signal. Thus, AMPfret seems to be appropriate for a more generalized energy-linked toxicity screening.

Fully Reversible Ratiometric Nanosensors for Continuously Quantifying Mitochondrial Glutathione Concentration in Living Cells.

Mitochondrial glutathione (mGSH) is both the cause of the oxidative damage and a mechanism for maintaining the redox homeostasis in mitochondria. To effectively measure mGSH dynamics in living cells, we have developed a new FRET-based nanosensor by immobilizing rhodamine B into dendritic mesoporous silica nanoparticles and installing GSH probes and mitochondria-targeting motifs onto the surface of nanoparticles. The result shows that these nanosensors show efficient FRET and a full reversibility and rapid response (<10 s) to GSH in the range of 0.5-20 mM, due to their unique nanostructure and well-overlapped spectra. The excellent photostability and low cytotoxicity make them an effective means for monitoring mGSH concentration in real time. When the mGSH nanosensors are used for quantitatively measuring mGSH variations under glucose deprivation stimulation in HeLa cells, they successfully prove themselves a useful tool for mitochondrial redox activity studies.

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor 'MolyProbe'.

Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or toxic symptoms within plants, fungi and animals. Recently, fluorescence resonance energy transfer (FRET) methods have emerged, monitoring cellular and subcellular molybdate distribution dynamics using a genetically encoded molybdate-specific FRET nanosensor, named MolyProbe. Here, we show that the MolyProbe system is a fast and reliable in vitro assay for quantitative molybdate determination. We added a Strep-TagII affinity tag to the MolyProbe protein for quick and easy purification. This MolyProbe is highly stable, resistant to freezing and can be stored for several weeks at 4 °C. Furthermore, the molybdate sensitivity of the assay peaked at low nM levels. Additionally, The MolyProbe was applied in vitro for quantitative molybdate determination in cell extracts of the plant Arabidopsis thaliana, the fungus Neurospora crassa and the yeast Saccharomyces cerevisiae. Our results show the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 and indicate that FRET-based molybdate detection is an excellent tool for measuring bioavailable Mo.

An array-based nanosensor for detecting cellular responses in macrophages induced by femtomolar levels of pesticides.

Environmental agents can induce cellular responses at concentrations far below the limits of detection for current viability and biomarker-based cell sensing platforms. Hypothesis-free cell sensor platforms can be engineered to maximize sensitivity to phenotypic changes, providing a tool for lowering the threshold for detecting cellular changes. Pesticides are one of the most prevalent sources of chemical exposure due to their use in food and agriculture fields. We report here a FRET-based nanosensor array engineered to maximize responses to changes at cell surfaces after pesticide exposure. This sensor array robustly detected macrophage responses to femtomolar concentrations of common pesticides-orders of magnitude lower concentrations than traditional toxicological and biomarker-based strategies. Significantly, this platform was able to classify these responses by pesticide class, demonstrating the ability to distinguish between changes induced by these different agents. Taken together, hypothesis-free cell surface sensing is a promising tool for detecting the effects of ultra-trace environmental chemicals on human health, as well as detecting threshold responses for use in drug discovery and diagnostics.

Capability of novel fluorescence DNA-conjugated CdTe/ZnS quantum dots nanoprobe for COVID-19 sensing

Urgent identification of COVID-19 in infected patients is highly important nowadays. Förster or fluorescence resonance energy transfer (FRET) is a powerful and sensitive method for nanosensing applications, and quantum dots are essential materials in FRET-based nanosensors. The QDs are conjugated to DNA or RNA and used in many applications. Therefore, in the present study, novel fluorescence DNA-conjugated CdTe/ZnS quantum dots nanoprobe designed for detection of Covid-19 after extracting their RNA from saliva of hesitant people. For achieving this purpose, the water-soluble CdTe/ZnS QDs-DNA prepared via replacing the thioglycolic acid (TGA) on the surface of QDs with capture DNA (thiolated DNA) throw a ligand-exchange method. Subsequently, by adding the different concentrations of complementary (target DNA) in a mixture of quencher DNA (BHQ2-labeled DNA) and the QDs-DNA conjugates at different conditions, sandwiched hybrids were formed. The results showed that the fluorescence intensity was decreased with increasing the concentration of target DNA (as a positive control). The linear equation and regression (Y = 40.302 X + 1 and R2 = 0.98) were obtained by using the Stern-Volmer relationship. The Limit of detection (LOD) was determined 0.000823 µM. The achieved results well confirm the outcomes of the RT-PCR method in real samples.

Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells

N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.

Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin

Nanobiotechnological improvements defined on the utilization of biological materials and principles have enormously partaken to revolutionize physical, chemical, and biological sciences. However, the exploration of plant nanobiotechnology is still in its outset. The search for novel tools to monitor plant biomolecules is an emerging issue for the nanobiotechnologists. Given this, a genetically encoded FRET-based nanobiosensor has been developed to monitor the popular plant cardiac glycoside – digoxin, which is used as the most common prescription drug for heart-related illnesses across the world. Digoxin is sourced from the leaves of the foxglove plant (Digitalis purpurea L.) and has a significant demand in the medical sector. Moreover, with the rising popularity of the herbal formulations in the global market, attention towards the authentication and quality control of the herbal drugs is important. Furthermore, digoxin has a very narrow therapeutic range, i.e., 0.6 nM - 2.6 nM. Therefore, strict monitoring of blood digoxin levels is necessary. Besides, previously used techniques for drug authentication and quantification of small-molecule drugs in blood samples are not the best choice available. The nanobiosensor is based on the FRET (Fluorescence Resonance Energy Transfer) mechanism, and it is constructed in such a way that it gives a changed FRET output in the presence of digoxin. Two fluorophores, enhanced cyan fluorescent protein (ECFP) and Venus, were attached on either end of the sensory domain - human nuclear receptor ROR-gamma (RORγt). The developed nanobiosensor was named as fluorescent indicator protein for digoxin, (FLIP-digoxin). The ligand binding affinity of FLIP-digoxin was calculated as 425 μM. Affinity mutants of the FLIP-digoxin were also generated to measure digoxin in wide concentration ranges. This sensor offers high-throughput qualitative analysis of digoxin in Digitalis preparations procured from local drug stores. It confirms the authenticity of the preparations through the detection of digoxin. The FLIP-1n was also able to monitor digoxin concentration in serum samples in lesser than 5 min. The nanobiosensor was found pH stable, digoxin-specific, non- interfered by the biological serum species and can perform high throughput screening of the Digitalis powder, infusion and tincture preparations.

Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor

Ajmalicine is one of the most popular antihypertensive drugs obtained from the root barks of Cathranthus roseus (L.) G. Don and Rauvolfia serpentine (L.) Benth. ex Kurz. It has also potential antimicrobial, cytotoxic, central depressant and antioxidant activities. As the demand for the alkaloid is significantly high, metabolic engineering approaches are being tried to increase its production in both homologous and heterologous systems. The metabolic engineering approach requires knowledge of the metabolic regulation of the alkaloid. For understanding the metabolic regulation, fluxomic analysis is important as it helps in understanding the flux of the alkaloid through the complicated metabolic pathway. The present study was conducted to analyse the flux analysis of the ajmalicine biosynthesis, using a genetically encoded Fluorescent Resonance Energy Transfer FRET-based nanosensor for ajmalicine (FLIP-Ajn). Here, we have silenced six important genes of terpenoid indole alkaloid (TIA), namely G10H, 10HGO, TDC, SLS, STR and SDG, through RNA-mediated gene silencing in different batches of C. roseus suspension cells, generating six silenced cell lines. Monitoring of the ajmalicine level was carried out using FLIP-Ajn in these silenced cell lines, with high spatial and temporal resolution. The study offers the rapid, high throughput real-time measurement of ajmalicine flux in response to the silenced TIA genes, thereby identifying the regulatory gene controlling the alkaloid flux in C. roseus suspension cells. We have reported that the STR gene encoding strictosidine synthase of the TIA pathway could be the regulatory gene of the ajmalicine biosynthesis.

Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor

Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability. The level of GSH varies across the cells, tissues and environmental conditions. However, our current understanding of physiological and pathological GSH changes at high spatial and temporal resolution is limited due to non-availability of practicable GSH-detection methods. In order to measure GSH at real-time, a ratiometric genetically encoded nanosensor was developed using fluorescent proteins and fluorescence resonance energy transfer (FRET) approach. The construction of the sensor involved the introduction of GSH binding protein (YliB) as a sensory domain between cyan fluorescent protein (CFP; FRET donor) and yellow fluorescent protein (YFP; FRET acceptor). The developed sensor, named as FLIP-G (Fluorescence Indicator Protein for Glutathione) was able to measure the GSH level under in vitro and in vivo conditions. When the purified FLIP-G was titrated with different concentrations of GSH, the FRET ratio increased with increase in GSH-concentration. The sensor was found to be specific for GSH and also stable to changes in pH. Moreover, in live bacterial cells, the constructed sensor enabled the real-time quantification of cytosolic GSH that is controlled by the oxidative stress level. When expressed in yeast cells, FRET ratio increased with the external supply of GSH to living cells. Therefore, as a valuable tool, the developed FLIP-G can monitor GSH level in living cells and also help in gaining new insights into GSH metabolism.

Facile construction of AIE-based FRET nanoprobe for ratiometric imaging of hypochlorite in live cells

Fluorescence resonance energy transfer (FRET) nanoprobes based on aggregated induced emission (AIE) molecules are promising for the monitoring of biomolecules in living cells, but this nanoprobe for reactive oxygen species (ROS) has rarely been reported. Here, a novel AIE-based FRET nanoparticles (NPs) is developed for ratiometric detection and imaging of hypochlorite in live cells. Different to common FRET-based nanosensors, the NPs is prepared via facile co-assembly strategy in water using amphiphilic tetraphenylethene (TPE1, energy donor) and thienyl-diketopyrrolopyrrole (NDPP, energy acceptor). This hybrid NPs are uniform spherical, with size of approximately 120 nm. NDPP maintains bright fluorescence inside the hybrid NPs and acts as the reactive unit for hypochlorite in this FRET nanosystem. As a ratiometric fluorescent nanoprobe, the hybrid NPs shows high selectivity to hypochlorite with a detection limit calculated to be 105 nM, thanks to its stable particle size in hypochlorite sensing. Furthermore, the successful ratiometric fluorescent imaging of ClO− in HeLa cells demonstrates that our NPs can effectively pass through the cell membrane and recognize the ClO− in living cells.

Flow Chamber Assay to Image the Response of FRET-Based Nanosensors in Pollen Tubes to Changes in Medium Composition.

Pollen tubes growing in the transmitting tract are presented with an extracellular matrix rich in a variety of substances. The expression of a multitude of genes for transport proteins in the pollen tube indicates that pollen tubes take up at least some of the components provided by the transmitting tract, for example nutrients, ions, or signaling molecules. FRET (Förster resonance energy transfer)-based nanosensors are perfectly suited to study the uptake of these molecules into pollen tubes. They are genetically encoded and can easily be expressed in Arabidopsis pollen tubes. Furthermore, the method is noninvasive and nanosensors for a wide range of substances are available. This chapter will describe the design of plasmids required to generate stable Arabidopsis lines with a pollen tube-specific expression of nanosensor constructs. We also present a method to germinate Arabidopsis pollen tubes in a flow chamber slide that allows the perfusion of the pollen tubes with liquid medium supplemented with the substrate of the nanosensor. Simultaneous evaluation of the FRET efficiency of the nanosensor by confocal microscopy reveals whether the substance is taken up by the pollen tubes. Together with the great number of available nanosensors this method can generate a detailed picture of the substances that are taken up during pollen tubes growth.

Conversion of Cytochrome P450 2D6 of Human Into a FRET-Based Tool for Real-Time Monitoring of Ajmalicine in Living Cells.

Ajmalicine is naturally present in the root bark of Catharanthus roseus L. and Rauvolfia serpentina (L.) Benth ex.Kurz. It has been extensively utilized in the treatment of hypertension across the world. The increased demand, overconsumption, and low content of the alkaloid in the plants have raised the issue of the depletion of natural sources. The metabolic engineering approach has not been successful in improving the content of the ajmalicine because the metabolic regulation of this metabolite is not known. The regulation of a metabolite in the metabolic pathway requires a tool that can carry out real-time measurement of the flux of the metabolite in living system. Given this, the present study was conducted to develop a genetically encoded FRET-based nanosensor by engineering human Cytochrome P450-2D6, an ajmalicine binding protein. The Cytochrome P450-2D6 was sandwiched between two FRET fluorophores. The design of the nanosensor brings two fluorescent proteins in conjunction with the ajmalicine binding protein, such that it undergoes FRET (Fluorescence Resonance Energy Transfer) upon binding of the ligand. The nanosensor, named as FLIP-Ajn (Fluorescence Indicator Protein for Ajmalicine), was pH stable and ajmalicine specific. The affinity of the FLIP-Ajn was 582 μM. The FLIP-Ajn successfully performed real-time measurement of ajmalicine in prokaryotic (bacteria) and eukaryotic systems (yeast, animal cell line, and plant suspension culture), thereby, establishing its biocompatibility in monitoring of ajmalicine in living cells. Besides, several affinity mutants of the nanosensor were generated through mutations in the ajmalicine binding protein to increase the detection range of the nanosensor at varying physiological scales. The non-invasiveness and high spatial and temporal resolution of the tool holds a great significance in the bio-imaging of a highly compartmentalized metabolic pathway. The flux study of ajmalicine will help in identifying the regulatory steps involved in the synthesis of the alkaloids and, hence, will improve the production rate of ajmalicine from its natural sources.

Engineering genetically encoded FRET-based nanosensors for real time display of arsenic (As3+) dynamics in living cells

Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As3+ sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (Kd) of 0.676 × 10−6 M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes in vivo in a non-invasive manner.

Astrocyte Specific Remodeling of Plasmalemmal Cholesterol Composition by Ketamine Indicates a New Mechanism of Antidepressant Action

Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases [cAMP]i in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP]i on vesicular secretion: a rise in [cAMP]i facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine’s rapid antidepressant action.

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

Nickel (Ni2+) is an essential mineral nutrient that is required in trace quantities, but exposure to excess amounts can be toxic or carcinogenic. Mechanisms underlying Ni2+ imbalance and toxicity are poorly understood because most analytical methods currently used to probe this metal are invasive or also have toxic effects. To address these problems, a genetically encoded FRET-based probe for nickel metal (FProNiM) was constructed for real-time detection of Ni2+ in live cells. FProNiM was constructed by sandwiching the bacterial periplasmic nickel-binding protein NikA between the fluorescent protein variants ECFP and Venus. Ni2+ binding to FProNiM induced a conformational change that could be detected by a change in fluorescence resonance energy transfer (FRET) ratio (acceptor/donor fluorescence). In vitro studies demonstrated that FProNiM is specific for Ni2+, insensitive to changes in pH from 5.0 to 8.0, and has an affinity (Kd) of 21.6 μM, which allows detection of Ni2+ concentrations from 0.1 to 5000 μM. The sensor variant FProNiM-5 was also expressed in Escherichia coli (E. coli), yeast, and mammalian cells. In each cell type, changes in Ni2+ concentrations were detected with subcellular resolution using confocal microscopy.

FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 μM and covers the physiological range of 500 nM to 12 μM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.

Study of energy transfer mechanism from CdS quantum dots to Rhodamine 101 in reverse micelle medium

In this study, fluorescence resonance energy transfer (FRET) between CdS quantum dots (QDs) and Rhodamine 101 was investigated in reverse micelles. CdS QDs as donor were synthesized by using reverse micelle method. The particle sizes of CdS QDs were found as 1.18 ± 0.05 for QD1 and 2.11 ± 0.25 nm for QD2 with TEM measurements. It was determined that the fluorescence intensity of the QDs quenched as the concentration of Rh101 increased. The effect of the concentration of dye on the quenching of the fluorescence intensity of QDs was evaluated by the Stern-Volmer approach and KSV and kq values were calculated. It was observed that the quenching was not diffusion controlled. The FRET parameters were also calculated by using fluorescence spectroscopy. The distances between donor-acceptor (r) were also calculated as 4.74 nm for QD1 and 2.26 nm for QD2. The steady-state transfer efficiencies were calculated as 0.24 and 0.53 for QD1 and QD2, respectively. The time-resolved transfer efficiencies were also determined as 0.11 for QD1 and 0.48 for QD2. The results demonstrated that the novel donor-acceptor pairs may play an important role in the applications of many research areas such as FRET-based nanosensors and light harvesting devices.