A molecular approach to rationally constructing specific fluorogenic substrates for the detection of acetylcholinesterase activity in live cells,mice brains and tissues

Acetylcholinesterase (AChE) is an extremely critical hydrolase tightly associated with neurological diseases. Currently, developing specific substrates for imaging AChE activity still remains a great challenge due to the interference from butyrylcholinesterase (BChE) and carboxylesterase (CE). Herein, we propose an approach to designing specific substrates for AChE detection by combining dimethylcarbamate choline with a self-immolative scaffold. The representative P10 can effectively eliminate the interference from CE and BChE. The high specificity of P10 has been proved via imaging AChE activity in cells. Moreover, P10 can also be used to successfully map AChE activity in different regions of a normal mouse brain, which may provide important data for AChE evaluation in clinical studies. Such a rational and effective approach can also provide a solid basis for designing probes with different properties to study AChE in biosystems and another way to design specific substrates for other enzymes.

In this work, a new approach was developed for designing the representative P10 with high selectivity and sensitivity for imaging AChE activity in the cells and normal mouse brain.     

A novel off-on fluorescent probe for specific detection and imaging of cysteine in live cells and in vivo

Cysteine(Cys) plays a pivotal role in many physiological and pathological processes,including detoxification and protein synthesis.The abnormal levels of Cys are linked to many diseases.In this study,a novel red-emitting off-on fluorescent probe Cys-TCF was masterly constructed for discriminative detection of Cys.After a series of experimental assessment,Cys-TCF displayed higher selectivity and sensitivity for Cys over other biothilols with a low detection limit(0.04 μmol/L).More notably,the pro...     

Organelle-specific zinc detection using zinpyr-labeled fusion proteins in live cells

A protein labeling approach is employed for the localization of a zinc-responsive fluorescent probe in the mitochondria and in the Golgi apparatus of living cells. ZP1, a zinc sensor of the Zinpyr family, was functionalized with a benzylguanine moiety and thus converted into a substrate (ZP1BG) for the human DNA repair enzyme alkylguaninetransferase (AGT or SNAP-Tag). The labeling reaction of purified glutathione S-transferase tagged AGT with ZP1BG and the zinc response of the resulting protein-bound sensor were confirmed in vitro. The new detection system, which combines a protein labeling methodology with a zinc fluorescent sensor, was tested in live HeLa cells expressing AGT in specific locations. The enzyme was genetically fused to site-directing proteins that anchor the probe onto targeted organelles. Localization of the zinc sensors in the Golgi apparatus and in the mitochondria was demonstrated by fluorescence microscopy. The protein-bound fluorescence detection system is zinc-responsive in living cells.     

A near-infrared emissive Al sensing platform for specific detection in solution,cells and probing DNase activity

A new tricarbocyanine-based chemosensor exhibited a dramatic Al³⁺-specific fluorescence turn-on response in the near-infrared (NIR) region. The receptor was found to be highly selective towards Al³⁺ over other metal ions in physiological condition. The sensor was non-toxic and could thus be employed as an imaging probe for detecting intracellular Al³⁺ in live cells. Interestingly, upon interaction with DNA in solution, the L–Al³⁺ ensemble rendered tracking of DNase activity in solution through a systematic reduction in the fluorescence emission intensity.     

Detection of mitochondrial caspase activity in real time in situ in live cells.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP-caspase 3 substrate-YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.     

Real-time PCR detection of telomerase activity using specific molecular beacon probes

Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The endogenous GABA analogue gamma-hydroxybutyric acid: Tritium labeling at high specific activity

2,3,4-³H] Gamma-hydroxybutyric acid (GHB, 4) was prepared by means of a catalytic tritium reduction of 2(5H)-furanone (2) followed by hydrolysis. It has proven useful as a tool to study the GHB receptor. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two dansyl fluorophores bearing amino acid for monitoring Hg in aqueous solution and live cells

A series of compounds (1-4) bearing one or two dansyl fluorophore(s) based on a Lys amino acid were synthesized in solid phase synthesis. Among them, two dansyl labeled Lys amino acid 3 detected Hg²⁺ in a 100% aqueous solution with high sensitivity (K_d=4.3 nM) via a turn-on response. Compound 3 was applied for monitoring Hg²⁺ in environmental and biological fields. 3 showed a hypersensitive response to Hg²⁺ without interferences from other metal ions and satisfied the requirements for monitoring the maximum allowable level (2 ppb) of mercury ions in drinking water demanded by EPA. In addition, 3 penetrated living HeLa cells and detected intracellular Hg²⁺. The organic spectroscopic data revealed the two sulfonamide and amide groups of 3 played a key role in stabilizing the 3-Hg²⁺ complex.     

Magneto-mechanical detection of nucleic acids and telomerase activity in cancer cells

The ultra-sensitive magneto-mechanical detection of DNA, single-base-mismatches in nucleic acids, and the assay of telomerase activity are accomplished by monitoring the magnetically induced deflection of a cantilever functionalized with magnetic beads associated with the biosensing interface. The analyzed M13phi DNA hybridized with the nucleic acid-functionalized magnetic beads is replicated in the presence of dNTPs that include biotin-labeled dUTP. The resulting beads are attached to an avidin-coated cantilever, and the modified cantilever is deflected by an external magnetic field. Similarly, telomerization of nucleic acid-modified magnetic beads in the presence of dNTPs, biotin-labeled dUTP, and telomerase from cancer cell extracts and the subsequent association of the magnetic beads to the cantilever surface results in the lever deflection by an external magnetic field. M13phi DNA is sensed with a sensitivity limit of 7.1 x 10(-20) M by the magneto-mechanical detection method.     

A two-photon excited luminescence of water-soluble rhodamine-platinum(II) complex: fluorescent probe specific for Hg2+ detection in live cell

The first example of cyclometalated platinum(II)-containing rhodamine probe (1) with two-photon induced luminescent properties was synthesized and investigated for mercury detection. A highly selective color change of 1, from light yellow to pink, is observed only in the presence of Hg²⁺ due to the formation of 1,3,4-oxadiazole ring in 2. This selectivity of Hg²⁺ with color changes can be observed easily by the naked-eye. Meanwhile, a remarkable turn-on and selective 20-fold fluorescent enhancement of 1 upon binding with Hg²⁺ over the other tested metal ions was observed. The water-soluble probe 1 was successfully applied in the visualizing of the site of Hg²⁺ accumulation as well as estimating of trace amounts of mercury ions in live HeLa cells by two-photon microscopy.     

A highly specific BODIPY-based fluorescent probe for the detection of hypochlorous acid

A fluorescent probe, HKOCl-1, has been successfully developed for the detection of hypochlorous acid on the basis of a specific reaction with p-methoxyphenol. The formation of HOCl has been successfully detected not only in an abiotic system but also in an enzymatic system (myeloperoxidase/H2O2/Cl(-) system) and in living macrophage cells upon stimulation. This new probe might be used as an efficient tool for probing the roles HOCl plays in biological systems.     

In situ detection of live cancer cells by using bioprobes based on Au nanoparticles

We fabricate the high-performance probes based on Au nanoparticles (AuNP) for detection of live cancer cell. AuNP were synthesized with narrow sized distribution (ca. 10 nm) by Au salt reduction method and deposited onto the aminated substrate as a cross-linker and hot spot. Herein, AuNP has enabled the easy and efficient immobilization of the antibody (Cetuximab), which can selectively interact with epidermal growth factor receptor (EGFR) on the surface of epidermal cancer, as detecting moiety onto the AuNP-deposited substrate without nanolithography process. After conjugation of Cetuximab with AuNP-deposited substrate, Cetuximab-conjugated probe as a live cancer cell detector (LCCD) could detect EGFR-highexpressed A431 cells related to epithelial cancer with 54-times larger specificity and sensitivity in comparison with EGFR-deficient MCF7 cells. This implies that AuNP-based probes demonstrate abundant potentials for detection and separation of small biomolecules, cells and other chemicals.     

Highly sensitive turn-on detection of Ag+ in aqueous solution and live cells with a symmetric fluorescent peptide

This communication presents a symmetric fluorescent peptide (K(d) = 17.4 nM) for hypersensitively detecting Ag(+) in 100% aqueous solution by turn-on response. The peptide penetrated live HeLa cells and detected intracellular Ag(+) by turn-on response.     

Extremely high and specific activity of DNA enzymes in cells with a Philadelphia chromosome

BACKGROUND: Chronic myelogenous leukemia (CML) results from chromosome 22 translocations (the Philadelphia chromosome) that creates BCR-ABL fusion genes, which encode two abnormal mRNAs (b3a2 and b2a2). Various attempts to design antisense oligonucleotides that specifically cleave abnormal L6 BCR-ABL fusion mRNA have not been successful. Because b2a2 mRNA cannot be effectively cleaved by hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to engineer specific cleavage of this chimeric mRNA. Nonspecific effects associated with using antisense molecules make the use of such antisense molecules questionable. RESULTS: The usefulness of DNA enzymes in specifically suppressing expression of L6 BCR-ABL mRNA in mammalian cells is demonstrated. Although the efficacy of DNA enzymes with natural linkages decreased 12 hours after transfection, partially modified DNA enzymes, with either phosphorothioate or 2'-O-methyl groups at both their 5' and 3' ends, remained active for much longer times in mammalian cells. Moreover, the DNA enzyme with only 2'-O-methyl modifications was also highly specific for abnormal mRNA. CONCLUSIONS: DNA enzymes with 2'-O-methyl modifications are potentially useful as gene-inactivating agents in the treatment of diseases such as CML. In contrast to conventional antisense DNAs, some of the DNA enzymes used in this study were highly specific and cleaved only abnormal BCR-ABL mRNA.     

"Click chemistry" inspired synthesis of pseudo-oligosaccharides and amino acid glycoconjugates

[reaction: see text] Various pseudo-oligosacchardies and amino acid glycoconjugates were synthesized via an intermolecular 1,3-dipolar cycloaddition ("click") reaction using easily accessible carbohydrate and amino acid derived azides and alkynes as building blocks. It is pertinent to mention that the conjugation reaction is highly regioselective and high yielding and can be carried out under mild reaction conditions.     

The colorimetric and ratiometric fluorescent detection of cyanide and sulfide in live cells,application for logic gate and bioimging

We described the design and synthesis of a colorimetric and ratiometric fluorescent probe based on a conjugated π-electron system that displayed differential fluorescence responses towards cyanide and sulfide and demonstrated its utility in intracellular ion imaging and logic gate.     

A monomethine cyanine dye Cyan 40 for two-photon-excited fluorescence detection of nucleic acids and their visualization in live cells

Monomethine cyanine dye 4-((1-methylbenzothiazolyliliden-2)methyl)-1,2,6-trimethylpyridinium perchlorate (Cyan 40) was investigated as a two-photon-excited fluorescence probe for nucleic acids (NA). Cyan 40 has been shown to demonstrate efficient two-photon-excited fluorescence in the presence of NA in vitro in contrast to solutions without NA. Two-photon confocal laser scanning microscopy (TPCLSM) and two-photon laser scanning microspectrofluorometry were used to check the possibility of using Cyan 40 as two-photon-excited fluorescence label for NA in living cells. Study of dye effect on viability of cells was also carried out. We ascertained that Cyan 40 is a cell-permeant dye, manifesting efficient two-photon-excited fluorescence when bound to NA in living cells, without any significant influence on viability of cells. TPCLSM images obtained from stained cells indicate preferential RNA staining by Cyan 40 compared with DNA.     

Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography.

Assessment of lactate metabolism is of particular interest during exercise and in disease states such as diabetes, shock, and absorptive abnormalities of short-chain fatty acids by the colon. We describe an analytical method that introduces radio-active tracers and high-performance liquid chromatography (HPLC) to simultaneously analyze concentrations and specific activities (SAs) of plasma lactate. The HPLC conditions included separation on a reversed-phase column (octadecylsilane) and an isocratic buffer (30% acetonitrile in water). [3H]Acetate served as an internal standard. Lactate and acetate were extracted from plasma samples with diethyl ether following a pH adjustment to less than 1.0 and back-extracted into a hydrophilic phase with sodium carbonate (2 mM, pH greater than 10.0). Lactate is detected in the ultraviolet range (242 and 320 nm) by derivatization with alpha-bromoacetophenone. Control plasma samples were studied after an overnight fast for precision and analytical recovery. Calibration curves were linear in the range 0.18-6.0 mM (r = 0.92). The precision was 3% and the analytical recovery was 87%. The detection limit of the method was 36 pmol. Determination of lactate metabolism was performed in a patient with chronic congestive heart failure who was administered primed-continuous L-[U-14C]lactate (10 microCi bolus and 0.3 microCi/min continuously) during a 60-min rest period. Mean arterial lactate concentration and SA were 1.69 +/- 0.2 mM and 253.8 +/- 22 dpm/mumol, respectively. Systemic lactate turnover was 25.65 mumol/kg per min. Lactic acid systemic turnover, organ uptake and release rates can be accurately determined by isocratic HPLC.     

抗坏血酸辅助碳纤维超微电极特异性检测特布他林

采用循环伏安法(CV)、差分脉冲伏安法(DPV)研究了特布他林与抗坏血酸共存的条件下在碳纤维超微电极上的电化学行为.通过优化抗坏血酸浓度,建立了一种电化学定量检测特布他林的方法.实验结果表明,在20 mmol/L pH 7.0的Tris-HCl缓冲溶液中,当共存的抗坏血酸浓度为1×10~(-4) mol/L时,对碳纤维电极检测特布他林具有较好的稳定作用.在优化条件下,采用DPV法对特布他林进行定量分析,特布他林的氧化峰电流与其浓度在1×10~(-6)～1×10~(-4) mol/L范围内呈良好的线性关系,线性相关系数为R~2=0.994 2,检测限达2.202×10~(-10) mol/L (S/N=3).该修饰方法重现性好,电极稳定性佳,可应用于生物样品中特布他林的高灵敏分析.