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Fluorescence catalytic beacons have emerged as a general platform for sensing applications. However, almost all such sensing systems need covalent modification of the DNAzymes with fluorophore-quencher pairs, which may require elaborate design of the synthetic routes and many heavy and complicated synthetic steps and result in increased cost and lower synthesis yield. Here we report the construction of fluorescent cascadic catalytic beacons. With separation of the molecular recognition module from the signal reporter, this new design both avoids DNAzyme modifications and improves sensitivity through an endonuclease-based cascadic enzymatic signal amplification. This allows detection of L-histidine with high sensitivity (LOD = 200 nM) and excellent specificity. The proposed sensing system has also been used for detection of L-histidine in cellular homogenate with satisfactory results.

On the basis of the remarkable difference in affinity of graphene (GO) with ssDNA containing a different number of bases in length, we for the first time report a GO-DNAzyme based biosensor for amplified fluorescence "turn-on" detection of Pb(2+). A FAM-labeled DNAzyme-substrate hybrid acted as both a molecular recognition module and signal reporter and GO as a superquencher. By taking advantage of the super fluorescence quenching efficiency of GO, our proposed biosensor exhibits a high sensitivity toward the target with a detection limit of 300 pM for Pb(2+), which is lower than previously reported for catalytic beacons. Moreover, with the choice of a classic Pb(2+)-dependent GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the newly designed sensing system also shows an obviously improved selectivity than previously reported methods. Moreover, the sensing system was used for the determination of Pb(2+) in river water samples with satisfying results.

In this paper, we unveil a novel naphthalimide-porphyrin hybrid based fluorescence probe (1) for ratiometric detection of Hg(2+) in aqueous solution and living cells. The ratiometric signal change of the probe is based on a carefully predesigned molecule containing two independent Hg(2+)-sensitive fluorophores with their maximal excitation wavelengths located at the same range, which shows reversibly specific ratiometric fluorescence responses induced by Hg(2+). In the new developed sensing system, the emissions of the two fluorophores are well-resolved with a 125 nm difference between two emission maxima, which can avoid the emission spectra overlap problem generally met by spectra-shift type probes and is especially favorable for ratiometric imaging intracellular Hg(2+). It also benefits from a large range of emission ratios and thereby a high sensitivity for Hg(2+) detection. Under optimized experimental conditions, the probe exhibits a stable response for Hg(2+) over a concentration range from 1.0 x 10(-7) to 5.0 x 10(-5) M, with a detection limit of 2.0 x 10(-8) M. The response of the probe toward Hg(2+) is reversible and fast (response time less than 2 min). Most importantly, the ratiometric fluorescence changes of the probe are remarkably specific for Hg(2+) in the presence of other abundant cellular metal ions (i.e., Na(+), K(+), Mg(2+), and Ca(2+)), essential transition metal ions in cells (such as Zn(2+), Fe(3+), Fe(2+), Cu(2+), Mn(2+), Co(2+), and Ni(2+)), and environmentally relevant heavy metal ions (Ag(+), Pb(2+), Cr(3+), and Cd(2+)), which meets the selective requirements for biomedical and environmental monitoring application. The recovery test of Hg(2+) in real water samples demonstrates the feasibility of the designed sensing system for Hg(2+) assay in practical samples. It has also been used for ratiometric imaging of Hg(2+) in living cells with satisfying resolution, which indicates that our novel designed probe has effectively avoided the general emission spectra overlap problem of other ratiometric probes.

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 novel fluorometric assay method based on target-induced signal on was developed for acetylcholinesterase (AChE) inhibitor with obviously improved detection sensitivity. In this method, the AChE molecules catalyzed the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, which in turn can specifically react with fluorescent squaraine derivative, a specific chemodosimeter for thiol-containing compounds, resulting in fluorescence quenching and offering a low fluorometric background for the further detection of AChE inhibitor. In the presence of AChE inhibitor, the catalytic hydrolysis of ATCl is blocked, and then the squaraine derivative remains intact and shows signal-on fluorescence. The amount of the remaining fluorescent squaraine derivative is positively correlated with that of the AChE inhibitor in solution. This new designed sensing system shows an obviously improved sensitivity toward target with a detection limit of 5 pg mL(-1) (0.018 nM) for the AChE inhibitor, comparing favorably with previously reported fluorometric methods. To our best knowledge, this new method is the first example of fluorometric enzymatic assay for AChE inhibitors based on such a signal-on principle and using a specific reaction, which has potential to offer an effective strategy for the detection of AChE inhibitors.

Fluorescence resonance energy transfer (FRET) strategy has been widely applied in designing ratiometric probes for bioimaging applications. Unfortunately, for FRET systems, sufficiently large spectral overlap is necessary between the donor emission and the acceptor absorption, which would limit the resolution of double-channel images. The through-bond energy transfer (TBET) system does not need spectral overlap between donor and acceptor and could afford large wavelength difference between the two emissions with improved imaging resolution and higher energy transfer efficiency than that of the classical FRET system. It seems to be more favorable for designing ratiometric probes for bioimaging applications. In this paper, we have designed and synthesized a coumarin-rhodamine (CR) TBET system and demonstrated that TBET is a convenient strategy to design an efficient ratiometric fluorescent bioimaging probe for metal ions. Such TBET strategy is also universal, since no spectral overlap between the donor and the acceptor is necessary, and many more dye pairs than that of FRET could be chosen for probe design. As a proof-of-concept, Hg(2+) was chosen as a model metal ion. By combining TBET strategy with dual-switch design, the proposed sensing platform shows two well-separated emission peaks with a wavelength difference of 110 nm, high energy transfer efficiency, and a large signal-to-background ratio, which affords a high sensitivity for the probe with a detection limit of 7 nM for Hg(2+). Moreover, by employing an Hg(2+)-promoted desulfurization reaction as recognition unit, the probe also shows a high selectivity to Hg(2+). All these unique features make it particularly favorable for ratiometric Hg(2+) sensing and bioimaging applications. It has been preliminarily used for a ratiometric image of Hg(2+) in living cells and practical detection of Hg(2+) in river water samples with satisfying results.

It is well-known that Zr(4+) could selectively bind with two phosphate-functionalized molecules through a coordinate covalent interaction to form a sandwich-structured complex (-PO(3)(2-)-Zr(4+)-PO(3)(2-)-). In this paper, we for the first time converted such interaction into fluorescence sensing systems for Zr(4+) via a target-triggered DNA molecular beacon strategy. In the new designed sensing system, two phosphorylated and pyrene-labeled oligonucleotides were chosen as both recognition and reporter units, which will be linked by target Zr(4+) to form a hairpin structure and bring the two labeled pyrene molecules into close proximity, resulting in a "turn-on" excimer fluorescence signal. Moreover, γ-cyclodextrin was introduced to afford an amplified fluorescence signal and, therefore, provided an improved sensitivity for the target Zr(4+). This allows detection of Zr(4+) with high sensitivity (limit of detection, LOD = 200 nM) and excellent selectivity. The proposed sensing system has also been used for detection of Zr(4+) in river water samples with satisfactory result.

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.

Mesoporous SBA-15 silica is an excellent support for constructing fluorescent surface sensors. In this letter, we reported a two-step surface reaction involved strategy to construct efficient fluorescent surface sensors for metal ions by clicking fluoroionophores onto azide-functionalized SBA-15. Our experimental results indicate that such a strategy exhibits an obviously higher loading efficiency within commercial SBA-15 than a previously reported strategy. As a proof-of-concept, a newly designed alkyne-functionalized Hg(2+) fluoroionophore was grafted onto SBA-15 to form a fluorescent Hg(2+) surface sensor. It shows improved sensitivity and selectivity than the fluoroionophore itself working in the solution phase with a detection limit of 2.0 x 10(-8) M for Hg(2+).

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.