A ratiometric fluorescent probe for hypoxanthine detection in aquatic products based on the enzyme mimics and fluorescence of cobalt-doped carbon nitride

A ratiometric fluorescent probe for hypoxanthine(Hx)detection was established based on the mimic enzyme and fluorescence characteristics of cobalt-doped graphite-phase carbon nitride(Co doped g-C_(3)N_(4)).In addition to emitting strong fluorescence,the peroxidase activity of Co doped g-C_(3)N_(4)can catalyze the reaction of O-phenylenediamine and H_(2)O_(2)to produce diallyl phthalate which can emit yellow fluorescence at 570 nm.Through the decomposition of Hx by xanthine oxidase,Hx can be indirectly detected by the generating hydrogen peroxide based on the measurement of fluorescent ratio I(F_(570)/F_(370)).The linear range was 1.7-272.2 mg/kg(R^(2)=0.997),and the detection limit was 1.52 mg/kg(3σ/K,n=9).The established method was applied to Hx detection in bass,grass carp,and shrimp,and the data were verified by HPLC.The result shows that the established probe is sensitive,accurate,and reliable,and can be used for Hx detection in aquatic products.

A pyrene-based ratiometric fluorescent probe with a large Stokes shift for selective detection of hydrogen peroxide in living cells

Hydrogen peroxide(H2O2)plays a significant role in regulating a variety of biological processes.Dysregulation of H2O2 can lead to various diseases.Although numerous fluorescent imaging probes for H2O2 have been reported,the development of H2O2 ratiometric fluorescent probe with large Stokes shift remains rather limited.Such probes have shown distinct advantages,such as minimized interference from environment and improved signal-to noise ratio.In this work,we reported a new pyrene-based compound Py-VPB as H2O2 fluorescent probe in vitro.The probe demonstrated ratiometric detection behavior,large Stokes shift and large emission shift.In addition,the probe showed high sensitivity and selectivity towards H2O2 in vitro.Based on these excellent properties,we successfully applied Py-VPB to the visualization of exogenous and endogenous H2O2 in living cells.Cell imaging study also showed that our probe was localized in the mitochondria.We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H2O2 in living systems.

Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

The global pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus has necessitated rapid,easy-to-use,and accurate diagnostic methods to monitor the virus infection.Herein,a ratiometric fluorescence enzyme-linked immunosorbent assay(ELISA)was developed using Si-fluorescein isothiocyanate nanoparticles(FITC NPs)for detecting SARSCoV-2 nucleocapsid(N)protein.Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane(APTES)-FITC as the Si source.This method did not need post-modification and avoided the reduction in quantum yield and stability.The p-nitrophenyl(pNP)produced by the alkaline phosphatase(ALP)-mediated hydrolysis of pnitrophenyl phosphate(pNPP)could quench Si fluorescence in Si-FITC NPs via the inner filter effect.In ELISA,an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody.ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs.The change in fluorescence intensity ratio could be used for detecting N protein,with a wide linearity range(0.01-10.0 and 50-300 ng/mL)and low detection limit(0.002 ng/mL).The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum.Moreover,this proposed method can accurately distinguish coronavirus disease 2019(COVID-19)and non-COVID-19 patient samples.Therefore,this simple,sensitive,and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.

Design and applications of carbon dots-based ratiometric fluorescent probes:A review

Ratiometric fluorescence(FL)probes can eliminate the background interference and provide more accurate detection results than single emission intensity-based nanoprobes.Recently,carbon dots(CDs)-based ratiometric FL probes have received extensive research attention due to their excellent biocompatibility,water solubility,and multi-emission capabilities.In this review,we firstly summarize the construction strategies of CDs-based ratiometric FL probes,including physical mixing,nanohybrid,and dual-emitting CDs strategies.Additionally,we classify the sensing types of CDs-based ratiometric FL probes into five categories according to the difference in spectral variation caused by analytes:“single-response-ON”,“single-response-OFF”,“doubleresponses-ON”,“double-responses-OFF”,and“double-responses-Reverse”types.Finally,a thorough overview of CDs-based ratiometric FL probe applications in ions,molecules,pH,and temperature sensing is provided.We believe this review can show the latest research progress of CDs-based ratiometric FL sensing fields and provide perspectives on future developments for the construction of CDs-based ratiometric FL probes and their potential applications.

Dual-emission carbonized polymer dots for ratiometric sensing and imaging of L-lysine and pH in live cell and zebrafish

It is highly desired to accurately and selectively detect and image intracellular L-lysine and pH in biological systems because they could act as the biomarkers in certain abnormal conditions and may give us a warning of the occurrence of diseases.It has been attracted more focuses to design new ratiometric fluorescent probe for monitoring L-lysine and pH to improve detection accuracy.Carbonized polymer dots(CPDs),which possess carbon/polymer hybrid structure rather than pure carbon structure and constitute of a carbon core and large amounts of functional groups/polymer chains on the surface,rise up as a new type of fluorescent nanomaterials and especially display many advantages for bioanalysis.In this study,o-phenylenediamine(o-PD)and poly(styrene-co-maleic anhydride)(PSMA)are used as the precursors to synthesize the desired CPDs through one-step hydrothermal amide method.The prepared CPDs display two well-resolved fluorescence emission bands,i.e.,a very weak emission centered at 470 nm in blue region and a strong emission centered at 558 nm in yellow region.It is found that the two emissions are both responsive to L-lysine based on the surface passivation mechanism,whereas,only the yellow emission is responsive to pH due to the protonation/deprotonation process of the amino groups.Based on the different responsive behaviors,ratiometric detection and imaging of L-lysine and pH are achieved.The prepared ratiometric CPDs probe is successfully applied for L-lysine and pH sensing and imaging at two emission channels in live cell and zebrafish with satisfactory results.

Ratiometric fluorescent Si-FITC nanoprobe for immunoassay of SARS-CoV-2 nucleocapsid protein

Coronavirus disease 2019(COVID-19)highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission.Here,we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles(NPs)for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)nucleocapsid protein(N protein).The synthesis of Si-FITC NPs did not need post-modification,which addressed the issue of quantum yield reduction during the coupling reaction.Si-FITC NPs showed two distinct peaks,Si fluorescence atλem=385 nm and FITC fluorescence atλem=490 nm.In the presence of KMnO_(4),Si fluorescence was decreased and FITC fluorescence was enhanced.Briefly,in the presence of N protein,catalase(CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates.Subsequently,hydrogen peroxide(H_(2)O_(2))and Si-FITC NPs/KMnO_(4)were injected into the microplate together.The decomposition of H_(2)O_(2)by CAT resulted in remaining of KMnO_(4),which changed the fluorescence intensity ratio of Si-FITC NPs.The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL,and the detection limit was 0.003 ng/mL,which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL.The N protein concentration can be accurately determined in human serum.Furthermore,the COVID-19 and non-COVID-19 patients were distinguishable by this method.Therefore,the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity.

Monitoring the pH fluctuation of lysosome under cell stress using a near-infrared ratiometric fluorescent probe

Cell stress responses are associated with numerous diseases including diabetes, neurodegenerative diseases, and cancer. Several events occur under cell stress, in which, are protein expression and organellespecific pH fluctuation. To understand the lysosomal pH variation under cell stress, a novel NIR ratiometric pH-responsive fluorescent probe(BLT) with lysosomes localization capability was developed.The quinoline ring of BLT combined with hydrogen ion which triggered the rearrangement of π electrons conjugated at low pH medium, meanwhile, the absorption and fluorescent spectra of BLT showed a red-shifts, which gived a ratiometric signal. Moreover, the probe BLT with a suitable p Kavalue has the potential to discern changes in lysosomal pH, either induced by heat stress or oxidative stress or acetaminophen-induced(APAP) injury stress. Importantly, this ratiometric fluorescent probe innovatively tracks pH changes in lysosome in APAP-induced liver injury in live cells, mice, and zebrafish. The probe BLT as a novel fluorescent probe possesses important value for exploring lysosomal-associated physiological varieties of drug-induced hepatotoxicity.

A benzothiazole-based ratiometric fluorescent probe with large Stokes shifts for quantitative profiling of sulfite in real samples and living cells

Rapid,selective,and quantitative profiling of SO_(3)^(2-)is a prerequisite for investigating its effect on different physiopathological processes.To address this challenge,our team prepared a ratiometric fluorescent probe HBTCS,based on 4-(benzothiazol-2-yl)-3-hydroxybenzaldehyde for specific detection of SO_(3)^(2-)under a singlewavelength excitation.The solution of probe HBTC-S itself exhibited orange fluorescence(λ_(em)=614 nm),and became green fluorescent(λ_(em)=471 nm)after the addition of SO_(3)^(2-).The vitro experiments suggested that the probe displayed high sensitivity,excellent selectivity,and a low detection limit(0.15μM).Moreover,probe HBTC-S was successfully applied for qualitative and quantitative monitoring of SO_(3)^(2-)in realistic samples and living HeLa cells in a ratiometric manner.

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。

Single-wavelength Excited Ratiometric Fluorescence pH Probe to Image Intracellular Trafficking of Tobacco Mosaic Virus

As a typical plant virus which has biocompatibility and high transfection efficiency,tobacco mosaic virus(TMV)has shown broad application potential in drug or gene delivery field.Elucidating its intracellular trafficking is of great importance in investigation of its cytotoxicity,targeting site,and delivery efficiency,and is advantageous to designing new TMV-based drug delivery systems with different targets.By taking advantage of the regulated pH value of different organelles in a mammalian cell,we exploit a pH detection strategy to investigate the intracellular trafficking pathway of TMV.Here,we report a single-wavelength excited ratiometric fluorescent pH probe.This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate(FITC)and pH-insensitive rhodamine B isothiocyanate(RBIRC)onto the inner surface of TMV.The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents.By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy,it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes.This work may provide important guidance on construction of TMV-based nano carriers.

基于镧系金属-有机骨架材料的比率型荧光探针研究进展

荧光探针能够快速、可靠的识别目标物,因此备受人们关注。与传统单发射中心型荧光探针相比,比率型荧光探针具有更高的灵敏度、高可靠性及可视化传感等优点。镧系金属-有机框架物(Ln-MOFs)具有优异的发光性质,在荧光传感、生物成像等方面具有广阔应用前景。通过合理选择镧系金属离子、有机配体及客体分子可以构筑具有优异发光性能的Ln-MOFs基双发射比率型荧光探针。对Ln-MOFs基双发射比率型荧光探针的构建方法、荧光传感机理及应用领域的最新进展进行综述,并对其未来发展进行了展望,以期为构筑Ln-MOFs基双发射比率型荧光探针提供借鉴。

基于Eu^(3+)-生物质碳量子点探针的比率型荧光传感体系检测盐酸四环素

该文以橙皮为原料制备的碳量子点(CDs)与铕离子(Eu^(3+))构建双发射比率型荧光探针(Eu^(3+)-CDs)并用于抗生素盐酸四环素的检测。在370 nm激发波长下,Eu^(3+)-CDs荧光探针在425 nm处出现较强的蓝色荧光峰,在617 nm处出现较弱的红色荧光峰。随着盐酸四环素浓度的逐渐增大,Eu3+与盐酸四环素螯合形成的配合物增多,在425 nm处的CDs荧光(I_(F425))逐渐减弱,而617 nm处铕配合物的荧光(I_(F617))逐渐增强,可基于荧光强度比I_(F617)/I_(F425)的变化对盐酸四环素进行测定。考察了Eu^(3+)的浓度、pH值和反应时间对探针荧光强度的影响。在最佳条件下,该双发射比率型荧光探针对盐酸四环素具有特异性识别能力,荧光强度比(I_(F617)/I_(F425))与盐酸四环素浓度在10~100µmol/L范围内存在良好的线性关系,相关系数r^(2)=0.9926,检出限为5.09µmol/L。该方法成功用于猪肉中盐酸四环素含量的检测,加标回收率为102%~110%,相对标准偏差为0.20%~2.4%,具有较好的实用性。

检测活性氧小分子比例荧光探针研究进展

活性氧物种(ROS)如过氧化氢(H_(2)O_(2))、次氯酸(HClO)、过氧化亚硝基阴离子(ONOO^(-))、超氧阴离子(O_(2)^(-))、羟基自由基(HO·)、单线态氧(^(1)O_(2))等,它们是广泛存在于生物体内具有高反应活性的含氧物质。活性氧物种在生物体内的时空分布在癌症、关节炎、阿尔兹海默症等疾病领域的研究得到空前的关注。而比例型活性氧荧光探针以其高灵敏度、准确度、多通道原位成像的优势为这一领域提供了有力的工具。比例型荧光探针的设计需要借助多种光物理特性,本文就光物理特性分类按照分子内电荷转移(ICT)、荧光共振能量转移(FRET)、激发态分子内质子转移(ESIPT)、跨键能量转移(TBET)等介绍活性氧比例型荧光探针的最新进展。

一种新型比率荧光毛细管pH传感器的制备及性能

实时便捷的pH检测对于环境监测和医学诊断等领域具有重要应用价值。本文通过溶胶-凝胶法制备了一种比率荧光毛细管pH传感器。该传感器以2.8-羟基芘-1,3,6-三磺酸三钠盐(8-hydroxy-pyrene-1,3,6-tri-sulfonate,HPTS)作为pH敏感的荧光探针,利用HPTS与十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide,CTAB)结合形成HPTS-IP离子对,然后将离子对分散于溶胶-凝胶中,并将其固定于毛细管内壁即制得比率荧光毛细管pH传感器。该传感器利用HPTS在双激发带下的发射强度比值实现比率荧光检测,当pH从5.0上升至8.0时,HPTS的荧光强度比率随pH值增加逐渐增强,pKa值为6.95,通过分析HPTS的比率荧光强度变化可间接监测pH波动。该传感器具有较好的pH敏感性、稳定性和可逆性,且可快速、灵活、便捷地进行实际操作,在环境保护和生物医学领域的pH监测分析方面拥有良好的应用前景。

一种用于过氧亚硝酸根检测的近红外比率荧光探针的研制

设计合成了一种基于半花菁的近红外比率荧光探针分子Cy-P,并将其用于活细胞中的外源性和内源性过氧亚硝酸根(ONOO-)荧光成像检测.研究结果表明:探针分子Cy-P自身具有较大的π共轭结构,其最大发射波长在近红外区域;加入ONOO-后,强氧化性的ONOO-会导致Cy-P的π共轭体系被破坏,并生成蓝色荧光物质,最大发射波长蓝移了268 nm;探针分子Cy-P对ONOO-线性检测范围为0~15μmol/L,检测下限为13 nmol/L;该探针分子对ONOO-的识别性能优于其他各种可能干扰的生物分析物;该探针分子具有低细胞毒性,适用于活细胞中的外源性和内源性ONOO-的成像检测.

Rare-earth ions coordination enhanced ratiometric fluorescent sensing platform for quantitative visual analysis of antibiotic residues in real samples

Levofloxacin(LVFX)as a representative drug of quinolone antibiotics is widely used in clinical,and its residues enriched in water bodies and sideline products seriously damage human health.It is imperative to develop a real-time/on-site sensing method for monitoring residual antibiotics.Here,we report a portable sensing platform by utilizing a composite fluorescent nanoprobe constructed by the cerium ions(Ce^(3+))coordination functionalized Cd Te quantum dots(QDs)for the visual and quantitative detection of LVFX residues.This fluorescent probe provides a distinct color variation from red to green,which shows a good linear relationship to LVFX residues concentrations in the range of 0-6.0μmol/L with a sensitive limit of detection(LOD)of 16.3 nmol/L.The smartphone platform with Color Analyzer App installed,which could accomplish quantified detection of LVFX in water,milk,and raw pork with a LOD of 27.9nmol/L.The facile sensing method we proposed realizes rapid visualization of antibiotics residual in the environment and provides a practical application pathway in food safety and human health.

生菜源比率型荧光碳量子点制备及其对生活用水中Cu^(2+)检测研究

Cu^(2+)的定量检测对于保护自然环境及维持人体健康具有十分重要的意义。目前,Cu^(2+)的荧光检测法大多为单峰检测,易受到背景信号干扰而影响检测结果。实验以生菜为生物质碳源,通过溶剂热法合成了Cu^(2+)特异性检测比率型碳点,线性范围为0.8~70.0 μmol·L^(-1),最低检出限为0.449 μmol·L^(-1),最低定量限为1.45 μmol·L^(-1)。合成的碳点在生活用水中的Cu^(2+)检测中显示出优异性能,回收率为96.5%~103.5%,相对标准偏差小于5%。

双发射荧光碳点的简易合成及其在pH检测中的应用

以绿茶茶叶渣为碳源,通过简单易操作的微波法制备了水溶液中呈现双发射的茶叶基碳点(T-CDs),两发光峰分别位于520nm和678nm附近,绿光520nm左右的发射峰随激发波长的增大而红移,红光发射峰随激发波长的改变不移动。研究发现,双峰对pH值的敏感程度不同,随着缓冲溶液pH值的增大,该碳点的绿色荧光逐渐增强,而红色荧光相对减弱。因此,利用T-CDs的双发射峰的强度比作为pH的测定信号构建了比率型pH荧光探针,结果表明,该荧光探针不仅亲水性好、响应快、检测范围宽(pH值4-10),而且可以实现裸眼可见的比色检测,在pH值检测领域具有广阔的应用前景。

A ratiometric fluorescent probe for the detection of hypochlorous acid in living cells and zebra fish with a long wavelength emission

A ratiometric fluorescence probe,NClO,for the rapid and selective detection of HClO had been designed and synthesized based on a 1,8-naphthalimide derivative.Probe NClO displayed a red emission(λmax=615 nm).In the presence of HClO,the solution of probe NClO gave off a strong green fluorescence(λem,max=520 nm)with a rapid response(within seconds).This probe had been applied to image HClO in living cells and zebra fish.

Revealing HOC1 burst from endoplasmic reticulum in cisplatin-treated cells via a ratiometric fluorescent probe

The reactive oxygen species(ROS) are tightly associated with endoplasmic reticulum(ER) stress.Thus,the deep and visual insight of aberrant ROS fluctuations in the ER can help us better investigate the ER stress-associated pathology.In this work,a fluorescent probe ERC for HOCI detection in the ER based on phenothiazine-derived coumarin platform was developed.In the presence of HOCI,ERC exhibited an emission change from 609 nm to 503 nm within seconds.It also showed high sensitivity(0.44 μmol/L)and superb photostability.Significantly,ERC displayed low cytotoxicity,good cell membrane permeability,and appreciable ER-targetability.Ultimately,the probe was successfully utilized to image exogenous and endogenous HOCl in living cells and reveal the HOCI burst in cisplatin-treated cancer cells.