Application and development of fluorescence probes in MINFLUX nanoscopy(invited paper)

The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.

Primary amine coupling on nanocarbon catalysts: Reaction mechanism and kinetics via fluorescence probe analysis

Non-metallic nanocarbon materials catalyzed coupling reactions of primary amines to produce imine is an efficient,green and sustainable synthetic route,which has a wide application prospect in fine chemicals or pharmaceutical molecules.In the present study,we show firstly the relatively high catalytic activity of graphene oxide in the reaction of oxidative coupling of benzylamine(OCB),which is even comparable with typical metal-based catalysts,indicating the great potential of nanocarbon materials in this reaction system.More importantly,a novel twophoton fluorescence probe molecule(N-propyl-4-hydrazinyl-1,8-naphthalimide,NA)with special chemical structure of hydrazine functionality was synthesized.The probe NA could selectively react with aldehyde or ketone compounds,leading to the photoluminescence enhancement via inhibition of photo induced electron transfer(PET)process.The synthesized NA was applied as probe in carbon catalyzed OCB system to predict the existence of reaction intermediate benzaldehyde(BA),indicating the reaction pathway of oxidation-deamination-condensation in nanocarbon catalyzed OCB process.The proposed luminescence-probe strategy for revealing the kinetics and mechanism may also shed light in other reaction systems concerning the intermediates or products of ketones or aldehydes.

An insight into the in vivo imaging potential of curcumin analogues as fluorescence probes

Curcumin and its derivatives have good electrical and optical properties due to the highly symmetric structure of delocalized π electrons. Apart from that, curcumin and its derivatives can interact with numerous molecular targets, thereby exerting less side effects on human body. The fluorescence emission wavelength and fluorescence intensity of curcumin can be enhanced by modifying its π-conjugated system and β-diketone structure. Some curcumin-based fluorescent probes have been utilized to detect soluble/insoluble amyloid-β protein, intracranial reactive oxygen species, cysteine, cancer cells, etc. Based on the binding characteristics of curcumin-based fluorescent probes with various target molecules, the factors affecting the fluorescence intensity and emission wavelength of the probes are analyzed, in order to obtain a curcumin probe with higher sensitivity and selectivity. Such an approach will be greatly applicable to in vivo fluorescence imaging.

Lighting up plants with near-infrared fluorescence probes

Fluorescence imaging is a non-invasive and highly sensitive bioimaging technique that has shown remarkable strides in plant science. It enables real-time monitoring and analysis of biological and pathological processes in plants by labeling specific molecular or cellular structures with fluorescent probes. However, tissue scattering and phytochrome interference have been obstacles for conventional fluorescence imaging of plants in the ultraviolet and visible spectrum, resulting in unsatisfactory imaging quality. Fortunately, advances in near-infrared(NIR) fluorescence imaging technology(650-900 nm) offer superior spatial-temporal resolution and reduced tissue scattering, which is sure to improve plant imaging quality. In this review, we summarize recent progress in the development of NIR fluorescence imaging probes and their applications for in vivo plant imaging and the identification of plant-related biomolecules. We hope this review provides a new perspective for plant science research and highlights NIR fluorescence imaging as a powerful tool for analyzing plant physiology, adaptive mechanisms, and coping with environmental stress in the near future.

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.

Activatable molecular fluorescence probes for the imaging and detection of ischemic stroke

The real-time, noninvasive, nonionizing, high spatiotemporal resolution, and flexibility characteristics of molecular fluorescence imaging provide a uniquely powerful approach to imaging and monitoring the physiology and pathophysiology of ischemic stroke. Currently, various fluorescence probes have been synthesized with the aim of improving quantitative and quantitative studies of the pathologic processes of ischemic stroke in living animals. In this review, we present an overview of current activatable fluorescence probes for the imaging and diagnosis of ischemic stroke in animal models. We categorize the probes based on their activatable signals from the biomarkers associated with ischemic stroke, and we present representative examples of their functional mechanisms. Finally, we briefly discuss future perspectives in this field.

Activatable fluorescent probes for imaging and diagnosis of rheumatoid arthritis

Rheumatoid arthritis(RA)is a systemic autoimmune disease that is primarily manifested as synovitis and polyarticular opacity and typically leads to serious joint damage and irreversible disability,thus adversely affecting locomotion ability and life quality.Consequently,good prognosis heavily relies on the early diagnosis and effective therapeutic monitoring of RA.Activatable fluorescent probes play vital roles in the detection and imaging of biomarkers for disease diagnosis and in vivo imaging.Herein,we review the fluorescent probes developed for the detection and imaging of RA biomarkers,namely reactive oxygen/nitrogen species(hypochlorous acid,peroxynitrite,hydroxyl radical,nitroxyl),pH,and cysteine,and address the related challenges and prospects to inspire the design of novel fluorescent probes and the improvement of their performance in RA studies.

A highly sensitive fluorescent probe RN-NA reveals peroxynitrite as a novel biomarker for primary open angle glaucoma

AIM:To directly quantify peroxynitrite(ONOO-)using a highly sensitive fluorescence resonance energy transfer probe RN-NA,investigate the association between ONOOand primary open angle glaucoma(POAG),and clarify whether RN-NA could be used as a potential tool for POAG diagnosis.METHODS:Plasma and aqueous humor(AH)samples were collected from POAG patients(n=100,age:59.70±6.87y)and age-related cataract(ARC)patients(n=100,age:61.15±4.60y)admitted to our hospital.Next,RN-NA was used to detect ONOO-in plasma and AH samples,and the relationship between ONOO-level and POAG was analyzed using binary logistic regression.Besides,Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO-with the patients’age,intraocular pressure(IOP),and mean deviation of visual field testing.The ONOO-scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine(SIN-1)-induced ocular hypertension in mice(n=7,6-8wk).Finally,the IOP and ONOO-in both eyes were measured 30min after the last drug treatment.RESULTS:ONOO-levels of AH and plasma were significantly higher in the POAG group than in the ARC group(P<0.01).Additionally,ONOO-levels were closely correlated with POAG in a binary logistic regression analysis[odds ratio(OR)=1.008,95%confidence interval(CI):1.002-1.013,P<0.01 for AH;OR=1.004,95%CI:1.002-1.006,P<0.001 for plasma].Pearson correlation analysis showed that ONOO-levels in AH or plasma were positively associated with visual field defects(R=0.51,P<0.01 for AH;R=0.45,P<0.001 for plasma),and ONOO-levels in plasma and AH were correlated in the POAG group(R=0.69,P<0.001).However,administering MnTMPyP to mouse eyes reversed the elevated IOP caused by SIN-1(P<0.05).CONCLUSION:ONOO-levels in AH and plasma,detected by RN-NA,are significantly related to POAG and positively correlated with visual field defects in POAG patients.Hence,ONOO-is a potential biomarker of POAG,especially advanced POAG.Besides,anti-nitration compounds may be novel ocular hypotensive agents based on the animal study.

A ratiometric benzothiazole-based fluorescence probe for selectively recognizing HClO and its practical applications

A ratiometric probe(HBT-HBZ)bearing 2-hydrazino benzothiazole and 3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde for sensing hypochlorous acid(HClO)with high selectivity and sensitivity is reported in this article.The fluorescence intensity ratios(I470nm/I572nm)of the probe with different concentrations of analyte showed excellent selectivity and a linear response to minor changes in HClO.The detection limit of 24 nmol/L suggests that the sensor is very sensitive to HClO.According to the series of performed experiments,HBT-HBZ has practical applications,such as the detection of HClO residues in tap water,which has been rarely reported.In addition,confocal laser microscopy experiments confirmed that HBT-HBZ can selectively recognize HClO in HeLa cells.A ratiometric probe(HBT-HBZ)for sensing HClO with high selectivity and sensitivity is reported in this article.The probe exhibited high selectivity for HClO among other ROS,RNS and anions.In addition,HBTHBZ has some practical applications such as the analysis of the HClO content in tap water.Furthermore,confocal fluorescence microscopy imaging showed that HBT-HBZ can be applied for detecting HClO in living cells.

In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

Mitochondrial DNA has a special structure that is prone to damage resulting in many serious diseases,such as genetic diseases and cancers.Therefore,the rapid and specific monitoring of mitochondrial DNA damage is urgently needed for biological recognition.Herein,we constructed an in situ hydrophobic environment-triggering reactive fluorescence probe named MBI-CN.The fluorophore was 2-styrene-1H-benzo[d]imidazole,and malononitrile was introduced as a core into a molecule to initiate the hydrolysis reaction in the specific environment containing damaged mitochondrial DNA.In this design,MBI-CN conjugates to mitochondrial DNA without causing additional damages.Thus,MBI-CN can be hydrolyzed to generate MBI-CHO in an in situ hydrophobic environment with mitochondrial DNA damage.Meanwhile,MBI-CHO immediately emitted a significative fluorescence signal changes at 437 and 553 nm within 25 s for the damaged mitochondria DNA.Give that the specific and rapid response of MBI-CN does not cause additional damages to mitochondrial DNA,it is a potentially effective detection tool for the real-time monitoring of mitochondrial DNA damage during cell apoptosis and initial assessment of cell apoptosis.

Fine-tailoring the linker of near-infrared fluorescence probes for nitroreductase imaging in hypoxic tumor cells

Imaging hypoxia using fluorescence probes for nitroreductase(NTR) have attracted much attention in last decade. At least three different linkers have been commonly used to connect the recognition unit and reporting unit in reported probes for NTR. Meanwhile, the linker is known to be a key factor for achieving best sensing performance. In this work, three near-infrared fluorescence probes CyNP-1, CyNP-2 and CyNP-3 were designed and synthesized from an aminocyanine dye CyNP. The three probes have the same recognition unit and same fluorescence reporting unit, but different linkers. CyNP-1 was found to have the best sensing performance for NTR with 40-fold of fluorescence enhancement. It is well investigated how the difference of the linkers brings out the different sensing performance by HPLC, MS and docking calculations. In the end, CyNP-1 was found to have good selectivity for NTR and used to imaging hypoxia in Hela cells.

Rational design of shortwave infrared(SWIR) fluorescence probe:Cooperation of ICT and ESIPT processes for sensing endogenous cysteine

Cysteine is well-known to be an important biothiol and related to many diseases. However, the in vivo detection of endogenous cysteine still suffers from lacking small-molecule fluorophores with both excitation and emission in the near-infrared(650-900 nm)/shortwave-infrared region. Herein, we report a molecular engineering strategy for shortwave infrared(SWIR, 900-1700 nm) sensing of cysteine, which integrated an excited-state intermolecular proton transfer(ESIPT) building block into the intramolecular charge transfer(ICT) scaffold. The obtained novel fluorophore SH-OH displays a maximum absorption at the NIR region, and emission at the SWIR region. We introduce the cysteine-recognition moiety to SH-OH structure, and demonstrate sensing of endogenous cysteine in living animals, using the SWIR emission as a reliable off-on fluorescence signal. This fluorophore design strategy of cooperation of ICT and ESIPT processes expands the in vivo sensing toolbox for accurate analysis in clinical applications.

Highly selective and turn-on fluorescence probe with red shift emission for naked-eye detecting Al^(3+)and Ga^(3+)based on metal-organic framework

A novel ZnII-based metal-organic framework with the formula of{[Zn_(2)(BBIP)_(2)(NDC)_(2)]·H_(2)O}n(JXUST-5)derived from 3,5-bis(benzimidazol-1-yl)pyridine(BBIP)and 1,4-naphthalenedicarboxylic acid(H_(2)NDC)has been synthesized.The adjacent Zn^(II)ions are linked through two BBIP ligands to form a[Zn_(2)(BBIP)_(2)]secondary building unit(SBU).The neighbouring SBUs are further connected by NDC^(2-)withμ2-η^(1):η^(1)andμ2-η^(1):η^(1):η^(1)bridging modes to form a two-dimensional(2D)framework.Topological analysis shows that JXUST-5 could be simplified as an uninodal fes topology with a point symbol of{4.8^(2)}.Furthermore,the 2D framework net could be extended through C-H···πinteraction to form the three-dimensional supramolecular structure.Luminescent experiments suggest that JXUST-5 could selectively and sensitively recognize Al^(3+)and Ga^(3+)through fluorescence enhancement effect along with a relatively large red shift.The detection limits for Al^(3+)and Ga^(3+)are 0.17 and 0.69 ppm,respectively.Interestingly,the sensing process for both Al^(3+)and Ga^(3+)could be directly observed with naked eyes under 365 nm UV lamp.Notably,JXUST-5 could be recycled at least five times as a fluorescent sensor toward Al^(3+)and Ga^(3+),which is the second example of turn-on MOF based fluorescent sensor toward Ga^(3+).

Carbon Nitride Quantum Dots:A Novel Fluorescent Probe for Non-Enzymatic Hydrogen Peroxide and Mercury Detection

The mercury species in the ocean(MeHg,Hg^(2+))will be enriched in marine organisms and threaten human health through the food chain.While the excessive H_(2)O_(2)in the metabolic process will produce hydroxyl radicals and accelerate the aging of human cells,causing a series of diseases.Hence,the cost-effective and rapid detection of mercury and H_(2)O_(2)is of urgent requirement and significance.Here,we synthesized emerging graphitic carbon nitride quantum dots(g-CNQDs)with high fluorescence quantum yield(FLQY)of 42.69%via a bottom-up strategy by a facile one-step hydrothermal method.The g-CNQDs can detect the H_(2)O_(2)and Hg^(2+)through the fluorescence quenching effect between g-CNQDs and detected substances.With the presence of KI,g-CNQDs show concentration-dependent fluorescence toward H_(2)O_(2),with a wide detection range of 1–1000μmolL^(-1)and a low detection limit of 0.23μmolL^(-1).The g-CNQDs also show sensitivity toward Hg^(2+)with a detection range of 0–0.1μmolL^(-1)and a detection limit of 0.038μmolL^(-1).This dual-function detection of g-CNQDs has better practical application capability compared to other quantum dot detection.This study may provide a new strategy for g-CNQDs preparation and construct a fluorescence probe that can be used in various systems involving H_(2)O_(2)and Hg^(2+),providing better support for future bifunctional or multifunction studies.

Synthesis of 3-hydroxyflavone fluorescent probes and study of their fluorescence properties

Direct measurement of dipole potential in biological membranes has been impossible and 3-hydroxyflavones(3HFs) have allowed detection of changes in dipole potential in biological systems.In the present study,sixteen derivatives of 3HF with aliphatic hydrocarbon chains of different lengths at 4′-position and 6-position were synthesized.The basic fluorescence properties of 3HFs are maintained in all the probes in terms of strong blue shift in maximum fluorescence emission wavelength and】100 fold increase in quantum yield in organic solvents and in dioleoylphosphatidylcholine(DOPC) small unilamellar vesicles(SUV) in comparison to in aqueous Hepes buffer(15 mmol/L,pH 7.4).More importantly,the ability of the new compounds to report dipole potential changes in biological systems are also maintained,since all the new probes showed spectrum properties that are similar to yet different from that of F4N1,which potentially may allow more sensitive measurement of the dipole potential change in membranes.

Visualization of endoplasmic reticulum viscosity in the liver of mice with nonalcoholic fatty liver disease by a near-infrared fluorescence probe

Nonalcoholic fatty liver disease(NAFLD)can cause serious liver damage.Early diagnosis and effective treatment of NAFLD can greatly improve treatment rates.The initiation and development of NAFLD has been closely linked to endoplasmic reticulum(ER)stress,which might cause ER viscosity variations.Therefore,if the internal relationship between ER viscosity and NAFLD is clarified,an effective approach for early diagnosis may result.Herein,we fabricated a novel near-infrared(NIR)fluorescence imaging probe,Er-V,for monitoring ER viscosity through a molecular rotor strategy.Er-V exhibited a strong NIR fluorescence signal(at 626 nm)when the environmental viscosity hindered the rotation of the malononitrile group.Using Er-V,we successfully observed a significant enhancement in viscosity in the liver of mice with NAFLD.Therefore,this imaging method based on Er-V is expected to provide a new approach for early detection and diagnosis of NAFLD.

A Fluorescent Probe based on a Carbazole Derivative for Detecting Brilliant Blue in Food

Here a fluorescent probe based on a carbazole derivative(CNS)was developed to increase the detection range and reduce the detection limit of brilliant blue.Characteristics of CNS are studied.Due to the quenching ability of colorants,CNS shows an excellent current response to brilliant blue(from 1 to 10μM)with a detection limit of 2.7×10^(-8)mol/L(3σ/k)in the conditions of a 1:1 volume ratio of water to tetrahydrofuran.And the stability and reproducibility of CNS in the detection of actual samples indicate great potential for application.

Evaluation of a resoruf in-based fluorescent probe for tyrosinase detection in skin pigmentation disorders

Purpose Skin pigmentation disorders,such as vitiligo and melasma,are difficult to diagnose in the early stages,but abnor-mal tyrosinase levels and tyrosinase activity are potential indicators.Some resorufin-based fluorescence probes(RBFPs)have been designed to detect tyrosinase in tumors,but they have not been used in skin pigmentation disorders.In this study,one of these RBFPs(synthesized by resorufin salt coupled with 3-(bromomethyl)phenol)was evaluated comprehensively.Methods The RBFP was tested in different kinds of mouse and human skin cells,as well as in in vivo models,including zebrafish,guinea pigs,and Sprague-Dawley rats.In addition,small interfering RNAs(siRNAs),kojic acid,and 1-phenyl-2-thiourea(PTU)were used to inhibit tyrosinase levels or tyrosinase activity.Results This probe successfully detected tyrosinase and emitted red fluorescence in melanoma cells and melanocytes.Fluorescence was also observable in zebrafish and on the skin of guinea pigs when using the RBFP.In mouse and human cells,the RBFP showed good selectivity to tyrosinase.Moreover,in the case of decreased tyrosinase levels or activity caused by siRNAs,kojic acid,or PTU,the probe was sensitive to these changes.Further,the RBFP showed no toxic effects at concentrations of<20μmol/L,both in vitro and in vivo.Conclusions Our findings indicate the value and limitations of the RBFP in tyrosinase detection,but suggest the need for further improvement of fluorescent probes in the diagnosis of skin pigmentation disorders.

Hydroxylated graphene quantum dots as fluorescent probes for sensitive detection of metal ions

Highly sensitive methods are important for monitoring the concentration of metal ions in industrial wastewater.Here,we developed a new probe for the determination of metal ions by fluorescence quenching.The probe consists of hydroxylated graphene quantum dots(H-GQDs),prepared from GQDs by electrochemical method followed by surface hydroxylation.It is a non-reactive indicator with high sensitivity and detection limits of 0.01μM for Cu2+,0.005μM for Al3+,0.04μM for Fe3+,and 0.02μM for Cr3+.In addition,the low biotoxicity and excellent solubility of H-GQDs make them promising for application in wastewater metal ion detection.

Responsive mechanism and coordination mode effect of a bipyridine-based two-photon fluorescent probe for zinc ion

The properties of one-photon absorption(OPA),emission and two-photon absorption(TPA)of a bipyridine-based zinc ion probe are investigated employing the density functional theory in combination with response functions.The responsive mechanism and coordination mode effect are explored.The structural fluctuation is illustrated by molecular dynamics simulation.The calculated OPA and emission wavelengths of the probe are consistent with the experimental data.It is found that the red-shift of OPA wavelength and the enhancement of TPA intensity are induced by the increased intra-molecular charge transfer mechanism upon metal binding.The structural fluctuation could result in the blue-shift of TPA wavelength and the decrease of the TPA cross section.The TPA properties are quite different among the zinc complexes with different coordination modes.The TPA wavelength of the complexes with two ligands is close to that of the probe,which is in agreement with the experimental observation.