Recent Advances in Dual-target-activated Fluorescent Probes for Biosensing and Bioimaging.

Fluorescent probes have been powerful tools for visualizing and quantifying multiple dynamic processes in living cells. However, the currently developed probes are often constructed by conjugation a fluorophore with a recognition moiety and given signal-output after triggering with one singly target interest. Compared with the single-target-activated fluorescent probes mentioned above, the dual-target-activated ones, triggering with one target under stimulus (such as photoirradiation, microenvironment) or another targets, have the advantages of advoiding nonspecific activation and "false positive" results in complicated environments. In recent years, many dual-target-activated fluorescent probes have been developed to detect various biologically relevant species. In view of the importance of a comprehensive understanding of dual-target- activated fluorescent probes, a thorough summary of this topic is urgently needed. However, no comprehensive and critical review on dual target activated fluorescent probes has been published recently. In this review, we focus on the dual-target-activated fluorescent probes and briefly outline their types and current state of development. In each type, the chemical structure, proposed responsive mechanism and application of probes are highlighted. At last, the challenges and prospective opportunities of every type were proposed.

A novel ratiometric near-infrared fluorescent probe for monitoring cyanide in food samples.

Cyanide is a highly toxic anion. Nonetheless, many food plants could produce endogenous cyanide, which causes great danger to human health. Thus, monitoring cyanide in food samples is critically significant. Herein, we rationally developed the first ratiometric near-infrared fluorescent probe for sensing cyanide in food samples. The probe displayed noticeable fluorescence in near-infrared region. Moreover, upon treatment with cyanide, the probe exhibited highly selective and sensitive ratiometric fluorescence response, with limit of detection determined to be 0.075 µM and limit of quantification determined to be 0.25 µM. The ratios of fluorescent intensities at 519 and 688 nm (I519/I688) was linear with added cyanide concentrations from 0 to 80 µM. The relative standard deviations for repeatability and reproducibility varied from 0.55 to 8.94 and from 1.17 to 9.46, respectively. Significantly, probe Hy has been successfully applied for monitoring cyanide in various food samples, such as almonds, sprouting potatoes, and bamboo shoots.

A novel fluorescent probe with extremely low background fluorescence for sensing hypochlorite in zebrafish.

Development of an efficient fluorescent probe for sensing hypochlorite in water samples and biological samples is highly demanded. However, the currently reported fluorescent probes for hypochlorite frequently suffered from the problem of high background fluorescence. Herein, based on the combined effect of two different fluorescence quenching groups, we rationally developed a novel fluorescent probe for hypochlorite with extremely low background fluorescence. Notably, due to the doubly quenching groups, the probe could even keep low background fluorescence in a solution with high viscosity. Furthermore, the probe displayed highly sensitive and selective response to hypochlorite, with the detection limits calculated to be 10.5 nM. Practical application demonstrated that the probe was able to quantitatively detect hypochlorite in various water samples with good recovery. Significantly, the probe showed extremely low background fluorescence in living cells and was capable of detecting minor variation of endogenous hypochlorite in RAW 264.7 cells. Moreover, the fluorescence imaging different concentration of hypochlorite in zebrafish has been successfully conducted. The probe developed herein will be widely used as a reliable tool to accurately monitor the variation of hypochlorite in living organism.

Simultaneous Discrimination of Hypochlorite and Single Oxygen during Sepsis by a Dual-Functional Fluorescent Probe.

Hypochlorite (ClO-) and singlet oxygen (1O2) commonly coexist in living systems and exert important interplaying roles in many diseases. To dissect their complex inter-relationship, it is urgently required to construct a fluorescent probe that can discriminate ClO- and 1O2 in living organisms. Herein, by taking the 3-(aliphaticthio)-propan-1-one group as the unique recognition unit for both ClO- and 1O2, we proposed the first fluorescent probe, Hy-2, to simultaneously discriminate ClO- and 1O2 with high sensitivity and selectivity. Probe Hy-2 itself showed fluorescence in blue channel. After treatment with ClO- and 1O2, respectively, pronounced fluorescence enhancements were observed in the green channel and red channel correspondingly. Moreover, upon development of the probe with aggregation-induced emission (AIE) characteristics, the probe could work well in a solution with high water volume fraction. Probe Hy-2 was also able to accumulate into mitochondria and was utilized as an effective tool to image exogenous and endogenous ClO- and 1O2 in mitochondria. Significantly, as the first trial, probe Hy-2 was employed to simultaneously monitor the variation of ClO- and 1O2 level in cecal tissues of rat in the cecal ligation and puncture (CLP)-induced polymicrobial sepsis model. The results demonstrated that the expressed ClO- and 1O2 levels were tightly correlated with the severity of sepsis, inferring that the overproduction of ClO- and 1O2 is an important factor in the pathogenesis of sepsis. The probe illustrated herein may provide a guide for further exploring the functions of ClO- and 1O2 in various diseases.

Determination of Cyanide in Water and Food Samples Using an Efficient Naphthalene-Based Ratiometric Fluorescent Probe.

Monitoring cyanide levels in water and food samples is crucial. Herein, we rationally developed a simple and efficient fluorescent probe for cyanide determination. The probe displayed selective ratiometric fluorescent response to cyanide. In addition, after treatment with cyanide, the fluorescence ratios (I 509/I 466) exhibited a good linearity with cyanide concentration in the range of 0-60 µM, and the detection limit was determined to be 0.23 µM (S/N = 3). Significantly, the practical application demonstrated that the probe was able to quantitatively detect cyanide concentration in natural water samples. Monitoring of endogenous cyanide in cherry nut by the probe was also successfully conducted. Notably, upon fabrication of test strips, the probe could be conveniently utilized for field measurement of cyanide in bitter almond without relying on sophistical instruments. Furthermore, the cyanide in potato tissues was determined for the first time by means of fluorescence imaging.

A novel and sensitive chemiluminescence immunoassay based on AuNCs@pepsin@luminol for simultaneous detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether and tetrabromobisphenol A mono(hydroxyethyl) ether.

A novel chemiluminescence immunoassay based on luminol-modified gold nanoclusters (AuNCs@Peps@luminol) was developed for simultaneous detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE) and tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA-MHEE), an important derivative and byproduct of tetrabromobisphenol A (TBBPA), respectively. In the system, alkaline phosphatase (ALP) was labeled on the second antibody (Ab2) for signal amplification. When ALP-Ab2 was captured by antigen-primary antibody (Ab1) complex, disodium phenyl phosphate (PPNa) generated massive phenol under the catalysis of ALP, markedly inhibiting the chemiluminescence intensity of AuNCs@Peps@luminol. Under the optimized conditions, the calculated detection of limit (LOD, 90% inhibition) was 0.078 µg/L for TBBPA-DHEE with a linear range of 0.23-9.32 µg/L, which was lower 9 times than that of conventional ELISA with the same antibody. In addition, our method showed satisfactory accuracy and precision (recoveries, 88.00-113.4%; CV, 2.75-8.14%), it can be applied to systematically investigate the concentration of the trace TBBPA-DHEE and TBBPA-MHEE in environmental and food samples.

A sensitive, colorimetric immunosensor based on Cu-MOFs and HRP for detection of dibutyl phthalate in environmental and food samples.

A sensitive and artful colorimetric immunosensor based on horseradish peroxidase (HRP) was designed by labelling metal-organic frameworks (Cu-MOFs) on the second antibody (Cu-MOFs@Ab2) as signal amplification for the detection of trace dibutyl phthalate (DBP). In this system, when Cu-MOFs@Ab2 was captured by antigen- primary antibody (Ab1) complex, tremendous Cu(II) will be released from Cu-MOFs in the presence of nitric acid (HNO3), and Cu(II) will be further reduced to Cu(I) after the addition of sodium ascorbate (SA), consequently, inhibiting the HRP to catalyse the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (ox TMB). Under the optimized conditions, the limit of detection (LOD) was 1 µg L-1, which was almost 60 times lower than that using a conventional ELISA with the same antibody. In addition, our method showed good accuracy and reproducibility (recoveries of 87.73-103.4%; CV values of 1.46-5.95%) through a spike-recovery analysis. The proposed immunosensor indicated great potential for trace DBP determination from environmental and food samples.

Cyclodextrin-functionalized hollow carbon nanospheres by introducing nanogold for enhanced electrochemical sensing of o-dihydroxybenzene and p-dihydroxybenzene.

In this work, hollow carbon nanospheres (HCNS) were prepared, followed by the introduction of gold nanoparticles (AuNPs) on the HCNS surface, and then functionalization with per-6-thio-ß-cyclodextrin (CD) based on the formation of "Au-S" bond, resulting in a novel cyclodextrin/carbon-based nanohybrid (CD-AuNPs/HCNS), which possesses the unique properties of HCNS (excellent electrochemical properties and large surface area), CD (high host-guest recognition and water-solubility) and AuNPs (excellent electrocatalytic activity). The obtained CD-AuNPs/HCNS nanohybrids were characterized by scanning electron microscopy, transmission electron microscopy, inductively coupled plasma-atomic emission spectroscopy, Fourier transform infrared spectroscopy and electrochemical methods. Furthermore, CD-AuNPs/HCNS were applied in the simultaneous electrochemical sensing of o-dihydroxybenzene (o-DHB) and p-dihydroxybenzene (p-DHB) (both o- and p-DHB have similar structures and coexist in environment; moreover, they are toxic to humans and difficult to degrade). Under the optimum conditions, the detection limits of o- and p-DHB obtained in this work are 0.01 and 0.02 µM, respectively.

Sensitive electrochemical sensing for polycyclic aromatic amines based on a novel core-shell multiwalled carbon nanotubes@ graphene oxide nanoribbons heterostructure.

Being awfully harmful to the environment and human health, the qualitative and quantitative determinations of polycyclic aromatic amines (PAAs) are of great significance. In this paper, a novel core-shell heterostructure of multiwalled carbon nanotubes (MWCNTs) as the core and graphene oxide nanoribbons (GONRs) as the shell (MWCNTs@GONRs) was produced from longitudinal partially unzipping of MWCNTs side walls using a simple wet chemical strategy and applied for electrochemical determination of three kinds of PAAs (1-aminopyrene (1-AP), 1-aminonaphthalene and 3,3'-diaminobiphenyl). Scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, thermogravimetric analysis and electrochemical methods were used to characterize the as-prepared MWCNTs@GONRs. Due to the synergistic effects from MWCNTs and GONRs, the oxidation currents of PAAs at the MWCNTs@GONRs modified glassy carbon (GC) electrode are much higher than that at the MWCNTs/GC, graphene/GC and bare GC electrodes. 1-AP was used as the representative analyte to demonstrate the sensing performance of the MWCNTs@GONRs/GC electrode, and the proposed modified electrode has a linear response range of 8.0-500.0 nM with a detection limit of 1.5 nM towards 1-AP.

Reaction-based fluorescent probe for detection of endogenous cyanide in real biological samples.

Herein, two compounds (1 a and 1 b) were rationally constructed as novel reaction-based fluorescent probes for CN(-) by making use of the electron-withdrawing ability of the cyano group that was formed from the sensing reaction. Notably, this design strategy was first employed for the development of fluorescent CN(-) probes. The experimental details showed that probe 1 a exhibited a fluorescence turn-on response to CN(-), whereas other anions, biological thiols, and hydrogen sulfide gave almost no interference. The detection limit of probe 1 a for CN(-) was found to be 0.12 µM. The sensing reaction product of 1 a with CN(-) was characterized by NMR spectroscopy and mass spectrometry. TD-DFT calculations demonstrated that the formed cyano group drives the intramolecular charge transfer (ICT) process from coumarin dye to the cyano group and thus the original strong ICT from the coumarin dye to the 3-position pyridyl vinyl ketone substituent is weakened, which results in recovery of coumarin fluorescence. The practical utility of 1 a was also examined. By fabricating paper strips, probe 1 a can be used as a simple tool to detect CN(-) in field measurements. Moreover, probe 1 a has been successfully applied for quantitative detection of endogenous CN(-) from cassava root.

Efficient fluorescence resonance energy transfer-based ratiometric fluorescent cellular imaging probe for Zn(2+) using a rhodamine spirolactam as a trigger.

This letter described the design and synthesis of a novel fluorescein-appended rhodamine spirolactam derivative and its preliminary application as a ratiometric fluorescent cellular imaging probe for Zn(2+). The ratiometric fluorescent signal change of the probe is based on an intramolecular fluorescence resonance energy transfer (FRET) mechanism modulated by a specific metal ion induced ring-opening process of the rhodamine spirolactam (acting as a trigger). In the new developed sensing system, the emission peaks of the two fluorophores are well-resolved, which can avoid the emission spectra overlap problem generally met by spectra-shift type probes and benefits for observation of fluorescence signal change at two different emission wavelengths with high resolution. It also benefits for a large range of emission ratios, thereby a high sensitivity for Zn(2+)detection. Under optimized experimental conditions, the probe exhibits a stable response for Zn(2+) over a concentration range from 2.0 x 10(-7) to 2.0 x 10(-5) M, with a detection limit of 4.0 x 10(-8) M. Most importantly, the novel probe has well solved the problem of serious interferences from other transition metal ions generally met by previously reported typical fluorescent probes for Zn(2+) with the di(2-picolyl)amine moiety as the receptor (in this case, the fluorescence response induced by Cd(2+)is even comparable to that of Zn(2+)) and shows a reversible and fast response toward Zn(2+). All these unique features make it particularly favorable for ratiometric cellular imaging investigations. It has been preliminarily used for ratiometric imaging of Zn(2+) in living cells with satisfying resolution.

Highly sensitive and selective colorimetric and off-on fluorescent chemosensor for Cu2+ in aqueous solution and living cells.

The design and synthesis of a novel rhodamine spirolactam derivative and its application in fluorescent detections of Cu(2+) in aqueous solution and living cells are reported. The signal change of the chemosensor is based on a specific metal ion induced reversible ring-opening mechanism of the rhodamine spirolactam. It exhibits a highly sensitive "turn-on" fluorescent response toward Cu(2+) in aqueous solution with an 80-fold fluorescence intensity enhancement under 10 equiv of Cu(2+) added. This indicates that the synthesized chemosensor effectively avoided the fluorescence quenching for the paramagnetic nature of Cu(2+) via its strong binding capability toward Cu(2+). With the experimental conditions optimized, the probe exhibits a dynamic response range for Cu(2+) from 8.0 x 10(-7) to 1.0 x 10(-5) M, with a detection limit of 3.0 x 10(-7) M. The response of the chemosensor for Cu(2+) is instantaneous and reversible. Most importantly, both the color and fluorescence changes of the chemosensor are remarkably specific for Cu(2+) in the presence of other heavy and transition metal ions (even those that exist in high concentration), which meet the selective requirements for biomedical and environmental monitoring application. The proposed chemosensor has been used for direct measurement of Cu(2+) content in river water samples and imaging of Cu(2+) in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems.

A ratiometric chemosensor for fluorescent determination of Hg(2+) based on a new porphyrin-quinoline dyad.

Quinolin-8-ol p-[10',15',20'-triphenyl-5'-porphyrinyl]benzoate (1) was synthesized for the first time and developed as a ratiometric fluorescent chemosensor for recognition of Hg(2+) ions in aqueous ethanol with high selectivity. The 1-Hg(2+) complexation quenches the fluorescence of porphyrin at 646nm and induces a new fluorescent enhancement at 603nm. The fluorescent response of 1 towards Hg(2+) seems to be caused by the binding of Hg(2+) ion with the quinoline moiety, which was confirmed by the absorption spectra and (1)H NMR spectrum. The fluorescence response fits a Hill coefficient of 1 (1.0308), indicating the formation of a 1:1 stoichiometry for the 1-Hg(2+) complex. The analytical performance characteristics of the chemosensor were investigated. The sensor shows a linear response toward Hg(2+) in the concentration range of 3x10(-7) to 2x10(-5)M with a limit of detection of 2.2x10(-8)M. Chemosensor 1 shows excellent selectivity to Hg(2+) over transition metal cations except Cu(2+), which quenches the fluorescence of 1 to some extent when it exists at equal molar concentration. Moreover, the chemosensor are pH-independent in 5.0-9.0 and show excellent selectivity for Hg(2+) over transition metal cations.

A fluorescent probe for zinc ions based on N-methyltetraphenylporphine with high selectivity.

N-methyl-alpha,beta,gamma,delta-tetraphenylporphine (NMTPPH) has been used to detect trace amount of zinc ions in ethanol-water solution by fluorescence spectroscopy. The fluorescent probe undergoes a fluorescent emission intensity enhancement upon binding to zinc ions in EtOH/H(2)O (1:1, v/v) solution. The fluorescence enhancement of NMTPPH is attributed to the 1:1 complex formation between NMTPPH and Zn(II) which has been utilized as the basis for the selective detection of Zn(II). The linear response range covers a concentration range of Zn(II) from 5.0x10(-7) to 1.0x10(-5)mol/L and the detection limit is 1.5x10(-7)mol/L. The fluorescent probe exhibits high selectivity over other common metal ions except for Cu(II).

A porphyrin derivative containing 2-(oxymethyl)pyridine units showing unexpected ratiometric fluorescent recognition of Zn2+ with high selectivity.

A porphyrin derivative (1), containing two 2-(oxymethyl)pyridine units has been designed and synthesized as chemosensor for recognition of metal ions. Unlike many common porphyrin derivatives that show response to different heavy metal ions, compound 1 exhibits unexpected ratiometric fluorescence response to Zn(2+) with high selectivity. The response of the novel chemosensor to zinc was based on the porphyrin metallation with cooperating effect of 2-(oxymethyl)pyridine units. The change of fluorescence of 1 was attributed to the formation of an inclusion complex between porphyrin ring and Zn(2+) by 1:1 complex ratio (K=1.04x10(5)), which has been utilized as the basis of the fabrication of the Zn(2+)-sensitive fluorescent chemosensor. The analytical performance characteristics of the proposed Zn(2+)-sensitive chemosensor were investigated. The sensor can be applied to the quantification of Zn(2+) with a linear range covering from 3.2x10(-7) to 1.8x10(-4) M and a detection limit of 5.5x10(-8) M. The experiment results show that the response behavior of 1 to Zn(2+) is pH-independent in medium condition (pH 4.0-8.0) and show excellent selectivity for Zn(2+) over transition metal cations.

A highly selective potentiometric sensor of molybdate based on a mu-oxo-bridged manganeseporphyrin dimer.

A novel high-selective potentiometric sensor for molybdate was prepared with a PVC membrane combining mu-oxo-bis[5,10,15,20-tetra(p-methylphenyl)porphinatomanganese(III)] [[Mn(p-Me)TPP](2)O] as an electroactive material and 2-nitrophenyl octyl ether (o-NPOE) as a plasticizer in the percentage ratio of 3:65:32, [Mn(p-Me)TPP](2)O:o-NPOE:PVC (w:w). The sensor exhibited a linear response with a Nernstian slope of 30.5 mV per decade within a concentration range of 2.1 x 10(-6) to 1.0 x 10(-1) M MoO4(2-), with a working pH range from 5.0 to 12.5, and a fast response time of less than 15 s. The electrode showed improved selectivity toward molybdate with respect to common coexisting anions compared to monometalloporphyrin counterparts. Several electroactive materials and solvent mediators were compared and the experimental conditions were optimized. The sensor is preliminary applied to the assay of MoO4(2-) in corrosion inhibitor samples with satisfactory results.

A wide pH range optical sensing system based on a sol-gel encapsulated amino-functionalized corrole.

The synthesis of a new compound, 10-(4-aminophenyl)-5,15-dimesitylcorrole, and its application for the preparation of optical chemical pH sensors is described. The dye materials were immobilized in a sol-gel glass matrix and characterised upon exposure to aqueous buffer solutions. The response of the sensor is based on the fluorescence intensity changing of corrole owing to multiple steps of protonation and deprotonation. Due to its containing several proton sensitive centers, the 10-(4-aminophenyl)-5,15-dimesitylcorrole based optode shows a wider response range toward pH than that of tetraphenylporphyrin (TPPH(2)) and 5,10,15-tris(pentafluorophenyl)corrole (H(3)(tpfc)). It shows a linear pH response in the range of 2.17-10.30. The effect of the composition of the sensor membrane has been studied and the experimental conditions were optimized. The optode showed good reproducibility and reversibility, and common co-existing inorganic ions did not show obvious interference to its pH measurement.

A fluorescent chemical sensor for Hg(II) based on a corrole derivative in a PVC matrix.

A fluorescent chemical sensor for Hg(II) using 5,10,15-tris(pentafluorophenyl)corrole (H(3)(tpfc)) as fluorophore is described in this paper. The response of the sensor is based on the fluorescence quenching of H(3)(tpfc) by coordination with Hg(II). H(3)(tpfc) based sensor shows a linear response towards Hg(II) in the concentration range from 1.2x10(-7) to 1.0x10(-4)M, with a working pH range from 5.0 to 8.0. The response time for Hg(II) concentration