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.

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.

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.

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.

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.

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。

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.

A carbon nanoparticle-peptide fluorescent sensor custom-made for simple and sensitive detection of trypsin

Herein,we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles(CNPs)modified by acid oxidation.The fluorescence of the fluorescein-labelled peptide was quenched by CNPs.The sensor reacted with trypsin to cleave the peptide,resulting in the release of the dye moiety and a substantial increase in fluorescence intensity,which was dose-and time-dependent,and trypsin could be quantified accordingly.Correspondingly,the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity,with a detection limit of 0.7 mg/mL.The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range,suitable for point-of-care testing.Furthermore,the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.

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。

用于血清胰岛素测定的荧光共振能量传递(FRET)免疫检测法的建立

目的研究探讨新型荧光染料Dylight(DL)和金纳米颗粒(AuNP)间荧光共振能量转移(FRET)效应,并将其与免疫竞争法结合构建一种新型胰岛素荧光免疫检测方法。方法分别将胰岛素抗原(Ag)和胰岛素抗体(Ab)与DL和AuNP进行偶联,形成DL-Ag偶联物和AuNP-Ab偶联物。利用它们间产生的FRET效应以及免疫竞争反应,建立针对胰岛素的新型荧光免疫检测方法。然后对该检测方法的检测性能进行评估和实际样品的应用研究。结果该荧光免疫检测方法不仅表现出较高的检测灵敏度(0.015 ng/mL),较快的检测时间(4 min)以及较好的检测特异性,而且成功地用于血清胰岛素的检测,回收率在96.9%~121.1%之间。结论新型荧光染料DL和AuNP间FRET效应可应用于血清胰岛素的荧光免疫检测。

基于四甲基联苯胺和吖啶橙间荧光共振能量转移的比率型荧光测定左氧氟沙星

基于3,3’,5,5’(四甲基联苯胺(3,3’,5,5’-tetramethylbenzidine,TMB)和吖啶橙(AO)之间荧光共振能量转移(FRET),建立了一种快速、低背景干扰、高灵敏度测定盐酸左氧氟沙星(LVF)的新型比率型荧光探针。在pH 5.0 NaAc-HCl缓冲溶液中,在310 nm的光激发下,TMB在350~500 nm处的荧光光谱和AO的吸收光谱重叠。以TMB作为能量供体,AO作为能量受体,构建了FRET体系。根据能量转移理论,该体系的荧光共振能量转移效率为62.5%,供体-受体间距离为2.17 nm,进一步说明TMB和AO之间发生了FRET。当在体系中加入LVF后,TMB将荧光能量转移给LVF,LVF又作为供体将能量转移给AO。LVF在TMB和AO之间起到桥梁作用,LVF将吸收的TMB荧光能量转移给AO,使得TMB荧光强度明显降低,AO的荧光强度则显著增加,从而提高了体系的FRET效率。在最优实验条件下,F546 nm与F402 nm之比与LVF浓度(2~80μmol·L^(-1))之间存在良好的线性关系,线性回归方程为F_(546 nm)/F_(402 nm)=87.916c+3.108,线性相关系数为0.9993,检出限(LOD)为15.7 nmol·L^(-1)。一些常见的阳离子(K^(+),Mg^(2+),Ca^(2+),Cu^(2+),Mn^(2+),Zn^(2+),Co^(2+),Ni^(2+),Cr^(3+)等)、阴离子(F^(-),Br^(-),NO_(3)^(-),IO_(3)^(-),CO_(3)^(2-),SO_(4)^(2-)等)、糖类(葡萄糖,蔗糖和淀粉)、药物(谷胱甘肽,抗坏血酸和异烟肼)和几种氨基酸(甘氨酸,亮氨酸,半胱氨酸等)均不干扰LVF的测定,表明该比率型荧光探针对LVF具有高选择性。该方法用于商用药物制剂中LVF含量的测定,加标回收率在93%~97%之间。该比率型荧光探针在临床研究中对LVF的检测具有较大的应用潜力,为开发一种简便、选择性和灵敏的检测药物制剂中LVF含量的传感器提供了较好的理论依据,同时为提高LVF临床用药的安全性与合理性水平提供了一定的方法。

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.

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.

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.

基于荧光共振能量转移效应的荧光传感器在真菌毒素检测中的应用

真菌毒素对人和动物具有剧毒和致癌性,且预防和控制其对食品造成的污染较为困难,因此人们对真菌毒素的关注度越来越高。检测食品中的真菌毒素十分必要,传统检测方法的检测结果准确、可靠,但所需设备昂贵、检测时间长,不符合快速检测真菌毒素的要求。因此,需要开发出快速、灵敏、准确且经济的真菌毒素检测方法。基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)效应的荧光传感器由于操作简单、反应速度快、结果可靠且成本低而广泛应用于检测行业。本文主要介绍了基于FRET效应荧光传感器的检测机制,综述了该传感器在真菌毒素检测中的应用情况,提出了目前荧光传感器仍存在的问题并对其未来的发展趋势进行了展望,以期为新型荧光传感器的设计及真菌毒素检测时灵敏度的优化提供参考。

FRET技术及其在蛋白质-蛋白质分子相互作用研究中的应用

简要综述了FRET方法在活细胞生理条件下研究蛋白质 蛋白质间相互作用方面的最新进展 .蛋白质 蛋白质间相互作用在整个细胞生命过程中占有重要地位 ,由于细胞内各种组分极其复杂 ,因此一些传统研究蛋白质 蛋白质间相互作用的方法 ,例如酵母双杂交、免疫沉淀等可能会丢失某些重要的信息 ,无法正确地反映在当时活细胞生理条件下蛋白质 蛋白质间相互作用的动态变化过程 .荧光共振能量转移 (fluorescenceresonanceenergytransfer ,FRET)是近来发展的一项新技术 ,此项技术的应用 ,为在活细胞生理条件下对蛋白质 蛋白质间相互作用进行实时的动态研究 。

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.