Monitoring the in vivo siRNA release from lipid nanoparticles based on the fluorescence resonance energy transfer principle

The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization.However,the in vivo distribution and release of siRNA still cannot be effectively monitored.In this study,based on the fluorescence resonance energy transfer(FRET)principle,a fluorescence dye Cy5-modified survivin siRNA was conjugated to nanogolds(Au-DR-siRNA),which were then wrapped with lipid nanoparticles(LNPs)for monitoring the release behaviour of siRNA in vivo.The results showed that once Au-DR-siRNA was released from the LNPs and cleaved by the Dicer enzyme to produce free siRNA in cells,the fluorescence of Cy5 would change from quenched state to activated state,showing the location and time of siRNA release.Besides,the LNPs showed a significant antitumor effect by silencing the survivin gene and a CT imaging function superior to iohexol by nanogolds.Therefore,this work provided not only an effective method for monitoring the pharmacokinetic behaviour of LNP-based siRNA,but also a siRNA delivery system for treating and diagnosing tumors.

A mini review and hypothesis for coronavirus detection using photonics: surface enhanced Raman scattering and fluorescence resonance energy transfer

COVID-19 has devastated numerous nations around the world and has overburdened numerous healthcare systems,which has also caused the loss of livelihoods due to prolonged shutdowns and further led to a cascading effect on the global economy.COVID-19 infections have an incubation period of 2–7 days,but 40 to 45%of cases are asymptomatic or show mild to moderate respiratory symptoms after the period due to subclinical lung abnormalities,making it more likely to spread the pandemic disease.To restrict the spread of the virus,on-site diagnosis methods that are quicker,more precise,and easily accessible are required.Rapid Antigen Detection Tests and Polymerase Chain Reaction tests are currently the primary methods used to determine the presence of COVID-19 viruses.These tests are typically time-consuming,not accurate,and,more importantly,not available to everyone.Hence,in this review and hypothesis,we proposed equipment that employs the properties of photonics to improve the detection of COVID-19 viruses by taking the advantage of typical binding of coronavirus with angiotensin-converting enzyme 2(ACE2)receptors.This hypothetical model would combine Surface-Enhanced Raman Scattering(SERS)and Fluorescence Resonance Energy Transfer(FRET)to provide great flexibility,high sensitivities,and enhanced accessibility.

Highly-efficient quantitative fluorescence resonance energy transfer measurements based on deep learning

Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.

Investigation of fluorescence resonance energy transfer ultrafast dynamics in electrostatically repulsed and attracted exciton-plasmon systems

Following the gradual maturation of synthetic techniques for nanomaterials,exciton-plasmon composites have become a research hot-spot due to their controllable energy transfer through electromagnetic fields on the nanoscale.However,most reports ignore fluorescence resonance energy transfer(FRET)under electrostatic repulsion conditions.In this study,the FRET process is investigated in both electrostatic attraction and electrostatic repulsion systems.By changing the Au:quantum dot ratio,local-field induced FRET can be observed with a lifetime of ns and a fast component of hundreds of ps.These results indicate that the intrinsic transfer process can only elucidated by considering both steady and transient state information.

Retraction Note:Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

Retraction note:Khan M,Rauf W,Habib F,Rahman M,Iqbal M.Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry.World J Hepatol 2020;12(11):976-992 PMID:33312423 DOI:10.4254/wjh.v12.i11.976.The online version of the original article can be found at https://www.wjgnet.com/1948-5182/full/v12/i11/976.htm.

A novel fluorescence sensor for milk clotting enzyme chymosin using peptide as substrate and covalent organic framework nanosheet as fluorescence quencher

Chymosin is one of the critical enzymes in cheese making.Herein,we proposed a novel fluorometric assay for chymosin determination.Firstly,covalent organic frameworks(COF)were synthesized and exfoliated to 2-dimensional COF nanosheets(COF NS)by ultrasound treatment.Gold nanoparticles(Au NPs)were loaded with COF NS to prepare AuNPs/COF NS(Au@COF NS).Secondly,rhodamine B(RhB)modified substrate peptide(Pep)for chymosin was linked with Au@COF NS to construct a Pep-Au@COF NS nanocomposite.For the sensing principle,fluorescence of RhB was quenched by Au@COF NS and the fluorescence intensity was weak due to the fluorescence resonance energy transfer between COF NS and RhB of Pep.However,in the presence of chymosin,the RhB was released by specific cleavage of the substrate peptide by chymosin and resulted in the recovery of fluorescence.The increased fluorescence intensity was proportional to the increase of chymosin concentration and thus a“turn on”fluorescent sensor for chymosin was constructed.The sensor showed a linear range in the concentration of 0.05-60.00μg/mL for the detection of chymosin with a detection limit of 20 ng/mL.The sensor was used to quantify chymosin in rennet product with good selectivity,which has the potential applications in cheese manufacturing.

基于FRET成像探究棕榈酰化修饰调节Fyn激酶活性

目的探讨棕榈酰化修饰调节非受体酪氨酸激酶Fyn活性的分子机制。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时检测细胞中的Fyn活性,并结合棕榈酰化位点缺失和共转染蛋白质酪氨酸激酶(C-terminal Src kinase,CSK)表达质粒研究其分子机制。结果实验发现,(C3,C6)任一位点的棕榈酰化缺失能引起Fyn的高活性表达,且C6位点影响更显著。已知CSK激活后发生膜转移,FRET检测证实其对细胞中的Fyn活性有下调作用,但不能有效调控(C3,C6)棕榈酰化位点缺失的Fyn(GSS)活性。结论本文结果初步支持了Fyn活性受细胞内的物理空间定位分布的一种调控机制假设,即棕榈酰化缺失的Fyn(GSS)受细胞膜上CSK抑制性的调节作用被减弱,从而促进了组成性的高活性表达。

Fluorescence resonance energy transfer-based nanomaterials for the sensing in biological systems

The applications of fluorescence resonance energy transfer(FRET)are coming to be one of the simplest and most accessible strategy with super-resolved optical measurements.Meanwhile,nanomaterials have become ideal for constructing FRET-based system,due to their unique advantages of tunable emission,broad absorption,and long fluorescence(FL)lifetime.The limitations of traditional FRET-based detections,such as the intrinsic FL,auto-FL,as well as the short FL lifetime,could be overcome with nanomaterials.Consequently,numbers of FRET-based nanomaterials have been constructed for precise,sensitive and selective detections in biological systems.They could act as both energy donors and/or acceptors in the optical energy transfer process for biological detections.Some other nanomaterials would not participate in the energy transfer process,but act as the excellent matrix for modifications.The review will be roughly classified into nanomaterial-involved and uninvolved ones.Different detection targets,such as nucleic acids,pathogenic microorganisms,proteins,heavy metal ions,and other applications will be reviewed.Finally,the other biological applications,including environmental evaluation and mechanism studies would also be summarized.

A Highly Selective and Sensitive Ratiometric Fluorescent Probe for pH Measurement Based on Fluorescence Resonance Energy Transfer

Fluorescein-rhodamine 6G（Flu-Rh） was synthesized and used as the fluorescence probe for pH measurement based on fluorescence resonance energy transfer（FRET）. In the probe, fluorescein fluorophore and pH-sensitive rhodamine 6G hydrazide were used as FRET donor and acceptor, respectively. The values of acidity constant（pKa） and fluorescence quantum yield（Ф） of Flu-Rh were 3.71 and 0.72, respectively. The fluorescence efficiency of Flu-Rh remains almost constant when the pH value of the sample solution changed 10 times in a range of 4.78-7.03 continuously. The present probe is simple and easy-to-use for the pH measurement in acidic media.

Exploring ratiometric endolysosomal pH nanosensors with hydrophobic indicators responding at the nanoscale interface and multiple fluorescence resonance energy transfers

Compared with conventional water-soluble fluorescence probes,pH-sensitive fluorescent nanosensors based on hydrophobic indicators remain largely unexplored.We report here the unique pH response of the nanosensors with a hydrophobic indicator(Ch3,a Nile Blue derivative)in polymeric nanoparticles(NPs).At the aqueous-organic interface of the NPs,spectral overlap and dye accumulation caused significant Förster resonance energy transfer(FRET)not only between the protonated and deprotonated Ch3(hetero-FRET),but also between the protonated and deprotonated Ch3 themselves(homo-FRET).The pH response was simulated according to an interfacial response mechanism and the dynamic range was found to depend on the size of the NPs and dye distribution(Kp).Therefore,adjusting the size of the NPs and the local dye concentration gave rise to a series of dynamic sensing ranges with apparent pKa values from 2.7 to 9.6 based on a single indicator.The nanosensors were successfully delivered to HeLa cells to monitor subcellular pH values in the endosomes and lysosomes.Based on cellular calibrations,the average pH in the organelles were determined to be ca.4.7.Moreover,the pH neutralization process during lysosome membrane permeabilization(LMP)induced by hydrogen peroxide stimulation was also successfully visualized with the nanosensors.

Protease-activated quantum dot probes based on fluorescence resonance energy transfer

A novel sensing system based on fluorescence resonance energy transfer (FRET) between CdTe quantum dots (QDs) and Rhoda-mine B (RB) was established for the detection of matrix metalloproteinases (MMOL/LPs). In this system, 535-nm-emitting quantum dots (QDs) were bound to Rhodamine B (RB) via a MMOL/LP-specific peptide. A 76% reduction in luminescence was achieved because of FRET. Release of RBs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 73% rise in luminescence over 60 min. The design platform of the nanosensor is flexible and can be fine-tuned for a wide array of applications such as the detection of biomarkers, early diagnosis of disease, and monitoring therapeutic efficacy simply by changing the sequence of the peptide linker.

A water-soluble fluorescence resonance energy transfer probe for hypochlorous acid and its application to cell imaging

A water-soluble fluorescence resonance energy transfer (FRET) probe for hypochlorous acid (HOCl), dansyl rhodamine B piperazinoacetohydrazide, was designed, synthesized and characterized. The dansyl moiety in the probe acted as a FRET donor and the rhodamine moiety acted as a FRET acceptor. The two moieties were connected by a HOCl-cleavable active bond, and cleavage of this linker decreased the FRET efficiency and increased the fluorescence intensity of the donor at 501 nm. The water solubility of the probe was improved compared with other probes by introduction of the cationic rhodamine fluorophore. As a result, the probe could be used to detect HOCl in aqueous biosystems with a linear range of 2-10 mol/L and a detection limit of 80 nmol/L (signal- to-noise = 3). The probe was successfully applied to fluorescence imaging of HOCl in HeLa cells.

Interaction of Surface-active Fluorescence Probes with Bovine Serum Albumin

The binding between three surface-active substituted 3H-indole fluorescence probes and bovine serum albumin (BSA) in aqueous solution was studied using fluorescence quenching. The binding constants of 3H-indole molecules with BSA were obtained. According to the F?rster resonance energy transfer theory, the distances between 3H-indole molecules and tryptophan of BSA were calculated. The results show that the oligoethyloxyethylene chain of 3H-indole molecules is longer, the binding between them is stronger, the energy transfer efficiency is higher, and the distance between tryptophan and 3H-indole is nearer.

Construction of triblock copolymer-gold nanorod composites for fluorescence resonance energy transfer via pH-sensitive allosteric

To explore the effects of microenvironmental adjustments on fluorescence,a pH-sensitive nanocomposite system based on fluorescence resonance energy transfer(FRET)was constructed.The model system included a modified triblock copolymer(polyhistidine-b-polyethylene glycol-b-polycaprolactone)and gold nanoparticles.A near-infrared dye was used as the donor,and spectrally matched gold nanorods,attached after C-terminus modification with α-lipoic acid,were used as the receptor to realize control of the FRET effect over the fluorescence intensity for two polymer configurational changes(i.e.,"folded"and"stretched"states)in response to pH.After synthesis and characterization,we investigated the self-assembly behavior of the system.Analysis by quartz crystal microbalance revealed the pH sensitivity of the polymer,which exhibited"folding"and"stretching"states with changes in pH,providing a structural basis for the FRET effect.Fluorescence spectrophotometry investigations also revealed the regulatory impact of the assembled system on fluorescence.

A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assaying the activity of alkaline phosphatase (AP) by using a phos-phate-binding tag molecule, Phos-tag {1,3-bis[bis(pyridine-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}, attached to a nonfluorescent 4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl (Dabcyl: λmax 475 nm) dye group. The fluorogenic biomolecule riboflavin 5’-phosphate (FMN: λem 525 nm) was used as an AP substrate. The Dabcyl-labeled Phos-tag specifically captured FMN to form a stable 1:1 complex, resulting in efficient fluorescence quenching. The quenching efficiency was more than 95% for a mixture of 12 μM FMN and 13.5 μM Dabcyl-labeled Phos-tag in aqueous solution at pH 7.4 and 25°C. When FMN was dephosphorylated with AP, riboflavin was released into the solution and fluorescence from the flavin moiety appeared. By using this quenching system, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.

利用FRET技术检测不同力学条件下T细胞中ERK活性的脉冲现象

目的探索Jurkat T细胞中胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)活性动力学以及基质刚度对ERK活性的影响。方法利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术实时观测Jurkat细胞中ERK活性的变化,或细胞处于I型胶原基质胶中检测其影响。结果部分Jurkat细胞中存在ERK活性脉冲现象,频率约为3次/h,FRET振幅变化约为20%。在抗体激活T细胞抗原受体(T-cell receptor,TCR)的条件下,ERK脉冲依然存在,频率和振幅无显著变化。当细胞处于I型胶原水凝胶中,随着胶基质刚度增加,脉冲频率有所下调。结论Jurkat T细胞中存在自发的ERK活性脉冲现象,初步实验显示其频率受基质刚度影响。而该信号波动的生理意义和分子机制仍有待探索。

Lineal DNA logic gate for microRNA diagnostics with strand displacement and fluorescence resonance energy transfer

Designing molecular logic gates to operate programmably for molecular diagnostics in molecular computing still remains challenging.Here,we designed a novel linear DNA logic gates for microRNA analysis based on strand displacement and fluorescence resonance energy transfer（FRET）.Two labeled strands closed each other produce to FRET through hybridization with a complementary strand to form a basic work unit of logic gate.Two indicators of heart failure（microRNA-195 and microRNA-21） were selected as the logic inputs and the fluorescence mode was used as the logic output.We have demonstrated that the molecular logic gate mechanism worked well with the construction of YES and AND gates.

Tuning of Förster Resonance Energy Transfer in Metal–Organic Frameworks: Toward Amplified Fluorescence Sensing

The assembly of Förster resonance energy transfer(FRET)donor and acceptor for amplified fluorescence sensing has been considered a big challenge.Herein,by using the multivariate approach,we report the design and synthesis of a series of FRET-based metal–organic frameworks(MOFs)with variable donor fluorophore-to-the-acceptor ratios.