Cationic cyanine as a near-infrared fluorescent probe for the determination of nucleic acids

A new method with a cationic near-IR cyanine as fluorescent probe was developed for the determination of nucleic acids. The near-IR cyanine shows maximum excitation and emission wavelengths at 765 and 790 nm, respectively, in aqueous solution. The method is based on the fluorescence decrease of near-IR cyanine in the presence of nucleic acids. Under optimal conditions, the ratio of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.10-1.2 microg/mL for CT (calf thymus) DNA or SM (salmon sperm) DNA, and 0.10-1.6 microg/mL for yeast RNA. The detection limits were 30 ng/mL for CT DNA, 25 ng/mL for SM DNA and 70 ng/mL for yeast RNA. The relative standard deviation (n = 6) was 2.1% for 500 ng/mL CT DNA, 2.4% for 500 ng/mL SM DNA and 2.7% for 500 ng/mL yeast RNA, respectively.     

Fluorescence enhancement method for the determination of nucleic acids using cationic cyanine as a fluorescence probe

A fluorescence enhancement method with a cationic cyanine as a probe was developed for the determination of nucleic acids. Under the experimental conditions, the fluorescence enhancement of cyanine (lambda(ex)/lambda(em)= 524/591.5 nm) was observed in the presence of DNA. The calibration graphs were linear over the range of 0.01-15 microg mL(-1) for both calf thymus DNA (CT DNA) and fish sperm DNA (FS DNA). The limits of detection were 0.005 and 0.007 microg mL(-1) for CT DNA and FS DNA, respectively. The method was applied to the determination of DNA in synthetic and real samples and satisfactory results were obtained. A possible fluorescence enhancement mechanism was also studied.     

Brilliant cresyl blue as a new red region fluorescent probe for determination of nucleic acids

A fluorescence quenching method was developed for determination of microamounts of nucleic acids by using brilliant cresyl blue (BCB) as a new red region fluorescent probe. In aqueous hexylmethylene tetramine solution, BCB showed maximum excitation and emission wavelengths at 626 and 670 nm, respectively, and the fluorescence of BCB could be greatly quenched by DNA (or RNA). Under optimal conditions, the calibration graphs are linear over the range of 0.02–0.80 μg/ml for SM DNA and 0.25–1.5 μg/ml for yeast RNA. The corresponding detection limits are 7 ng/ml for SM DNA and 25 ng/ml for yeast RNA, respectively. SM DNA can be determinated in the presence of 40% (w/w) RNA, and the relative standard deviation of six measurements is 2.5% for 500 ng/ml SM DNA. The result of the determination of golden staphylococcus DNA by this method was satisfactory.     

Application of magdala red as a fluorescence probe in the determination of nucleic acids

A fluorescence quenching method was developed for the rapid determination of DNA and RNA using magdala red as fluorescence probe. In weakly acidic ¶medium, the fluorescence of magdala red (λex>lem = 54055 nm) can be largely quenched by DNA or RNA. The calibration graphs are linear over the range 0.01–¶1.2 μg/mL for both calf thymus DNA (CT DNA) and salmon DNA (SM DNA), and 0.015–1.0 μg/mL for yeast RNA, respectively. The corresponding detection limits are ¶6.0 ng/mL for CT DNA, 7.0 ng/mL for SM DNA and ¶15.0 ng/mL for yeast RNA, respectively. CT DNA could be determined in the presence of 20% (w/w) yeast RNA, and the relative standard deviation of six replicate measurements is 3.18% for 400 ng/mL of CT DNA. Interference from coexisting substances in the determination of DNA was also examined. Real samples were determined with satisfactory results.     

Application of magdala red as a fluorescence probe in the determination of nucleic acids

A fluorescence quenching method was developed for the rapid determination of DNA and RNA using magdala red as fluorescence probe. In weakly acidic medium, the fluorescence of magdala red (lambdaex/lambdaem = 540/555 nm) can be largely quenched by DNA or RNA. The calibration graphs are linear over the range 0.01-1.2 microg/mL for both calf thymus DNA (CT DNA) and salmon DNA (SM DNA), and 0.015-1.0 microg/mL for yeast RNA, respectively. The corresponding detection limits are 6.0 ng/mL for CT DNA, 7.0 ng/mL for SM DNA and 15.0 ng/mL for yeast RNA, respectively. CT DNA could be determined in the presence of 20% (w/w) yeast RNA, and the relative standard deviation of six replicate measurements is 3.18% for 400 ng/mL of CT DNA. Interference from coexisting substances in the determination of DNA was also examined. Real samples were determined with satisfactory results.     

Two near-infrared highly sensitive cyanine fluorescent probes for pH monitoring

Two near-infrared(NIR) p H-activated heptamethine indocyanine probes with quaternary ammonium unit were designed and synthesized. The absorption and emission titrations indicate that cationic structure improves the cyanine dye's aqueous solubility and these two probes exhibit highly sensitive response to p H in acid condition. Their fluorescence intensities both gradually increase about 25-fold from p H 7.60 to 3.00 with p Ka values of 4.72 and 4.45 respectively, which are suitable for studying acidic organelles in living cells. Moreover, their fluorescence intensities are linearly proportional to p H values in the range of 5.50–4.00. These results are probably attributed to the protonation of the indole nitrogen atoms, which are verified by 1H NMR spectra. Furthermore, these two probes can achieve real-time imaging of cellular p H and detection of p H in situ in living He La cells due to their excellent properties,including good reversibility, desirable photostability, high selectivity, low cytotoxicity and remarkable membrane permeability.     

Application of functionalized CdS nanoparticles as fluorescence probe in the determination of nucleic acids

Nanometer-sized fluorescent particles were successfully synthesized. The nanoparticles have a narrow, tunable, symmetric emission spectrum and a broad, continuous excitation spectrum. They are also photochemically stable. A synchronous fluorescence method was developed for the rapid determination of DNA with functionalized CdS as a fluorescence probe, based on the synchronous fluorescence quenching of functionalized CdS in the presence of DNA. Maximum fluorescence is produced at pH 7.0, with maximum excitation and emission wavelengths of 360 and 620 nm, respectively. The maximum emission wavelength of synchronous fluorescence is 354 nm when delta lambda = 260 nm. Under optimum conditions, the calibration graphs are linear over the range 0-3.5 microg mL(-1) for calf thymus DNA (CT-DNA) and 0.2-3.0 microg mL(-1) for fish sperm DNA. The corresponding detection limit is 0.01 microg mL(-1) for CT-DNA and 0.02 microg mL(-1) for fish sperm DNA. The relative standard deviation of seven replicate measurements is 2.2% for 1 microg mL(-1) calf thymus DNA and 2.4% for 1 microg mL(-1) fish sperm DNA. The method is simple, rapid and sensitive. The recovery and relative standard deviation are very satisfactory.     

Local viscosity analysis of triblock copolymer micelle with cyanine dyes as a fluorescent probe

The local viscosity of Pluronic F127 triblock copolymer micelles in water was determined with cyanine dyes as fluorescent probes. These dyes show very weak fluorescence at a low temperature, but show enhanced fluorescence at a temperature higher than the critical micellization temperature (T(cm)). This is because a viscous environment within the micelle suppresses the formation of a nonradiative twisted intramolecular charge transfer (TICT) excited state of the dyes. The good correlation between the fluorescence quantum yields of the dyes and the viscosity and the temperature of the media allows a determination of local viscosity of micelle based on the fluorescence quantum yields. The local viscosity of both core and corona regions of micelles increases at >T(cm) and shows a maximum at a temperature 7-9 °C higher than T(cm), and decreases at higher temperature due to the increased fluidity. The core viscosity is larger than that of the corona, and the corona viscosity increases toward the micelle center. The polymer concentration has different effects on the core and corona viscosity: the corona viscosity increases with a polymer concentration increase at the entire temperature range, whereas the core viscosity increases only at a low temperature. The corona viscosity increase is due to the condensation of a large number of polyethylene oxide (PEO) blocks. In contrast, the dehydration degree of polypropylene oxide (PPO) blocks in the core scarcely changes, and the core has a similar composition regardless of polymer concentration. The larger polymer concentration promotes a micelle formation at lower temperature where the fluidity increase is very weak, resulting in larger core viscosity.     

Strongly fluorescent cysteamine-coated copper nanoclusters as a fluorescent probe for determination of picric acid

Microchimica Acta - A fluorometric assay is described for doxycycline detection. It is based on the use of nitrogen-doped carbon quantum dots (NCQDs) coated with molecularly imprinted polymers...     

Synthesis of novel near-infrared cyanine dyes for metal ion determination

Four heptamethine cyanine dyes 10, 19–21 containing an ortho-hydroxy-carboxy functionality for metal ion complexation and absorbing at λ max (methanol) 761±1 nm have been synthesized.     

Cationic liposomes and nucleic acids

During the past decade, cationic lipids have emerged as the primary choice for gene delivery in vitro, i.e. transfection of cultured cells. A number of lipids with cationic head groups have been synthesized and evaluated. However, their success in vivo for gene therapy has been limited. To date, simple electrostatic complexes of cationic lipid mixtures with DNA have been hampered in numerous aspects: lack of colloidal stability, relatively low efficiency observed as expression levels or % of transfected cells, short duration of expression, and most importantly, non-specific interactions with many cells and tissues. Appreciation of the complexity of in vivo requirements, and especially opposing requirements for extra- and intracellular trafficking, is leading to engineered designs of gene delivery vectors containing cationic lipids. These designs attempt to assemble layered colloidal systems that accommodate the multiple functions required to traverse the various extra- and intracellular barriers. Successful development of such systems will depend on the ability to characterize and optimize each step rather than rely only on reporter gene expression, in addition to the obvious need to characterize the layered nature of the complexes. Importantly, many pharmacological aspects must be considered, especially control of the biodistribution and toxicity. Initial reports on such systems appear to provide at least a proof of the concept.     

Development of a hypoxia-selective near-infrared fluorescent probe for non-invasive tumor imaging

A near-infrared fluorochrome, GPU-311, was designed, synthesized and evaluated for its application in non-invasive imaging of tumor hypoxia. Efficient synthesis was achieved by nucleophilic substitution and click chemistry ring using the bifunctional tetraethylene glycol linker 2 containing thiol and azide groups for the conjugation of the propargylated nitroimidazole 1 and the heptamethine cyanine dye 3 bearing a 2-chloro-1-cyclohexenyl ring. GPU-311 exhibited long excitation and emission wavelength (Ex/Em=785/802?nm) and a decent quantum yield (0.05). The water solubility and hydrophilicity of GPU-311 increased. After in vitro treatment of SUIT-2/HRE-Luc pancreatic cancer cells with GPU-311, a higher level of fluorescence was observed selectively in hypoxia than in normoxia. However, in vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination through the liver.     

Interaction of cyanine dyes with nucleic acids. XVII. Towards an aggregation of cyanine dyes in solutions as a factor facilitating nucleic acid detection

Spectral properties of newly synthesized cyanine dyes, namely 1-[6-(4-[6-[2,6-dimethyl-4-(3-methyl-2,3-dihydro-1,3-benzothiazol- 2-ylidenmethyl)-1-pyridiniumyl]hexanoyl]piperazino)-6- oxohexyl]-2,6-dimethyl-4-(3-ethyl-2,3-dihydro-1,3-benzothiazol+ ++-2-ylidenmethyl)pyridinium (K-6) (bichromophoric dye) and 1-[5-di(3-[5-[2,6-dimethyl-4-(3-methyl-2,3-dihydro-1,3-benzothiazol++ +-2-ylidenmethyl)-1-pyridiniumyl]pentylcarboxamido]pro pyl) carbamoylpentyl]-2,6-dimethyl-4-(3-methyl-2,3-dihydro-1,3-benzo thiazol-2-ylidenmethyl) pyridinium (K-T) (trichromophoric dye) in solutions in the presence of and without deoxyribonucleic acid (DNA) were studied within a wide concentration range. It has been established that absorption, as well as fluorescence of investigated dye solutions, without DNA are mainly determined by H-aggregates of dye molecules. On the contrary, the fluorescence of dye solutions in the presence of DNA gives an intrinsic dye molecular fluorescence. H-aggregates are broken because of binding dye molecules with DNA. It has been suggested that both K-T and K-6 molecules bind mainly with DNA via the interaction of two chromophores. As the ratio of the number of dye molecules to that of DNA base pairs increases with an increase in dye concentration, a formation of dye molecule H-aggregates on DNA molecules are observed. Such aggregates have a different structure than those formed in the solutions without DNA. On the grounds of the data obtained, it is concluded that it is possible to use a dye aggregation capable of obtaining higher values for fluorescence enhancement of the DNA stains.     

Preparation and application of cysteine-capped ZnS nanoparticles as fluorescence probe in the determination of nucleic acids

Cysteine-capped ZnS nanometer-sized fluorescent particles were produced by a colloidal aqueous synthesis. The functionalized nanoparticles are water-soluble and suitable for biological application. A synchronous fluorescence method has been developed for the rapid determination of DNA with functionalized nano-ZnS as a fluorescence probe, based on the synchronous fluorescence enhancement of cysteine-capped nano-ZnS in the presence of DNA. When Deltalambda =190 nm, maximum synchronous fluorescence is produced at 267 nm at pH 5.12. Under optimum conditions, the synchronous fluorescence intensity is proportional to the concentration of nucleic acids in the range 0.1-1.2 microg ml(-1) for calf thymus DNA, 0.1-0.6 microg ml(-1) for fish sperm DNA. The corresponding detection limit is 32.9 ng ml(-1) for calf thymus DNA and 24.6 ng ml(-1) for fish sperm DNA. This method is simple, inexpensive, rapid and sensitive. The recovery and relative standard deviation are satisfactory.     

Novel magnetic and fluorescent nanocomposite as a sensitive probe for the determination of proteins

The Fe(3)O(4)/(sodium oleic acid/ethyltrimethyl ammonium bromide)(n)/4-aminobenzoic acid (Fe(3)O(4)/(NaOL/CTAB)(n)/PABA) nanocomposites have been prepared by a layer-by-layer self-assembly approach. This kind of nanocomposites have fluorescent, magnetic and water-soluble properties. Taking advantage of the magnetic property of nanocomposites, we can separated them from solution easily by using a permanent magnet. By using their strong fluorescence, we can detect proteins. At pH 6.98, the fluorescence of Fe(3)O(4)/(NaOL/CTAB)(n)/PABA nanocomposites can be enhanced by the proteins. Under optimal conditions, the linear ranges of calibration curves were 0.2-20, 0.2-13, 0.2-10 microg mL(-1) for gamma-globulin (gamma-IgG), human serum albumin (HSA), and bovine serum albumin (BSA), respectively. The detection limits were 0.02, 0.01, 0.02 for gamma-IgG, HSA and BSA, respectively. The method has been applied to analyze the total proteins in human samples and the results were in good agreement with those reported by the hospital. This method is sensitive, simple and potential in many areas.     

Poly(o-phenylenediamine) colloid-quenched fluorescent oligonucleotide as a probe for fluorescence-enhanced nucleic acid detection

In this Letter, we demonstrate that chemical oxidation polymerization of o-phenylenediamine (OPD) by potassium bichromate at room temperature results in the formation of submicrometer-scale poly(o-phenylenediamine) (POPD) colloids. Such colloids can absorb and quench dye-labeled single-stranded DNA (ssDNA) very effectively. In the presence of a target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the POPD surface, leading to recovery of dye fluorescence. With the use of an oligonucleotide (OND) sequence associated with human immunodeficiency virus (HIV) as a model system, we demonstrate the proof of concept that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch.     

Application of linear-sweep voltammetry to the determination of nucleic acids using crystal violet as an electrochemical probe

A new linear-sweep voltammetric assay of nucleic acids (NAs) based on their interaction with crystal violet (CV) is proposed. In a pH 3.5 Britton—Robinson (B-R) buffer solution, CV had an irreversible voltammetric reductive peak at −0.77 V and the peak current greatly decreased by the addition of NAs. Under the experimental conditions, the decrease in the peak current was used for the NAs assay 0.5–18.0 μg/mL of fish sperm DNA, 0.6–15.0 μg/mL of calf thymus DNA, and 0.8–12.0 μg/mL of yeast RNA. The detection limits (3σ) were 0.32, 0.47, and 0.61 μg/mL for fsDNA, ctDNA, and yRNA, respectively. The binding reaction can be completed after mixing DNA with CV within 10 min and the electrochemical response is stable for 2 h. There are seldom interferences in this method and three synthetic samples were analyzed with satisfactory results. The stoichiometry of the supramolecular complex with the binding number 3 and the binding constant 2.78 × 10¹⁴ is calculated using electrochemical data. The text was submitted by the authors in English.     

Determination of nucleic acids using calcein-neodymium complex as a fluorescence probe.

A novel fluorometric method has been developed for rapid determination of DNA and RNA with calcein-neodymium complex as a fluorescence probe. The method is based on the fluorescence enhancement of calcein-Nd(III) complex in the presence of DNA or RNA, with maximum excitation and emission wavelength at 489 nm and 514 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0.5 - 3.0 microg/ml for both DNA and yeast RNA, 0.4 - 2.0 microg/ml for fish sperm DNA (FS DNA) and 0 - 3.0 microg/ml for calf thymus DNA (CT DNA). The corresponding detection limits are 15.1 ng/ml for DNA, 21.2 ng/ml for yeast RNA, 10.5 ng/ml for FS DNA and 8.9 ng/ml for CT DNA. The interaction mechanism for the binding of calcein-Nd(III) complex to DNA is also studied. The results of absorption spectra, fluorescence polarization measurements and thermal denaturation experiments, suggested that the interaction between calcein-Nd(III) complex and DNA is an electrostatic interaction.     

Terbium fluorescence as a sensitive,inexpensive probe for UV-induced damage in nucleic acids

Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb³⁺). Single-stranded oligonucleotides greatly enhance the Tb³⁺ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb³⁺/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb³⁺, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb³⁺/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb³⁺/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.