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.

Engineering an asymmetric rhodamine dye suitable for developing ratiometric fluorescent probe

Fluorescent probes based on rhodamine skeleton are extensively used in biological imaging.However,the construction of ratiometric fluorescent probes based on the rhodamine skeleton without introducing additional fluorophores is still challenging.Herein,we propose an effective method to construct a rhodamine-based ratiometric fluorescent probe through the regulation of electron cloud density.A ratiometric fluorescent probe RDQF-RB-NTR was successfully constructed for the detection of nitroreductase(NTR).RDQF-RB-NTR exhibits good sensitivity,high selectivity,and ratiometric response to NTR.Cell imaging experiments showed that RDQF-RB-NTR can rapidly and accurately detect the fluctuation of NTR in cells and difference of NTR levels between normal cells and cancer cells.In addition,RDQF-RB-NTR was successfully applied to the imaging of NTR in liver tissue slices,and we found that the level of NTR was upregulated in liver cirrhosis.

Design of quinoline-based fluorescent probe for the ratiometric detection of cadmium in aqueous media

A new fluorescent probe,DQCd2,based on 4-piperidinoquinoline has been synthesized as a fluorescent probe for Cd^2＋.It can ratiometrically respond to Cd^2＋ in PBS buffer by a remarkable emission intensity enhancement（88-fold） and wavelength shift（70 nm）.

An activatable ratiometric near-infrared fluorescent probe for hydrogen sulfide imaging in vivo

Ratiometric fluorescent probes hold great promise for in vivo imaging;however,stimuli-activatable ratiometric probes with fluorescence emissions in near-infrared(NIR)region are still very few.Herein,we report a hydrogen sulfide(H_2S)-activatable ratiometric NIR fluorescent probe(1-SPN)by integrating a H_2S-responsive NIR fluorescent probe 1 into a H_2S-inert poly[2,6-(4,4-bis-(2-ethylhexyl)-4 H-cyclopenta[2,1-b;3,4-b′]dithiophene)-alt-4,7(2,1,3-benzothiadiazole)](PCPDTBT)-based NIR semiconducting polymer nanoparticle(SPN).1-SPN shows"always on"PCPDTBT fluorescence at 830 nm and weak probe 1 fluorescence at 725 nm under excitation at 680 nm.The ratio of NIR fluorescence intensities between 725 and 830 nm(I_(725)/I_(830))is small.Upon interaction with H_2S,the fluorescence at 725 nm is rapidly switched on,resulting in a large enhancement of I_(725)/I_(830),which is allowed for sensitive visualization and quantification of H_2S concentrations in living cells.Taking advantage of enhanced tissue penetration depth of NIR fluorescence,1-SPN is also applied for real-time ratiometric fluorescence imaging of hepatic and tumor H_2S in living mice.This study demonstrates that activatable ratiometric NIR fluorescent probes hold great potential for in vivo imaging.

Ir(Ⅲ)-based Ratiometric Hypoxic Probe for Cell Imaging

Two new ratiometric hypoxia probes(Ir-C343 and Ir-GFP)are synthesized by covalently incorporating florescent internal standard molecules coumarin 343(C343)and green fluorescent protein(GFP)into bis[1-(9,9-dimethyl-9H-fluoren-2-yl)-isoquinoline](succinylacetone)Ir(Ⅲ)(Ir-fliq),respectively.After connecting with internal standard molecules,the Ir-fliq moiety still exhibits high sensitivity to oxygen concentration,while the fluorescence intensity of the internal standard remains relatively constant under different oxygen concentrations.As a result,a ratiometric response is realized that is only related to oxygen concentration.In addition,Ir-GFP shows more promising applications in the ratiometric hypoxia imaging of cells due to its long excitation wavelength,good water solubility,high biocompatibility,and low relative fluorescence intensity compared with the phosphorescent emitter Ir-fliq.

A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging.Often,a small probe-product spectral separation leads to crosstalk,but the rational development of ratiometric probes with zero probe-product crosstalk remains challenging.Harnessing the recent progress on photophysical modulation of xanthenoid fluorochromes,we propose a powerful and versatile probe design principle,that is,“bridging-group modification,”and developed totalROX,a robust probe for monitoring the total cellular oxidative capacity.

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.

Ratiometric and selective two-photon fluorescent probe based on PET-ICT for imaging Zn^(2+)in living cells and tissues

A two-photon fluorescent probe TPZn was developed for specific ratiometric imaging Zn2＋ in living cells and tissues. Significant ratiometric fluorescence change was based on photoinduced electron transfer and intramolecular charge transfer. The synthetic method of TPZn was simple. It was successfully used to selectively image Zn2＋ based on the higher binding affinity for Zn2＋ than for Cd2＋. TPZn was easily loaded into the living cell and tissues with high membrane permeability in a complex biological environment. TPZn could clearly visualize endogenous Zn2＋ by TP ratiometric imaging in hippocampal slices at a depth of 120 μm. Thus, TPZn is a useful tool to image of Zn2＋ in living cells and tissues without interference from Cd2＋.

A highly specific ratiometric two-photon fluorescent probe to detect dipeptidyl peptidase Ⅳ in plasma and living systems

Subject Code:H30With the support by the National Natural Science Foundation of China and National Basic Research Program of China,the group led by Prof.Ge Guangbo(葛广波)and Prof.Yang Ling(杨凌)from the Laboratory of Pharmaceutical Resource Discovery,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,reported a highly specific ratiometric two-photon fluorescent probe to detect

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.

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.

Multiplexed intracellular detection based on dual-excitation/dualemission upconversion nanoprobes

Multiplexed intracellular detection is desirable in biomedical sciences for its higher eficiency and accuracy compared to the single-analyte detection.However,it is very challenging to construct nanoprobes that possess multiple fluorescent signals to recognize the different intracellular species synchronously.Herein,we proposed a novel dual-excitation/dual-emission upconversion strategy for multiplexed detection through the design of upconversion nanoparticles(UCNP)loaded with two dyes for sensitization and quenching of the upconversion luminescence(UCL),respectively.Based on the two independent energy transfer processes of near-infrared(NIR)dye IR845 to UCNP and UCNP to visible dye PAPS-Zn,CIO-and Zn2+were simultaneously detected with a limit of detection(LOD)of41.4 and 10.5 nM,respectively.By tilizing a purpose built 830/980 nm dual-laser confocal microscope,both intrinsic and exogenous CIO and Zn2+in live MCF-7 cells have been accurately quantified.Such dual-excitation/dual-emission ratiometric UCL detection mode enables not only monitoring multiple intracellular analytes but also eliminating the detection deviation caused by inhomogeneous probe distribution in cells.Through modulation of NIR dye and visible dye with other reactive groups,the nanoprobes can be extended to analyze various intraellular species,which provides a promising tool to study the biological activities in live cells and diagnose diseases.

Activatable nanoscale metal-organic framework for ratiometric photoacoustic imaging of hydrogen sulfide and orthotopic colorectal cancer in vivo

Nanoscale metal-organic frameworks(nano MOFs)have emerged as a promising biomedical nanoplatform because of their unique properties.However,the exploration of nano MOFs in photoacoustic(PA)imaging is still limited.Here,a novel hydrogen sulfide(H2 S)-activated nano copper-based MOF(Cu-MOF)was developed as a near-infrared(NIR)ratiometric PA probe for in vivo monitoring of endogenous H2 S level and orthotopic colorectal cancer imaging via in situ reaction of nano Cu-MOFs with endogenous H2 S that is closely associated with tumor growth and proliferation in colon cancer.The synthesized nano Cu-MOFs displayed excellent PA responsiveness towards tumor H2 S level with high selectivity and rapid kinetics.The result suggests the developed probe may provide a unique opportunity to investigate the malignant behaviors of H2 S-associated events in vivo.

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.

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.

Establishment of a new molecular model for mercury determination verified by single crystal X-ray diffraction,spectroscopic analysis and biological potentials

A wide variety of molecular probes have been developed for real-time analysis,but most of organic fluorophores possess small Stokes shifts and self-absorption or inner filter effect that could not be avoided.In this study,a new dicyanoisophorone-based derivative(E)-0-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)diphenylphosphinothioate(λ_(ex)=405 nm,X_(em)=551 nm,denoted as ICM-S) with strong push-pull electron effect has been afforded and it exhibits red shift for absorption from 407 nm to 426 nm with distinct color change from pale yellow to deep yellow upon exposure to Hg~(2+).Moreover,an easily distinguishable fluorescence color change follows the route from green,yellow to red in the presence of Hg~(2+) over the range of 0-90 μmol/L(detection limit=137 nmol/L)can be observed by the naked eye under a UV lamp irradiation.Chlorodiphenylphosphine and sublimedsulfur are incorpo rated as re s ponsive sites and P-O bond has been cleaved upon the addition of mercu ry ions.During the recognition process,such dicyanoisophorone dye(ICM-S) has been evolved to 2-(3-(4-hydroxystyryl)-5,5-dimethylcyclohex-2-enylidene) malononitrile(ICM-OH).Clear evidences in the chemical processes can be identified via single crystal X-ray diffraction,spectroscopic analysis,photophysical studies and titration experiments.With the aim of exploring its potential in biological systems,its in vitro responses to Hg~(2+) have been evaluated in 293 T cells and the effectiveness in zebrafish model has also been verified.