Fluorescence Resonance Energy Transfer Acceptor Research Articles

Fluorescent Selectivity-Enhanced FRET Based on 3D Photonic Crystals for Multianalyte Sensing.

Fluorescence resonance energy transfer (FRET) finds widespread utility in biochemical sensing, single-molecule experiments, cell physiology, and various other domains due to its inherent simplicity and high sensitivity. Nevertheless, the efficiency of energy transfer between the FRET donor and acceptor is significantly contingent on the local photonic environment, a factor that limits its application in complex systems or multianalyte detections. Here, a fluorescent selectivity-enhanced acridine orange (AO)-aflatoxins (AFs) FRET system based on a range of 3D topological photonic crystals (PCs) was developed with the aim of enhancing the selectivity and discrimination capabilities of FRET. By exploring the angle-dependent characteristics of the photonic stopband, the stopband distribution across different 3D topological PCs pixels was investigated. This approach led to selective fluorescence enhancement in PCs that matched the stopbands, enabling the successful discrimination of six distinct aflatoxins and facilitating complex multianalysis of moldy food samples. In particular, the stopband, which was strategically positioned within the blue-purple structural color range, exhibited a strong alignment with the fluorescence peaks of both the FRET donor and acceptor. This alignment allowed the 3D three-pointed star PCs to be effectively employed for the identification of mixed samples containing six distinct aflatoxins as well as the detection of real aflatoxin samples present in moldy potatoes, bread, oats, and peanuts. Impressively, this approach achieved a remarkable accuracy rate of 100%. This innovative strategy not only presents a novel avenue for developing a multitarget discrimination analysis system but also offers a convenient pretreatment method for the quantitative detection of various aflatoxins.

Green Nanoformulations of Polyvinylpyrrolidone-Capped Metal Nanoparticles: A Study at the Hybrid Interface with Biomimetic Cell Membranes and In Vitro Cell Models.

Noble metal nanoparticles (NP) with intrinsic antiangiogenic, antibacterial, and anti-inflammatory properties have great potential as potent chemotherapeutics, due to their unique features, including plasmonic properties for application in photothermal therapy, and their capability to slow down the migration/invasion speed of cancer cells and then suppress metastasis. In this work, gold (Au), silver (Ag), and palladium (Pd) NP were synthesized by a green redox chemistry method with the reduction of the metal salt precursor with glucose in the presence of polyvinylpyrrolidone (PVP) as stabilizing and capping agent. The physicochemical properties of the PVP-capped NP were investigated by UV-visible (UV-vis) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies, dynamic light scattering (DLS), and atomic force microscopy (AFM), to scrutinize the optical features and the interface between the metal surface and the capping polymer, the hydrodynamic size, and the morphology, respectively. Biophysical studies with model cell membranes were carried out by using laser scanning confocal microscopy (LSM) with fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) techniques. To this purpose, artificial cell membranes of supported lipid bilayers (SLBs) made with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) dye-labeled with 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD, FRET donor) and/or lissamine rhodamine B sulfonyl (Rh, FRET acceptor) were prepared. Proof-of-work in vitro cellular experiments were carried out with prostate cancer cells (PC-3 line) in terms of cytotoxicity, cell migration (wound scratch assay), NP cellular uptake, and cytoskeleton actin perturbation.

Glutathione-Responsive Tannic Acid-Assisted FRET Nanomedicine for Cancer Therapy.

In cancer combination therapy, a multimodal delivery vector is used to improve the bioavailability of multiple anti-cancer hydrophobic drugs. Further, targeted delivery of therapeutics along with simultaneous monitoring of the drug release at the tumor site without normal organ toxicity is an emerging and effective strategy for cancer treatment. However, the lack of a smart nano-delivery system limits the application of this therapeutic strategy. To overcome this issue, a PEGylated dual drug, conjugated amphiphilic polymer (CPT-S-S-PEG-CUR), has been successfully synthesized by conjugating two hydrophobic fluorescent anti-cancer drugs, curcumin (CUR) and camptothecin (CPT), through an ester and a redox-sensitive disulfide (-S-S-) linkage, respectively, with a PEG chain via in situ two-step reactions. CPT-S-S-PEG-CUR is spontaneously self-assembled in the presence of tannic acid (TA, a physical crosslinker) into anionic, comparatively smaller-sized (~100 nm), stable nano-assemblies in water in comparison to only polymer due to stronger H-bond formation between polymer and TA. Further, due to the spectral overlap between CPT and CUR and a stable, smaller nano-assembly formation by the pro-drug polymer in water in presence of TA, a successful Fluorescence Resonance Energy Transfer (FRET) signal was generated between the conjugated CPT (FRET donor) and conjugated CUR (FRET acceptor). Interestingly, these stable nano-assemblies showed a preferential breakdown and release of CPT in a tumor-relevant redox environment (in the presence of 50 mM glutathione), leading to the disappearance of the FRET signal. These nano-assemblies exhibited a successful cellular uptake by the cancer cells and an enhanced antiproliferative effect in comparison to the individual drugs in cancer cells (AsPC1 and SW480). Such promising in vitro results with a novel redox-responsive, dual-drug conjugated, FRET pair-based nanosized multimodal delivery vector can be highly useful as an advanced theranostic system towards effective cancer treatment.

Force-Induced Shuttling of Rotaxanes Controls Fluorescence Resonance Energy Transfer in Polymer Hydrogels

The molecular shuttling function of rotaxanes can be exploited to design mechanoresponsive reporter molecules. Here, we report a new approach to such rotaxane-based mechanophores, in which the fluorescence resonance energy transfer (FRET) between a donor-acceptor pair is mechanically controlled. A cyclic molecule containing a green-light-emitting FRET donor connected to a red-light-emitting FRET acceptor was threaded onto an axle equipped with a quencher at its center and two stoppers in the peripheral positions. In the force-free state, the green emitter is located near the quencher so that charge transfer interactions or photo-induced electron transfer between the two moieties suppress green emission and prevent the FRET from the green to the red emitter. The mechanophore was covalently incorporated into a linear polyurethane-urea (PUU), and stretchable hydrogels were prepared by swelling this polymer with water. Upon deformation of the PUU hydrogels and under an excitation light that selectively excites the donor, the intensity of the red fluorescence increases, as a result of a force-induced separation of the green emitter from the quencher, which enables the FRET. The switching contrast is much more pronounced in the gels than in dry films, which is due to increased molecular mobility and hydrophobic effects in the hydrogel, which both promote the formation of inclusion complexes between the ring containing the green emitter and the quencher.

Fluorogenic response from DNA templated micrometer range self-assembled gold nanorod.

Plasmonic gold nanorod (AuNR) on a macromolecular matrix exhibits an end-to-end (ETE) long-range self-assembly (AuNR)n with n > 100. In the case of small molecules as a template, the pre-synthesized macromolecular matrix is missing and this brings a synthetic challenge in directed long-range assembly of AuNR. Self-assembly with thiol-modified small DNA and AuNR shows a much short-range ETE assembly with n < 25 via a simple evaporation technique on a solid surface. In this study, the introduction of two short amine modified probe DNAs (∼2.5 nm) and one 22-mer complementary single strand (ss)-DNA template (∼7 nm) show the long-range ETE self-assembly of (AuNR)n with n > 130. In the solution state, the zigzag arrangement within the assembled structure controls the typical change in the absorption behavior for (AuNR)n ETE assembly. The formation of this long-range ETE self-assembly in a solution state was verified from the combined effect of fluorescence resonance energy transfer (FRET) and hotspot-induced fluorescence enhancement. The probe DNAs and templated DNA concentration on fluorescence enhancement have been varied to monitor the effect of (AuNR)n with n = ∼5-130 in ETE self-assembly. Primarily quenched FRET acceptor in the presence of AuNR decisively exhibits remarkable fluorogenic response in ETE self-assembly with maximum n value. Although the FRET efficiencies among the fluorophores are comparable, the fluorogenic boost in ETE AuNR is due to the increased number of intrinsic navigated hotspots in these assemblies.

A versatile cellulose nanocrystal‑carbon dots architecture: Preparation and environmental/biological applications

In this work, a versatile cellulose nanocrystal‑carbon dots (CNC-CDs) architecture is prepared and applied in environmental remediation and biological sensing. The citric acid (CA) is employed to extract CNCs and in-situ synthesize blue-emissive CDs. The carboxyl-rich surface of CNC-CDs enables the chelation of Hg2+ and fluorescence quenching of CDs, realizing the detection and adsorption of Hg2+ with a film comprised of the architecture. The limit of detection (LOD) is measured as 12.8 nM with satisfying retention efficiency. The versatility of CNC-CDs is demonstrated by the fabrication of a fluorescence resonance energy transfer (FRET) sensor using m-tetrakis(4-sulfonatophenyl)porphine (TSPP) as FRET acceptor and Fe3+ receptor. Fluorescence of the sensor is responsive to Fe3+ in a ratiometric manner, which significantly contributes to the visualized and sensitive Fe3+ detection (6.9 nM). In combination with excellent biocompatibility, the FRET sensor is capable of imaging intracellular Fe3+.

An imidazo[1,5-α]pyridines-based ratiometric fluorescent probe for sensing sulfur dioxide derivatives in real samples based on a FRET mechanism

A novel fluorescence resonance energy transfer (FRET)-based ratiometric emission fluorescent probe AT was designed and developed in which the imidazo[1,5-α]pyridine was served as a FRET donor and tricyanofuran (TCF) as the FRET acceptor to detect SO32-/HSO3- based on the Michael addition reaction. Probe AT had a high energy transfer efficiency (95%) and a large pseudo-Stokes shift (259 nm) in EtOH/PBS buffer (5/5, v/v). It also possessed good selectivity and quick response to SO32-/HSO3-. There was good linearity between the ratio of fluorescence intensity (F499/F645) and the concentrations of SO32-/HSO3- in the ranges of 1.5–7.5 μM and 9–20 μM, with calculated detection limits (LOD) of 55 nM. In addition, the probe could also detect the concentrations of SO32-/HSO3- in real samples such as environmental water and sugar, allowing the probe to be used in a variety of applications.

Dual Detection of Hemagglutinin Proteins of H5N1 and H1N1 Influenza Viruses Based on FRET Combined With DNase I.

Influenza A viruses (IAV) are classified based on their surface proteins hemagglutinin (HA) and neuraminidase (NA). Both pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses pose a significant threat to public health. Effective methods to simultaneously distinguish H1N1 and H5N1 are thus of great clinical value. In this study, a protocol for detection of HA proteins of both H1N1 and H5N1 was established. Specifically, we designed an aptasensor for HA using fluorescence resonance energy transfer (FRET) strategy combined with DNase I-assisted cyclic enzymatic signal amplification. HA aptamers of H1N1 and H5N1 IAVs labeled with various fluorescent dyes were used as probes. Graphene oxide (GO) acted as a FRET acceptor for quenching the fluorescence signal and protected aptamers from DNase I cleavage. The fluorescence signal was recovered owing to aptamer release from GO with HA protein. DNase I-digested free aptamers and HA proteins were able to further interact with more fluorescent aptamer probes, resulting in increased signal amplification. The limits of detection (LOD) of H5N1 HA and H1N1 HA were 0.73 and 0.43 ng/ml, respectively, which were 19 and 27 times higher than LOD values obtained with the DNase I-free system. The recovery rate of HA protein in human serum samples ranged from 88.23 to 117.86%, supporting the accuracy and stability of this method in a complex detection environment. Our rapid, sensitive, and cost-effective novel approach could be expanded to other subtypes of IAVs other than H1N1 and H5N1.

Imaging specific cell-surface sialylation using DNA dendrimer-assisted FRET

Sialylation plays a vital role in multiple different physiologic processes, aberrant sialylation is highly related to disease development. Especially in cancer development, changed states of specific cell-surface sialylation implies rich cancer-related information. Therefore, it is necessary to image specific cell-surface sialylation for better understanding biological functions of sialylation. To meet this purpose, we designed a DNA dendrimer-assisted fluorescence resonance energy transfer (FRET) strategy in this work. By labeling multiple FRET donors and acceptors on the target molecules through metabolic oligosaccharide engineering (MOE) and targeted recognition of aptamer-tethered DNA dendrimer, the FRET was significantly improved. With the DNA dendrimer-assisted FRET strategy, specific imaging of cell-surface sialylation on SMMC-7721 and CEM cells were successfully achieved. The obtained FRET signal intensity was approximately four times higher than the control without the assistance of DNA dendrimer. Moreover, this method is competent to monitor changed states of PTK7-specific sialylation induced by tunicamycin. The proposed imaging strategy may provide a powerful tool to explore the physiological roles of specific cell-surface sialylation and the related mechanism of diseases.

Development of a fluorescence sensing platform for specific and sensitive detection of pathogenic bacteria in food samples

Pathogenic bacteria cause disease outbreaks and threaten human health, which prompts the research on advanced detection assays. Herein, based on the fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) combined with aptamers and black hole quencher 1 (BHQ-1) ligated with cDNA, a fluorescence sensing platform was developed for specific and sensitive detection of pathogenic bacteria. The desirable distance (less than 10 nm) between UCNPs (donor) and BHQ-1 (acceptor) for FRET was achieved via the base pairing of aptamer and cDNA. Furthermore, the fluorescence emission spectrum of UCNPs and the absorption spectrum of BHQ-1 overlapped well, which enabled the occurrence of FRET and caused fluorescence quenching. Stable stem-loop structures might be the structural basis for the aptamer to specifically recognize target bacteria. In the presence of target bacteria, UCNPs-aptamer preferentially captured the bacteria, which decreased the UCNPs-aptamer-cDNA-BHQ-1 complex and caused fluorescence recovery. Using this sensing platform, we successfully detected one strain of S. aureus, ATCC 29213, with a limit of detection (LOD) of 6 cfu/mL in the linear range of 36–3.6 ⅹ107 cfu/mL and have verified this method in relevant food samples with good recoveries. In the future, the developed assay could also be used to detect other targets by selecting proper aptamers, which further broadens the potential applications of this method.

Highly efficient fluorescence resonance energy transfer in co-encapsulated BODIPY nanoparticles

Fluorescence resonance energy transfer (FRET) is a powerful tool used in a wide range of applications due to its high sensitivity and many other advantages. Co-encapsulation of a donor and an acceptor in nanoparticles is a useful strategy to bring the donor-acceptor pair in proximity for FRET. A highly efficient FRET system based on BODIPY-BODIPY (BODIPY: boron-dipyrromethene) donor-acceptor pair in nanoparticles was synthesized. Nanoparticles were formed by co-encapsulating a green emitting BODIPY derivative (FRET donor, lmax = 501 nm) and a red emitting BODIPY derivative (FRET acceptor, lmax = 601 nm) in an amphiphilic polymer using the precipitation method. Fluorescence measurements of encapsulated BODIPY in water following 501 nm excitation caused a 3.6 fold enhancement of the acceptor BODIPY emission at 601 nm indicating efficient energy transfer between the green emitting donor BODIPY and the red emitting BODIPY acceptor with a 100 nm Stokes shift. The calculated FRET efficiency was 96.5%. Encapsulated BODIPY derivatives were highly stable under our experimental conditions.

Gold-Bipyramid-Based Nanothernostics: FRET-Mediated Protein-Specific Sialylation Visualization and Oxygen-Augmenting Phototherapy against Hypoxic Tumor.

Despite several attempts, incorporating biological detection that supplies necessary biological information into therapeutic nanotheranostics for hypoxic tumor treatments is considered to be in its infancy. It is therefore imperative to consolidate biological detection and desirable phototherapy into a single nanosystem for maximizing theranostic advantages. Herein, we develop a versatile nanoprobe through combined fluorescence resonance energy transfer (FRET) and oxygen-augmenting strategy, namely APT, which enables glycosylation detection, O2 self-sufficiency, and collaborative phototherapy. Such APT nanoprobes were constructed by depositing platinum onto gold nano-bipyramids (Au NBPs), linking FITC fluorophore-labeled AS1411 aptamers for introducing FRET donors, and by conjugating G-quadruplex intercalated with TMPyP4 to their surfaces via the SH-DNA chain. By installing FRET acceptors on the glycan of targeted EpCAM glycoprotein using the metabolic glycan labeling and click chemistry, FRET signals appear on the cancerous cell membranes, not normal cells, when donors and acceptors are within an appropriate distance. This actualizes protein-specific glycosylation visualization while revealing glycan-based changes correlated with tumor progression. Interestingly, the deposited platinum scavenges excessive H2O2 as artificial nanoenzymes to transform O2 that alleviates tumor hypoxia and simultaneously elevates singlet oxygen (1O2) for inducing cancer cell apoptosis. Notably, the significant hyperthermia devastation was elicited via APT nanoprobes with phenomenal photothermal therapy (PTT) efficiency (71.8%) for thermally ablating cancer cells, resulting in synergistically enhanced photodynamic-hyperthermia therapy. Consequently, APT nanoprobes nearly actualized thorough tumor ablation while demonstrating highly curative biosafety. This work offers a new paradigm to rationally explore a combined FRET and oxygen-augmenting strategy with a focus on nanotheranostics for hypoxic tumor elimination.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Design of a ratiometric fluorescence sensor based on metal organic frameworks and Ru(bpy)32+-doped silica composites for 17β-Estradiol detection

17β-Estradiol (E2), an important endocrine disrupting compound, could be quantitatively detected by fluorescence resonance energy transfer (FRET) aptasensor, designed in this paper. Metal organic frameworks have large specific surface area and easily modifiable groups, which are helpful for the construction of aptasensor. Specifically, streptavidin was immobilized on the synthesized MIL-53-NH2 by covalent bonding, and further linked with the biotin modified E2 aptamer (apt) through specific bonding between avidin and biotin to obtain the FRET donor probe (MIL-53-apt). Meanwhile, complementary DNA (cDNA) modified Ru(bpy)32+-doped silica nanoparticles (RuSiO2-cDNA) were prepared through covalent bonding. They acted as the FRET acceptor probe, since its absorption spectrum showed large overlap with the emission spectrum of MIL-53-apt. In the presence of E2, aptamer modified donor probes tended to bind with E2, owing to their higher selectivity and affinity. Therefore, the optimal distance between FRET pairs was broken, resulting in the fluorescence emission recovery of donor and the fluorescence emission of acceptor decreased. Under optimal conditions, this proposed aptasensor displayed sensitive detection of E2 ranging from 0.5 to 1000 nM with a detection limit of 0.2 nM. Furthermore, the sensor provides a promising method for rapid and sensitive detection of other small biological molecules.

Amyloid-β Peptides Disrupt Interactions Between VAMP-2 and SNAP-25 in Neuronal Cells as Determined by FRET/FLIM.

Synaptic dysfunction prevalent in Alzheimer's disease (AD) brain is closely associated with increased accumulation of amyloid-β (Aβ) peptides in the brain parenchyma. It is widely believed that Aβ peptides trigger synaptic dysfunction by interfering with the synaptic vesicular fusion and the release of neurotransmitters, primarily facilitated by the SNARE protein complexes formed by VAMP-2, SNAP-25, and syntaxin-1. However, Aβ interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood. Our objective is to elucidate mechanisms by which Aβ peptides perturb SNARE complexes. Intensity (qualitative) and lifetime (quantitative) based measurements involving Forster (fluorescence) resonance energy transfer (FRET) followed by fluorescence lifetime imaging microscopy (FLIM) were employed to investigate the effect of Aβ peptides on dynamic interactions between VAMP-2, labeled with cerulean (Cer) at the N-terminus (FRET donor), and SNAP-25 labeled with citrine (Cit) on the N-terminus (FRET acceptor). The FRET and FLIM interactions at the exocytosis locations on the pre-synaptic membrane were recorded under spontaneous and high potassium evoked conditions. Moreover, cellular accumulation of fluorescein labeled Aβ (F-Aβ) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy. The F-Aβ40 and F-Aβ42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aβ40 and Aβ42 decrease interactions between the N-termini of Cer-VAMP2 and Cit-SNAP25 in N2A cells, as determined by FRET/FLIM. By perturbing the N-terminal interactions between VAMP-2 and SNAP-25, Aβ40 and Aβ42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.

PH-responsive intramolecular FRET-based self-tracking polymer prodrug nanoparticles for real-time tumor intracellular drug release monitoring and imaging.

An intramolecular fluorescence resonance energy transfer (FRET)-based macromolecular theranostic prodrug was designed by directly conjugating Doxorubicin (DOX) as the FRET acceptor onto the naphthalimide side groups in the fluorescent copolymer PPEGMA20-PNAP8 as the FRET energy donor via an acid-labile imine bond, without a fluorogenic linker. The proposed PPEGMA20-PNAP8-DOX theranostic prodrug showed a high DOX content of 24.3% owing to a conjugation efficiency of>93% under mild conjugation conditions. It could easily self-assemble into unique theranostic nanoparticles with a Dh of 71nm. The theranostic nanoparticles showed excellent pH-triggered DOX release performance with very low premature drug leakage of 6.3% in normal physiological medium over 129h, while>91% of the conjugated DOX was released in the acidic tumor intracellular microenvironment. MTT assays indicated the enhanced antitumor efficacy of the proposed theranostic nanoparticles compared with free DOX. Furthermore, because drug release was triggered by pH, orange fluorescence was restored to the blue fluorescence of the backbone copolymer. Such self-tracking pH-responsive colorful fluorescence variations during intracellular drug delivery and release are expected to allow real-time tumor intracellular drug release monitoring and imaging diagnosis.

A FRET-based near-infrared ratiometric fluorescent probe for detection of mitochondria biothiol

A new fluorescent probe A with BODIPY as FRET donor and near-infrared rhodamine as FRET acceptor is constructed through disulfide bonding and use for ratiometric fluorescence detection of biothiol. Due to the efficient fluorescence resonance energy transfer (FRET) from BODIPY donor to near-infrared rhodamine acceptor, Probe A only displays near-infrared rhodamine fluorescence (λem = 656 nm) under BODIPY excitation at 480 nm. The presence of biothiol leads to BODIPY fluorescence increases (λem = 511 nm) and near-infrared rhodamine fluorescence decreases since the disulfide bond of the probe is broken by biothiols, effectively separating the donor from the acceptor, thus inhibiting the FRET process. Probe A exhibits remarkable high selectivity and excellent linear relationship from 10 μM to 100 μM of GSH, with low detection limit as 0.26 μM. Cellular imaging experiments shows that the probe is predominantly present in mitochondria and has been successfully applied to detect biothiol concentrations changes in mitochondria of living cells.

Dual function of Eosin Y in miniemulsion polymerization: Stabilizer and FRET acceptor

Recent studies have shown that water-soluble fluorescent dyes can be used as miniemulsion stabilizers to avoid a series of deficiencies in traditional stabilizers. However, visual means to demonstrate the stabilizing performance of water-soluble dyes are still scarce. In this study, the principle of fluorescence resonance energy transfer (FRET) is combined with dual emission of fluorescent polystyrene nanoparticles prepared by miniemulsion polymerization. For this purpose, 4-(1,2,2-triphenylethyl) phenylundecene-10-enoate (TPE-Aly) was employed as aggregation induced emission (AIE) dye and donor and Eosin Y as acceptor and stabilizer. The polymer molecular weight, particle size and size distribution of the nanoparticles were optimized by regulating the amount of Eosin Y and ultrasonication time, respectively. The average number of Eosin Y molecules per nanoparticle was firstly estimated by combining dialysis and Lambert-Beer law. FRET between TPE-Aly and Eosin Y indicates the presence of Eosin Y on the surface of the polymer nanoparticles. With increasing concentration of Eosin Y, the as-prepared dual-emission nanoparticles display a decreasing particle size along with an enhancing FRET efficiency. Moreover, various stability tests show that Eosin Y can be used as an excellent stabilizer for miniemulsion polymerization and has broad application prospects in the field of functional materials research.

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.

Gated Mesoporous Silica Nanocarriers for Hypoxia-Responsive Cargo Release.

Mesoporous silica nanocarriers (MSNs) are appealing in terms of their large cavity surface area and high loading capacity, but they have been suffering from premature cargo release. Herein, we report a gated smart MSN that is sensitive to low oxygen concentration (i.e., hypoxia) via taking advantage of the superior electron-accepting ability of the azobenzene moiety. The azobenzene polymer was employed as the responsive gate-keeper that was deposited on the MSN surface, followed by coating with amphiphilic Pluronic F68 for steric stabilization. The obtained nanocarriers were less than 200 nm. The in vitro polymer degradation was spectrophotometrically witnessed via the employment of a reducing agent, namely, sodium dithionite, with astrong electron-donating ability. The hypoxia-responsive cargo release from the gated MSN was quantitatively demonstrated in breast cancer cells (MCF-7) using the fluorescence resonance energy transfer (FRET) technique where coumarin 6 and rhodamine B was selected as the FRET donor and acceptor, respectively. The FRET ratio was used as the index and decreased linearly over time under hypoxia, whereas it almost remained steady under normoxia. In addition, a model photosensitizer, namely, chlorin e6, was also loaded in the gated MSN whose toxicity under hypoxia was verified. This study developed a hypoxia-responsive MSN with the azobenzene polymer as the removable gate-keeper, which would expand the application of MSNs in pharmaceutical and biomedical areas since thelow oxygen concentration is a unique trigger in many pathological conditions.