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.

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.

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 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.

High sensitivity ratiometric fluorescence temperature sensing using the microencapsulation of CsPbBr3 and K2SiF6:Mn4+phosphor

A dual emission sensing film has been prepared for colorimetric temperature sensing using CsPbBr_(3)perovskite nanocrystals(CsPbBr_(3)NCs)and manganese doped potassium fluorosilicate(K_(2)SiF_(6):Mn^(4+),KSF)encapsulated in polystyrene by a microencapsulation strategy.The CsPbBr_(3)-KSF-PS film shows good temperature sensing response from 30℃to 70℃,with a relative temperature sensitivity(Sr)up to 10.31%℃^(−1) at 45℃.Meanwhile,the film maintains more than 95%intensity after 6 heating-cooling cycles and keeps its fluorescence characteristics after 3 months.The film can be used to monitor temperature change by naked eye under a UV lamp.In particular,the temperature discoloration point of the sensing film can be controlled by the ratio change of CsPbBr_(3):KSF to expand its applications.The study of the CsPbBr_(3)-KSF-PS sensing mechanism in this work is helpful to provide effective strategies for the design of reliable,high sensitivity and stable temperature sensing system using CsPbBr_(3)NCs.

Enhanced oxidase-like activity of g-C_(3)N_(4) nanosheets supported Pd nanosheets for ratiometric fluorescence detection of acetylcholinesterase activity and its inhibitor

The undesirable enzymatic activity of nanozymes under near neutral p H condition and the traditional single signal output always restrict the analytical application of nanozyme-based biosensors.Herein,graphitic carbon nitride nanosheets supported palladium nanosheets composite (Pd/g-C_(3)N_(4)) with both oxidase-like activity and fluorescent property is synthesized.Notably,Pd/g-C_(3)N_(4)exhibits enhanced oxidase-like activity compared to Pd NSs under p H 7.4.By combining Pd/g-C_(3)N_(4)with o-phenylenediamine(OPD),a ratiometric fluorescence assay for acetylcholinesterase (ACh E) activity detection is developed.Pd/g-C_(3)N_(4)can catalyze oxidation of nonfluorescent OPD to fluorescent oxidized OPD (ox OPD,Em=565nm),which can quench fluorescence of g-C_(3)N_(4)supporter (Em=441 nm) through fluorescence resonance energy transfer (FRET).However,in presence of ACh E,acetylthiocholine can be hydrolyzed into thiocholine,which will block the oxidase-like activity of Pd/g-C_(3)N_(4)and then hamper the FRET process.This ratiometric fluorescence assay is also viable to screen ACh E inhibitor.This work will guide design of ratiometric fluorescence assay based on nanozymes with improved enzymatic activity.

Field-portable ratiometric fluorescence imaging of dual-color label-free carbon dots for uranyl ions detection with cellphone-based optical platform

Under the public spotlight,uranyl(UO22+)ions has attracted considerable attention for the extreme radioactive and chemical toxicity to ourselves and our environment.Herein,we present a simple and effective ratiometric fluorescence imaging method for the visualizing and quantitative detection UO22+ions by cellphone-based optical platform.The sensing solution was prepared by mixing label-free red carbon dots(r-CDs)and blue carbon dots(b-CDs)together with a fixed photoluminescence intensity ratio of 4:1.When UO22+ions were added,the fluorescence of r-CDs can be selectively quenched,while the fluorescence of b-CDs remains stable without spectral cha nges.With the gradually increase the amounts of UO22+ions,the different response of dual-color CDs resulted in a signification color evolution from deep red to dark purple under the ultraviolet(UV)light illumination.Then,a cellphone-based optical platform was constructed for directly imaging the color change of the samples,and the built-in Colorpicker APP quickly output the red,green and blue(RGB)channel values of these images within one second.Interesting,there was a linear relationship between the ratio of red and blue(R/B)channel values and UO22+ions concentration from 0μmol/L to 30.0μmol/L(R^2=0.92804)with the detection limit of^8.15μmol/L(signal-to-noise ratio of 3).In addition,the optical platform has also been applied to the quantification of UO22+ions in tap water and river water sample.With the advantage of low-cost,portable,easy to operation,we anticipate that this method would greatly improve the accessibility of UO22+ions detection even in resource-limited areas.

A Bifunctional-Blocker-Aided Hybridization Chain Reaction Lighting-Up Self-calibrating Nanocluster Fluorescence for Reliable Nucleic Acid Detection

In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5′-CAC CGC T-3′and 5′-ATT TGC CTT TTG GGG ACG GATA-3′)at two terminals creates a red-emitting AgNC nucleation sequence(rNS,5′-CAC CGC TAT TTG CCT TTT GGG GAC GGATA-3′).We found that the presence of a toehold fragment(5′-TGCCC-3′)in HP1 could silence the rNS.Upon the addition of a target nucleic acid,HCR of HP1 and hairpin probe 2(HP2)could be initiated,resulting in the formation of long chain of DNA duplexes with multibranched rNS.As the toehold fragment in HP1participated in generating duplexes,a strong emission of rNS-templated AgNCs was observed at 670 nm.More significantly,a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting AgNCs nucleation sequence.On the one hand,the blocker could increase the signal-to-noise-ratio of the constructed biosensor,and on the other hand,the blocker also helped to prepare ratiometric HCR-AgNCs assay with self-calibrating ability to strengthen its reproducibility.Compared with the traditional HCR-AgNCs sensors,the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability,which is important for the fabrication of biosensors in various fields for practical biosensing applications.

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.

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.

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.

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.

Eu^(3+)-doped Bovine Serum Albumin-derived Carbon Dots for Ratiometric Fluorescent Detection of Tetracycline

The abuse of antibiotics has become a global concern,thus it is significant to develop simple antibiotic assays.Herein,a novel type of lanthanide-doped bovine serum albumin(BSA)derived carbon dots(BCDs)dubbed Eu-BCDs has been unveiled for ratiometric fluorescent detection of tetracycline(TC),which was formed from the one-step hydrothermal reaction of Eu^(3+)and BSA.Upon challenge with TC,the blue fluorescence of Eu-BCDs at 448 nm was gradually quenched due to the internal filtration effect,while the characteristic red fluorescence of Eu^(3+)at 618 nm was significantly enhanced owning to the antenna effect,leading to the ratiometric fluorescent detection of TC with fast response(2 min),a wide linear dynamic range(0-80μmol/L),and high selectivity and sensitivity(limit of detection(LOD)of 3 nmol/L).Moreover,Eu-BCDs can be successfully applied for the ratiometric fluorescent detection of TC in environmental and biological samples,such as river water,milk,honey and serum.The Eu-BCDs sensing platform would have great application potential in view of its simple preparation,rapid response,high sensitivity and good selectivity.

Fluorescent MOF-based nanozymes for discrimination of phenylenediamine isomers and ratiometric sensing of o-phenylenediamine

Although peroxidase-like nanozymes have made great progress in bioanalysis,few current nanozymebased biosensors are constructed for discriminating isomers of organic compounds.Herein,fluorescent metal-organic framework(MOF)-based nanozyme is utilized for phenylenediamine isomers discrimination and detection.NH_(2)-MIL-101(Fe),as a member of Fe-based MOFs,functions as not only fluorescent indicator but also peroxidase mimics.In the presence of H_(2)O_(2),NH2-MIL-101(Fe) can catalyze the oxidation of o-phenylenediamine(OPD) and p-phenylenediamine(PPD) into their corresponding oxidation products(OPDox and PPDox),which in turn quench its intrinsic fluorescence at 445 nm via inner filter effect(IFE).Differently,a new fluorescence peak at 574 nm is observed for OPDox.Thus,a ratiometric fluorescence method for the detection of OPD can be designed with the fluorescence intensity ratio F_(574)/F_(445)as readout.This proposed strategy displays excellent discrimination ability for three phenylenediamines and may open new applications of MOFs in environmental science.

A Novel Ratiometric Fluorescent Probe for Cyanide Anion with High Selectivity and Its Application in Cell Imaging

A novel hybrid acenaphthene-hemicyanine dye（Acc） was designed and synthesized as a colorimetric and ratiometric fluorescent sensor for cyanide detection. The nucleophilic addition of cyanide anion resulted in the blocking of the intramolecular charge transfer（ICT）. Thus, the sensor exhibited high sensitivity and selectivity to- wards cyanide anion among many anions in dimethylsulphoxide（DMSO）-water media, with the emission wavelength displaying a very large shift（186 nm）. Moreover, the color changes of the solution could be observed by the naked eyes and it had a short response time. Under optimized conditions, the detection limit for cyanide was calculated to be 1.3μmol/L（R2=0.9958）. In addition, the probe had excellent biocompatibility and could be utilized to monitor cyanide in living cells.

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）.

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＋.