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.

A Biomimetic Membrane-Coated Nanoprobe for the Ratiometric Fluorescence Detection of Phospholipase A_(2)

Phospholipase A_(2)(PLA_(2))is a critical biomarker of many diseases.Among various fluorescent probes,the ratiometric ones have shown great promise because they can provide built-in calibration to avoid interference from analyte-independent factors by simultaneously monitoring two or more fluorescence peaks.Herein,we develop a biomimetic membrane-coated ratiometric fluorescent nanoprobe for the detection of PLA_(2).In this design,silica nanoparticles modified with fluorescein isothiocyanate(FITC)are functionalized with a layer of biomimetic membrane that is loaded with Dil dye.Therefore,when the nanocomposite is illuminated by 488 nm incident light,in addition to the fluorescence of FITC centered at 522 nm,that of Dil peaked at 568 nm originating from the energy transfer from FITC to Dil can also be monitored,forming a ratiometric fluorescent nanoprobe.When the target PLA_(2)is added,FITC and Dil will no longer be in close proximity because it hydrolyzes the biomimetic membrane,cutting off the energy flow between the two dyes.Consequently,the ratiometric fluorescent signal of the nanoprobe is altered to quantitatively reflect the content of PLA_(2).This study provides an effective approach to PLA_(2)detection,and the biomimetic membrane-mediated ratiometric design is promising in developing a new generation of biosensors.

Development of ratiometric DNA biosensors with improved accuracy,precision,and signal-to-noise ratio

Ratiometric DNA biosensors,which utilize DNA as the recognition element and integrate dual signals from diverse sensing platforms including fluorescence,electrochemistry,and surface-enhanced Raman scattering(SERS),have demonstrated remarkable proficiency in detecting a wide range of targets.In this review,we showcase the significant progress achieved by ratiometric biosensors.Firstly,ratiometric biosensors have made notable advancements in analyzing real samples.These include the analysis of pH values near cancer cells,quantification of miRNA in human cell lysates,detection of human telomerase RNA in cell extracts,and performing DNA logicgated in situ bioimaging on cell membranes.Secondly,excellent sensitivity has been attained through the utilization of effective amplification methods such as RCA,HCR,and CHA,among others.Thirdly,the construction of stable reference signals has resulted in significantly improved precision for ratiometric biosensors.This breakthrough has overcome matrix effects,enabling reliable detection in real samples with high selectivity.This review provides a comprehensive overview of recent advancements in strategies employed by ratiometric DNA biosensors.We present three types of biosensors based on distinct sensing platforms:fluorescent,electrochemical,and SERS biosensors.Additionally,we discuss future directions and primary challenges in the development of ratiometric DNA biosensors.

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.

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.

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.

SciFinder-guided rational design of fluorescent carbon dots for ratiometric monitoring intracellular pH fluctuations under heat shock

Intracellular pH plays a significant role in various biological processes, including cell proliferation,apoptosis, metabolism, enzyme activity and homeostasis. In this work, a novel design strategy for the preparation of pH responsive carbon dots(CDs-pH) for ratiometric intracellular imaging was reported. By using SciFinder database, fluorescent CDs-pH with the required p Kavalue of 6.84 were rationally designed, which is vital important for precise sensing of intracellular pH. As a result, the synthesized CDspH demonstrated robust ability to test pH fluctuations within the physiological range of 5.4-7.4. The CDspH was further utilized for fluorescent ratiometric imaging of pH in living HeLa cells, effectively avoided the influence of autofluorescence from native cellular species. Moreover, real-time monitoring of intracellular pH fluctuation under heat shock was successfully realized. This SciFinder-guided design strategy is simple and flexible, which has a great potential to be used for the development of other types of CDs for various applications.

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.

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.

N-Positive ion activated rapid addition and mitochondrial targeting ratiometric fluorescent probes for in vivo cell H_(2)S imaging

Heterocyclic compound quinoline and its derivatives exist in natural compounds and have a broad spectrum of biological activity.They play an important role in the design of new structural entities for medical applications.Similarly,indoles and their derivatives are found widely in nature.Amino acids,alkaloids and auxin are all derivatives of indoles,as are dyes,and their condensation with aldehydes makes it easy to construct reaction sites for nucleophilic addition agents.In this work,we combine these two groups organically to construct a rapid response site(within 30 s)for H_(2)S,and at the same time,a ratiometric fluorescence response is presented throughout the process of H_(2)S detection.As such,the lower detection limit can reach 55.7 nmol/L for H_(2)S.In addition,cell imaging shows that this probe can be used for the mitochondrial targeted detection of endogenous and exogenous H_(2)S.Finally,this probe application was verified by imaging H_(2)S in nude mice.

A ratiometric fluorescent sensor for tracking Cu(Ⅰ) fluctuation in endoplasmic reticulum

A two-photon ratiometric fluorescent sensor for Cu^+ in endoplasmic reticulum(ER), CNSB, was developed via coumarin/ASBD integration based on FRET mechanism. In solution, CNSB shows reversible, highly-specific ratiometric response to Cu^+ .Moreover, CNSB exhibits suitable K_d value, suggesting the possibility of detecting Cu^+ in the living cells. The probe can enter the MCF-7 cells easily and specifically locates in the ER. The highly specific ratiometric response of CNSB toward Cu^+ in MCF-7 cells provides the sensor the capacity to visualize both exogenous and endogenous Cu^+ in the ER via fluorescence imaging.Next, CNSB was utilized to detect the fluctuation and distribution of Cu^+ under ER stress in MCF-7 cells, which confirmed directly the relationship between Cu^+ enhancement and ER stress. Meanwhile, the two-photon ability of coumarin facilitated the sensor to visualize Cu^+ fluctuation via two-photon fluorescence imaging. In addition, the spatial distribution of Cu^+ in the heart slice of the 14-day-old rat was demonstrated using CNSB. This study demonstrates the promising potential of CNSB in clarifying the Cu^+ -dependent signaling in the ER stress-related diseases.

Single-wavelength-excited fluorogenic nanoprobe for accurate realtime ratiometric analysis of broad pH fluctuations in mitophagy

Mitophagy has a critical role in maintaining cellular homeostasis through acidic lysosomes engulfing excess or impaired mitochondria,thereby pH fluctuation is one of the most significant indicators for tracking mitophagy.Then such precise pH tracking demands the fluorogenic probe that has tailored contemporaneous features,including mitochondrial-specificity,excellent biocompatibility,wide pH-sensitive range of 8.0–4.0,and especially quantitative ability.However,available molecular probes cannot simultaneously meet all the requirements since it is extremely difficult to integrate multiple functionalities into a single molecule.To fully address this issue,we herein integrate two fluorogenic pH sensitive units,a mitochondria-specific block,cellpenetrating facilitator,and biocompatible segments into an elegant silica nano scaffold,which greatly ensures the applicability for real-time tracking of pH fluctuations in mitophagy.Most significantly,at a single wavelength excitation,the integrated pHsensitive units have spectra-distinguishable fluorescence towards alkaline and acidic pH in a broad range that covers mitochondrial and lysosomal pH,thus enabling a ratiometric analysis of pH variations during the whole mitophagy.This work also provides constructive insights into the fabrication of advanced fluorescent nanoprobes for diverse biomedical applications.