Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution

Oxygen(O_(2))-sensing matrices are promising tools for the live monitoring of extracellular O_(2) consumption levels in long-term cell cultures.In this study,ratiometric O_(2)-sensing membranes were prepared by electrospinning,an easy,low-cost,scalable,and robust method for fabricating nanofibers.Poly(ε-caprolactone)and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)dichloride,which was used as the O_(2)-sensing probe,and rhodamine B isothiocyanate,which was used as the reference dye.The functionalized scaffolds were morphologically characterized by scanning electron microscopy,and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy,thermogravimetric analysis,and water contact angle measurement.The sensing capabilities were investigated by confocal laser scanning microscopy,performing photobleaching,reversibility,and calibration curve studies toward different dissolved O_(2)(DO)concentrations.Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5%to 20%and good stability under ratiometric imaging.In addition,the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines,namely metastatic melanoma cell line SK-MEL2,breast cancer cell line MCF-7,and pancreatic ductal adenocarcinoma cell line Panc-1,thus recreating a suitable biological environment in vitro.These O_(2)-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O_(2)content during drug testing/validation and tissue regeneration processes.

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.

Ratiometric Fluorescence Detection of 6-Mercaptopurine Based on the Nanohybrid of Fluorescence Carbon Dots and Gold Nanoclusters

The development of a simple and accurate quantitative method for the determination of 6-mercaptopurine (6-MP) is of great importance because of its serious side effects. Ratiometric fluorescence (RF) sensors are not subject to interference from environmental factors, and exhibit enhanced precision and accuracy. Therefore, a novel RF sensor for the selective detection of 6-MP was developed. The present work reports a sensitive and selective RF sensor for the detection of 6-mercaptopurine, by hybridizing carbon nanodots (CDots) and gold nanoclusters (AuNCs) capped with bovine serum albumin (BSA). The CDots serve as the reference signal and the AuNCs as the reporter. On addition of the 6-MP, AuNCs formed aggregates, because the existing cross-links within the AuNCs and BSA structure were broken in favour of the Au-S bonds, which can enhance the fluorescence of AuNCs, while the fluorescence of CDots is stable against 6-MP, leading to distinct ratiometric fluorescence changes when exposed to 6-MP. 6-MP could be detected in the range of 0 - 30.22 μM with a detection limit of 54 nM. The developed sensor was applied for the determination of 6-MP in human serum samples and satisfactory results were obtained.

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.

Terpyridine-derived Zn^(2+) Selective Fluorescence Sensor: Ratiometric Fluoroionophoric Behavior Study

A sensitive and selective zinc ion ratiometric fluorescence sensor has been synthesized and characterized. This material displays dual fluorescence. After the material was bonded to a closed-shell metal ion, such as Zn2＋, the recovery of the local excited fluorescence of the material-Zn2＋ complex, largely at the expense of the intramolecular charge transfer fluorescence, is consistent with the difference between selected orbital transitions of the free dye and the metal-chelated complex. For instance, the contribution of the πtpy→πtpy, transition becomes more prominent. This is also consistent with the results of the fluorescence decay behavior, measured via a time-correlated single photon counting setup. In contrast, the corresponding open shell Ni2＋ -bound complex quenches both kinds of photoluminescence, due to spin-orbit coupling.

Photoluminescence and ratiometric thermo-response of Eu^(2+)and Eu^(3+)in BaAl_(2)B_(2)O_(7):Eu^(2+),Eu^(3+)phosphor materials

A novel Eu^(2+)-Eu^(3+)co-activated ratiometric thermo-sensitive phosphor was developed and synthesized by solid-state reaction.The valence state of Eu,photoluminescence and thermo-sensitive performance of the phosphor prepared either in ambient air or carbothermally were investigated and discussed.The phosphor shows high sensitivity(Sa=0.0173 K^(-1),S_(r)=0.461%/K)and superior signal discriminability(Δν∼10380 cm^(−1)).The thermo-sensitive performance is subject to the dual effects of different thermo-responses by Eu^(2+)versus Eu^(3+)combined with energy transfer from Eu^(2+)to Eu^(3+),so that the sensitivity of the phosphor in the temperature range presents a non-monotonic trend.The development of the BaAl_(2)B_(2)O_(7):Eu^(2+),Eu^(3+)phosphor is not only expected to be relevant for application in the field of temperature sensing,but also of reference significance for improving the sensitivity by means of energy transfer between co-activator ions over a wider temperature range of Eu^(2+)-Eu^(3+)co-activated ratiometric thermo-sensitive phosphors.

Ratiometric fluorescence probe for accurate detection of Concanavalin A by coupling fluorescent microsphere with boric acid functionalized carbon dots

Accurate and sensitive strategies for Concanavalin A(Con A)sensing are conducive to the better cognition of various important biological and physiological processes.Here,by designing dextran-functionalized fluorescent microspheres(DxFMs)and boric acid-modified carbon dots(BCDs)as recognition unit and built-in signal reference respectively,a ratiometric fluorescent detection platform was proposed for Con A detection with high reliability.In this protocol,the BCDs/DxFMs precipitation was formed due to the covalent interactions between cis-diol of DxFMs and boronic acid groups of BCDs,thus only fluorescence of BCDs could be detected in the supernatant.When Con A was presented,it could bind to DxFMs through its carbohydrate recognition ability and suppress the subsequent assembly between DxFMs and BCDs,leading to the simultaneous capture of DxFMs and BCDs fluorescence in the supernatant.Since the BCDs content was superfluous,their fluorescence intensities were basically constant in all cases.Based on the unchanged BCDs fluorescence signal and target-dependent DxFMs fluorescence signal in supernatant,the ratiometric detection of Con A was realized.Under optimized conditions,this ratiometric fluorescent platform displayed a linear detection range from 0.125μg/mL to 12.5μg/mL with a detection limit of 0.089μg/mL.Moreover,satisfied analytical outcomes for Con A detection in serum samples were obtained,manifesting huge application potential of this ratiometric fluorescent platform in clinical diagnosis.

The Mn/Yb/Er triple-doped CeO_(2) nanozyme with enhanced oxidase-like activity for highly sensitive ratiometric detection of nitrite

Long-term excessive intake of nitrite(NO_(2)^(-))poses a great threat to human health,needing a simple and fast method to detect NO_(2)-in food.Herein,via a simple and feasible strategy,Mn/Yb/Er triple-doped CeO_(2) nanozyme(Mn/Yb/Er/CeO_(2))was synthesized for highly sensitive ratiometric detection of nitrite.By doping Mn,Yb,Er into CeO_(2) lattice structure,Mn/Yb/Er/CeO_(2) nanozyme showed enhanced oxidase-like activity,obtaining a higher density of oxygen vacancy and a higher ratio of Ce^(3+)to Ce~(4+)than that of CeO_(2).The 3,3,5,5-tetramethylbenzidine(TMB)can be effectively oxidized by Mn/Yb/Er/CeO_(2) to produce the oxidized TMB(ox TMB),showing a significant absorption signal at 652 nm.Additionally,nitrite can react with ox TMB to produce yellow diazotized ox TMB,which is accompanied by an elevated absorption signal at 445 nm and a decreased absorption signal at 652 nm.Thus,based on the oxidase-mimetic activity of Mn/Yb/Er/CeO_(2) and the diazotization reaction between NO_(2)^(-)and ox TMB,a ratiometric colorimetric assay was established for NO_(2)^(-)detection in food.Furthermore,by integrating Mn/Yb/Er/CeO_(2) with a smartphone,a colorimetric smartphone-sensing platform was successfully fabricated for visualization and quantitative detection of NO_(2)^(-).Notably,this two-detection mode showed excellent sensitivity,selectivity,reliability and practicability in monitoring the NO_(2)^(-)in real samples,impling its great potential for food safety.

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.

Incorporation of Fluorescent Carbon Quantum Dots into Metal-Organic Frameworks with Peroxidase-Mimicking Activity for High-Performance Ratiometric Fluorescent Biosensing

A new ratiometric fluorescent sensor based on the bifunctional carbon quantum dots(CQDs)@metal-organic framework(MOF)nanocomposite possessing peroxidase-mimicking catalytic and luminescent characteristics was developed for hydrogen peroxide(H_(2)O_(2))and cholesterol detection.The incorporation of fluorescent CQDs into the cavities of MIL-101(Fe)MOF with peroxidase-like activities endows the nanocomposite with bifunctional properties.The CQDs@MOF can oxidize o-phenylene-diamine to 2,3-diaminophenolazine by H_(2)O_(2)with yellow fluorescence(556 nm).Meantime,the intrinsic fluorescence signal(455 nm)of CQDs@MOF is inhibited due to the inner filter effect.Therefore,the ratio of the fluorescent intensity is employed as the signal output to construct the H_(2)O_(2)ratiometric biosensor.In addition,the cholesterol can be determined by the ratiometric sensor with high sensitivity.In addition,the total cholesterol in human serum is determined with high accuracy using our ratiometric biosensor.This ratiometric fluorescent platform based on the bifunctional CQDs@MOF provides new insights in the field of bio-sensing.

Lanthanide-based ratiometric luminescence nanothermometry

Luminescent nanothermometry can precisely and remotely measure the internal temperature of objects at nanoscale precision,which,therefore,has been placed at the forefront of scientific attention.In particular,due to the high photochemical stability,low toxicity,rich working mechanisms,and superior thermometric performance,lanthanide-based ratiometric luminesencent thermometers are finding prevalent uses in integrated electronics and optoelectronics,property analysis of in-situ tracking,biomedical diagnosis and therapy,and wearable e-health monitoring.Despite recent progresses,it remains debate in terms of the underlying temperature-sensing mechanisms,the quantitative characterization of performance,and the reliability of temperature readouts.In this review,we show the origin of thermal response luminescence,rationalize the ratiometric scheme or thermometric mechanisms,delve into the problems in the characterization of thermometric performance,discuss the universal rules for the quantitative comparison,and showcase the cutting-edge design and emerging applications of lanthanide-based ratiometric thermometers.Finally,we cast a look at the challenges and emerging opportunities for further advances in this field.

A fluorogenic probe for SNAP-tag protein based on ESPT ratiometric signals

Protein self-labeling tags achieve selective fusion and labeling of target proteins through genetic coding technology,but require exogenous fluorescent probes with fluorogenicity for protein tag binding to have the performance of wash-free fluorescence imaging in live cells.In this paper,we reported a fluorogenic probe 1 capable of ratiometric fluorescence recognition of SNAP-tag proteins.In this probe,the O6-benzylguanine derivative of 3-hydroxy-1,8-naphthalimide underwent a selective covalent linkage reaction with SNAP-tag protein.The hydroxyl group on the naphthalimide fluorophore formed a hydrogen bond with the functional group near the protein cavity.The excited state proton transfer occurred after illumination,to obtain the ratio fluorescence signal from blue emission to red emission,realizing the wash-free fluorescence imaging of the target proteins.

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-ratiometric magnetic resonance tunable nanoprobe with acidic-microenvironment-responsive property to enhance the visualization of early tumor pathological changes

The development of microenvironment-responsive nanoprobes has shown great promise for use in magnetic resonance imaging(MRI),with the advantage of significantly improved specificity and good biocompatibility.However,the clinical application of responsive probes is hampered by a lack of biological sensitivity for early molecular diagnostics and visualizing microenvionment of metabolism reprogramming in tumor progression.Here,we report on a dual-ratiometric magnetic resonance tunable(DMRT)nanoprobe designed by crosslinking different ratios of transferrin chelating gadolinium and superparamagnetic nanoparticles,complexed to a pH responsive biocompatible polymer.This dually activatable nanoprobe enables pH-dependent tumor microenvironment visualization,providing exceptional quantitative pathophysiological information in vitro and in vivo.When used in combination with dual-contrast enhancement triple subtraction imaging technique(DETSI),this smart nanoprobe guarantees the diagnosis of early-stage diseases.We envisage that this novel integrated nanoplatform will provide a new paradigm for the clinical translation of robust DMRT nanoprobes for early disease detection and staging,as well as microenvironment visualization and disease progression monitoring.

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.

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.

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 Fluorescein Derivative Chemosensor Combined with Triplet-Triplet Annihilation Upconversion System for Ratiometric Sensing of Cysteine

A derivative of fluorescein,fluorescein O,O-diacrylate(FODA),was utilized in a triplet-triplet annihilation upconversion(TTA-UC)system to develop a composite ratiometric chemosensor capable of detecting cysteine(Cys).FODA acted as both the probe for Cys and the energy acceptor for upconversion(UC)emission,thereby making UC a responsive signal to Cys.In addition,the phosphorescence signal of the sensitizer in the TTA-UC system remained constant and did not respond to Cys,making it an ideal internal reference signal for constructing a ratiometric sensor.Through this simple strategy,traditional fluorescent probes can be combined with TTA-UC system to establish a ratiometric sensing platform,which can be applied in more scenarios due to the longer wavelength excitation.

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.