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.

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.

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.

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。

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。

荧光共振能量转移上转换适配体探针法检测牛奶中的痕量氯霉素

本研究合成了亲水性稀土掺杂的上转换荧光纳米颗粒(UCNPs)作为荧光能量的供体,金纳米颗粒(AuNPs)作为荧光能量的受体,基于荧光共振能量转移(FRET)体系,建立了检测痕量氯霉素(CAP)的适配体探针方法。UCNPs在波长980 nm激发下,在波长547 nm处有特征发射光。柠檬酸钠还原法制备的AuNPs在波长519 nm处有吸收峰。UCNPs通过链霉亲和素-生物素放大系统连接CAP适配体,形成荧光供体探针(UCNPs-apt),AuNPs与修饰巯基的CAP互补链连接,形成荧光受体探针(AuNPs-cDNA)。由于适配体和cDNA的碱基互补配对,发生FRET导致UCNPs荧光猝灭,加入CAP后部分荧光恢复。在最优的检测条件下,CAP浓度与荧光恢复值具有良好的线性关系,线性范围为0.01~10 ng/mL,检测限为5 pg/mL。采用本方法对牛奶样品进行加标回收实验,回收率在93.5%~99.4%之间,相对标准偏差为1.18%~2.84%。该方法具有简单、特异性强、灵敏度高、抗荧光背景干扰能力强等特点。

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 。

量子点:FRET的新发展

荧光共振能量转移(FRET)技术作为一种高效的光学“分子尺”,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。但是许多有机染料吸收光谱较窄而发射光谱较宽,并且光漂白现象比较严重,使得FRET的应用受到了限制,因此迫切需要寻找新的能量供-受体对。由于量子点(QDs)相对于有机染料有很多优点,可以较好地应用于FRET,可能成为FRET领域发展的一个有意义的新方向,近来已引起了人们的关注。本文就FRET的原理以及量子点应用于FRET的最新进展情况做了评述。

用FRET技术研究TLR4和MD-2的相互作用

目的利用荧光共振能量转移(FRET)技术在活体细胞研究人Toll样受体(TLR)4与髓样细胞分化蛋白2(MD-2)的相互作用。方法用PCR方法扩增TLR4编码序列和MD-2编码序列(不包括信号肽序列)并分别亚克隆入带TLR4信号肽的增强型青色荧光蛋白和增强型黄色荧光蛋白表达载体(pECFP-C1-SP,pEYFP-C1-SP)中,重组质粒经酶切、序列鉴定分析后分别或共同转染HEK293细胞,用荧光显微镜观察荧光蛋白表达和分布情况,并用常规的方法和受体光漂白的方法对共表达青色荧光蛋白(CFP)-TLR4和黄色荧光蛋白(YFP)-MD-2的细胞进行FRET分析。结果重组质粒在HEK293细胞中得到表达,仅转染pECFP/TLR4或pEYFP/MD-2的细胞可见青色或黄色荧光主要分布在胞质内,以核周较多;而共转染pECFP/TLR4和pEYFP/MD-2的细胞可同时检测到青色和黄色荧光,主要分布在细胞膜,同时胞质也有少量表达。无论是用常规的方法还是受体光漂白的方法,对共表达CFP-TLR4和YFP-MD-2的细胞进行FRET分析结果表明有FRET现象的发生,提示TLR4和MD-2有相互作用并形成复合物。结论本研究为TLR4和MD-2在活体细胞中的相互作用提供了直接的证据。

利用FRET技术在活细胞内观察EGF对PKA作用的时空成像

cAMP依赖的蛋白激酶(protein kinase A, PKA)在细胞生长与分化过程中扮演重要角色,特别是在调节Ras信号通路引起的细胞增殖效应中起着重要作用。为了在活细胞内动态观察表皮生长因子(epidermal growth factor, EGF)对PKA的作用,采用一种可以检测PKA酶活性的报告蛋白(A-kinase activity reporter, AKAR)——这种报告蛋白是利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理设计的,使其在人类肺癌细胞(ASTC-a-1)中稳定表达。加入EGF刺激因子后,随时间变化的成像分析显示出在活细胞生理条件下被EGF作用的PKA酶活性变化的时空信息。这些资料为EGF作用PKA提供了直接的实时证据。

FRET的理论基础及应用

The structural and functional study of protein is a major topic of current functional genomics. Fluorescence resonance energy transfer (FRET) is one of few tools available for measuring nanometer scale distances and changes in distances in vivo . FRET is an ideal technology for detection of protein conformation and protein-protein interaction by using fluorescence protein, traditional organic dyes and other dyes as probes. It uses fluorescence protein, traditional organic dyes and other dyes as its probes. The application of FRET in the determination of intracellular events would be helpful for us to understand the structure and function of biology molecules. [

在活细胞中应用FRET技术研究TGFβ/TβRI信号传导

转移生长因子β(TGFβ)信号传导通路参与调节细胞的增殖、分化、凋亡、细胞迁移等一系列细胞过程,与骨代谢疾病的发病机制密切相关.本研究根据荧光共振能量转移(FRET)技术原理,构建包含CFP-TβRI-YFP融合蛋白的TβRI生物传感器,转染293T细胞,观察转染效率.以TGFβ1为诱导剂,激活TGFβ/TβRI信号传导通路,在活细胞生理条件下,动态监测TβRI生物传感器的FRET效应.结果表明,成功构建了TβRI生物传感器,转染细胞效率达50%,在TGFβ1诱导刺激6min后,FRET效率增加并达到最大值,该过程经历9 min后,随时间的延长,FRET效率下降.研究结果表明:在活细胞生理条件下,TGFβ1/TβRI信号转导过程存在一定的时间特异性.

玄参水提取物对高糖暴露条件下INS-1细胞AMPK的激活作用

目的研究玄参水提取物(AESN)对高糖(HG)条件下INS-1细胞腺苷酸活化蛋白激酶(AMPK)活性的影响。方法将INS-1细胞培养于HG培养基中,并给予不同浓度AESN共孵育处理;利用细胞计数试剂-8(CCK-8)法检测AESN干预对细胞增殖/活力和焦亡小体形成的影响,使用Western blotting法观察AESN对细胞内AMPK表达及磷酸化水平的影响;利用时间分辨-荧光共振能量转移(TR-FRET)实验检测AESN对AMPK激酶活性的影响。结果CCK-8检测结果显示同正常培养条件相比,HG暴露显著降低INS-1细胞增殖/活力并增加焦亡小体形成数量,Western blotting检测结果表明HG暴露可导致细胞内AMPK磷酸化水平下降;而AESN共孵育可呈浓度依赖性地增加INS-1细胞增殖/活力、抑制焦亡小体形成并激活AMPK。TR-FRET实验结果表明,AESN可浓度依赖性增加AMPK激酶活性。结论AESN对HG暴露条件下INS-1细胞AMPK具有激活作用。