Interaction of a novel red-region fluorescent probe, Nile blue, with DNA and its application to nucleic acids assay

A novel fluorimetric method was developed for the rapid determination of DNA and RNA based on their quenching effect on the cationic red-region fluorescent dye Nile Blue (NB). In the investigation of the interaction of NB with DNA by steady-state polarization measurements, thermal denaturing study, determination of absorption and fluorescence characteristics, salt effect study and electrophoresis experiments, the results supported the suggestion that NB served as an intercalator to the stack base pairs of nucleic acids. Further evidence showed that the quenching could be ascribed to the static quenching mode. A binding constant of about 10(6) M-1 and a binding site size of about three base pairs were obtained by spectral methods. Under optimum conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL-1-2.0 micrograms mL-1 and 27 ng mL-1-10 micrograms mL-1, respectively. The detection limits were 3.0 ng mL-1 for CT DNA and 27 ng mL-1 for RNA. The relative standard deviation (n = 6) was within 2.1% in the middle of the linear range. Interferences from some interesting co-existing substances in the determination of DNA were also examined.     

Synthesis and investigation on the interaction with calf thymus deoxyribonucleic acid of a novel fluorescent probe 7-oxobenzo[b][1,10]phenanthroline-12(7H)-sulfonic acid

In this paper, the synthetic route of a potential antitumor reagent, benzo[b][1,10] phenanthrolin-7(12H)-one (BPO), was improved. A sulfonic group was introduced to BPO to form a new compound, 7-oxobenzo[b][1,10]phenan-throline-12(7H)-sulfonic acid (OPSA), in order to enhance its water-solubility. The molecular structure of OPSA has been confirmed by IR, UV, MS, ¹H NMR and elements analysis. It was proved in our experiments that DNA could quench the fluorescence of OPSA and the maximum quenched intensity appeared at 408 nm (λ_ex = 284 nm). The quenched fluorescence intensity was proportional to the concentration of DNA. Based on this phenomenon, OPSA had been used as the fluorescent probe for detection of calf thymus DNA (ct-DNA) and the corresponding linear response range was from 1.0 to 150.0 μg mL⁻¹ and the limit of detection (LOD) was 3.8 ng mL⁻¹. Its interaction with ct-DNA was investigated by fluorescence, absorption and viscosity measurements. When binding to ct-DNA, OPSA showed obvious fluorescence quenching and the quenched intensity was stable with the presence and absence of NaCl. The absorption spectra of OPSA had no evidence of increasing or decreasing when ct-DNA was added. The viscosity of OPSA and ct-DNA mixture showed no obvious change comparing with the viscosity of ct-DNA along. The results suggested that the interaction between OPSA and ct-DNA was groove binding in nature. Scatchard plots constructed from fluorescence titration data gave a binding constant of 8.9 × 10⁵ L mol⁻¹ and a binding site size of 0.35 base pairs per bound drug molecule.     

A novel 1,8-naphthalimide probe: synthesis and interactions with nucleic acid and its precursor

Three novel, water-soluble N-substituted 1,8-naphthalimides as the spectroscopic probes of nucleic acid, N-(2-hydroxyethyl)-1,8-naphthalimide (NI1), 1,8-naphthalimide-N-acetic acid (NI2) and 1,8-naphthalimide-N-caproic acid (NI3), were synthesized and photophysically characterized. The steady-state fluorescence quenching of the NI probes with nucleic acids (NA) and their precursors (nucleobases and nucleosides) were studied by Stern–Volmer correlation. The rate constants for bimolecular quenching were obtained in Tris buffer solution. The transient absorption spectroscopy by nanosecond laser flash photolysis were explored to identify the transient species and to determine the kinetics. The dynamic interaction mechanism was attributed to electron transfer (ET) and energy transfer via ³NI.     

Linear sweep voltammetric studies on the interaction of brilliant cresyl blue with nucleic acids and its analytical application

In this paper, the interaction of brilliant cresyl blue (BCB) with nucleic acids was studied and further applied for the microdetermination of nucleic acids. In aqueous Britton-Robinson (B-R) buffer solution, BCB can be easily reduced on the hanging mercury drop electrode (HMDE) and had a sensitive voltammetric reduction peak at -0.09 V (vs. SCE). The reduction peak current of BCB could be greatly decreased by the addition of DNA. The results of voltammetric measurements had indicated that a binding reaction was occurred between BCB and DNA and a new supramolecular complex was formed, which resulted in the decrease of the diffusion coefficient of the reaction solution and the decrease of the reduction peak current correspondingly. The conditions of interaction and the electrochemical detection were carefully investigated. Under the selected conditions, the calibration curves for the detection of fish sperm (fs)DNA, calf thymus (ct)DNA and yeast (y)RNA were established. The linear range of this assay was 1.0-30.0 microg/mL for fsDNA, 1.0-45.0 microg/mL for ctDNA and 1.0-25.0 microg/mL for yRNA, respectively. The detection limits were 0.38 microg/mL fsDNA, 0.43 microg/mL ctDNA, 0.64 microg/mL yRNA. The interaction parameters such as the equilibrium constant and the binding number were calculated by electrochemical method. The results showed that the 2:3 type of complex was formed in the fsDNA-BCB complex with the binding constant as 2.51 x 10(7). The proposed method was further applied to the synthetic samples determination with satisfactory results.     

Resonance Rayleigh scattering study of the interaction of bleomycinA_5 with nucleic acids and its analytical application

The interaction of bleomycinA5 with nucleic acids has been investigated by using resonance Rayleigh scattering (RRS), molecular absorption and fluorescence spectra. The result shows that in near pH 2.2 buffer medium and absence of any metal ions, nucleic acids are capable of binding with bleomycinA5 (BLMA5) to form complexes which can remarkably enhance the RRS intensity and result in batho- chromic and hyperchromic molecular absorption of nucleic acids and fluorescence quenching of bleomycinA5. The RRS spectral characteristics for the binding products of bleomycinA5 with various DNA and RNA are similar, and the maximum RRS peaks are at 301 nm for ctDNA and sDNA, 370 nm for hsDNA, 310 nm for RNAtypeVI and RNAtypeIII, respectively. The increments of RRS intensity are greatly different in which DNA enhances greatly and RNA enhances lightly. In this work, the optimum condi- tions of the interaction and some influencing factors have been investigated. The reaction mechanism and a binding model for the interaction of BLMA5 with the nucleic acids are discussed. In addition, a highly sensitive, simple and rapid new method for the determination of DNA has been developed. The detection limits (3σ) are 5.7 ng/mL for ctDNA, 7.4 ng/mL for sDNA and 9.2 ng/mL for hsDNA, respectively. The method can be applied to determination of trace amounts of DNA.     

功能性Cu-PVK纳米微球与核酸相互作用的共振光散射光谱及分析应用

在超声辐射下,H2O2氧化降解聚乙烯醇（PVA）,制得与Cu^2＋形成配位体的纳米尺寸的β-二酮型高分子微球（Cu-PVK）。对Cu-PVK的共振散射光（RLS）性质研究发现Cu-PVK与核酸形成缔合物时将导致Cu-PVK本身RLS信号急剧增加,基于此建立一种用RLS信号测定痕量核酸的新方法。方法的抗干扰能力强,已用于合成样品的分析。     

Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells

A BODIPY-based ratiometric fluorescent probe for HOCl has been designed based on the transduction of thioether to sulfoxide function. This probe features a marked absorption and emission blue-shift upon the HOCl-promoted rapid transduction, enabling the highly selective and ratiometric detection. In addition, the probe works excellently within a wide pH range of 4–10, addressing the existing pH dependency issue. Living cells studies demonstrate that the probe is cell membrane permeable and can be employed successfully to image endogenous HOCl generation in macrophage cells.     

Study on the interaction between oxolinic acid aggregates and protein and its analytical application

It was found that oxolinic acid (OA) at high concentration can self-assemble into nano- to micro- meter scale OA aggregates in Tris-HCl (pH 7.48) buffer solution. The nanoparticles of OA were adopted as fluorescence probes in the quantitative analysis of proteins. Under optimum conditions, the fluorescence quenching extent of nanometer scale OA aggregates was in proportion to the concentration of albumins in the range of 3.0 × 10⁻⁸ to 3.0 × 10⁻⁵ g mL⁻¹ for bovine serum albumin (BSA) and 8.0 × 10⁻⁸ to 8.0 × 10⁻⁶ g mL⁻¹ for human serum albumin (HSA). The detection limits (S/N = 3) were 3.4 × 10⁻⁹ g mL⁻¹ for BSA, and 2.6 × 10⁻⁸ g mL⁻¹ for HSA, respectively. Samples were satisfactorily determined. The interaction mechanism of the system was studied using fluorescence, UV-vis, resonance light scattering (RLS) and transmission electron microscope (TEM) technology, etc., indicating that the nonluminescent complex was formed between serum albumin molecular and OA, to disaggregate the self-association of OA, which resulted in the dominated static fluorescence quenching in the system.     

Synthesis and application of rhodamine-based fluorescent probe dyes with spacer linker arm

When rhodamine-based fluorescent probe dyes are used to track target molecules they always perturb the behavior of target molecules because of steric hindrance effect. In order to minimize potential steric problems, a kind of rhodamine-based fluorescent probe dye with spacer linker arm was designed and synthesized and its application in immunofluorescence histochemistry was investigated.     

Molecular beacons: a novel DNA probe for nucleic acid and protein studies

A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.     

Study on electron transfer of fluorescent probe lumichrome and nucleic acid by laser flash photolysis

Laser flash photolysis (LFP) of lumichrome (LC) has been studied upon laser excitation at 355 nm. The transient absorption spectra of LC in aqueous solution displayed two bands at 380 nm and 540–700 nm for the triplet ³LC, and a wide band at 430–530 nm for the radical anion LC^.. The triplet-state lifetime was measured up to 12 μs. It was found that the self-quenching of ³LC by the ground state of LC occurred to produce LC^. during LFP. The transient spectra of LC in the presence of electron-donating triphenylamine (TPA), nucleosides and nucleic acids showed that the absorbance of ³LC was strongly quenched by these substrates and the formation of LC^. was enhanced in a great extent simultaneously. The bimolecular quenching kinetics were studied and the rate constants of ³LC by a series of quenchers, including TPA, LC, nucleosides and nucleic acids were determined to be 10⁸–10⁹ M⁻¹ s⁻¹. The photo-induced electron-transfer mechanism for these processes involving the triplet ³LC was proposed.     

A novel near-infrared fluorescent probe was synthesized and its application in imaging of living cells

A novel near-infrared fluorescent probe, IR-736, was synthesized and applied to living cells. The probe exhibited good response fluorescent characteristic, and cells experiments showed the probe had high affinity and without apparent cytotoxicity. Fluorescent image experiments in living L929 cells, MCF-7 cells, HepG2 cells, further demonstrated its potential applications in biological systems. The probe effectively prevented the influence of autofluorescence and native cellular species in biological systems. It also exhibited good photostability, and excellent cell membrane permeability.     

3-羟基邻苯二甲酰亚胺全水溶性次氯酸荧光探针及其应用

利用二甲基硫代氨基甲酸酯对次氯酸(HOCl)的特异性和吡啶盐的水溶性,以4-羟基异苯并呋喃-1,3-二酮作为原料,设计合成了一种检测HOCl的全水溶性激发态分子内质子转移(ESIPT)荧光探针.由于二甲氨基硫代甲酸酯对羟基的保护,探针分子内的ESIPT作用被阻碍,自身无荧光;当加入HOCl时,HOCl氧化二甲氨基硫代甲...     

Investigating the binding interaction of azur A with hyaluronic acid via spectrophotometry and its analytical application

The interaction between azur A (AA) and hyaluronic acid (HA) at AA concentrations from 3.430×10^–5 to 8.575×10^–5 M and sodium chloride concentrations from 0 to 0.01 M was investigated spectrophotometrically at 620 nm at temperatures from 0 to 50 °C. AA was shown to be a useful spectroscopic probe for detecting carboxyl groups in HA macromolecules. The interaction between AA and HA was temperature sensitive and little AA–HA interaction was observed at temperatures higher than 30 °C. The interaction of HA with AA was seen to be electrostatic in nature. The maximum binding number decreased with decreasing NaCl concentration, and the absorbance sensitivity decreased with increasing NaCl concentration in aqueous solution. Self-interference from the AA in the AA–HA interaction caused an overestimate of the molar mass of hyaluronic acid. An improved method was proposed to estimate the molar mass of HA, and a molar mass of 1.219×10⁶ Da was obtained with this improved method for HA.     

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.     

Studies on the interaction of protein with acid chrome blue K by electrochemical method and its analytical application

An electrochemical investigation on the interaction of acid chrome blue K (ACBK) with protein on the mercury electrode with different electrochemical methods such as cyclic voltammetry and linear sweep voltammetry was reported in this paper. In pH 3.0 Britton-Robinson (B-R) buffer solution, ACBK has an irreversible voltammetric reductive peak at -0.23 V (vs. SCE). The addition of human serum albumin (HSA) into the ACBK solution resulted in the decrease of reductive peak currents without the change of the peak potential and no new peaks appeared on the cyclic voltammogram. In the absence and presence of HSA, the electrochemical parameters such as the formal potential E0, the electrode reaction standard rate constant k(s) and the charge transfer coefficient alpha of the interaction system were calculated and the results showed that there were no significant changes between each other. Thus, the interaction of ACBK with protein forms an electro-inactive supramolecular bio-complex, which induces the decrease of the free concentration of ACBK in the reaction solution, and the decrease of the reductive peak current of ACBK. The binding constant and the binding ratio are calculated as 1.29 x 10(8) and 1:2, respectively, and the interaction mechanism is discussed. Based on the binding reaction, this new electrochemical method is further applied to the determination of HSA with the linear range from 3.0-20.0 mg/L and the linear regression equation as deltaIp"(nA) = 10.08 + 19.90 C (mg/L). This method was further applied to determinate the content of protein in the healthy human serum samples with the results in good agreement with the traditional Coomassie brilliant blue G-250 spectrophotometric method.     

Study on the interaction between silver nanoparticles and nucleic acids in the presence of cetyltrimethylammonium bromide and its analytical application

A novel method is proposed in this paper, that is the silver nanoparticle (nanoAg)-cetyltrimethylammonium bromide (CTMAB) is used as the probe of resonance light scattering (RLS) for the determination of nucleic acids. Under optimum conditions, there are linear relationships between the quenching extent of RLS and the concentration of nucleic acids in the range of 4.0x10(-9)-2.0x10(-6)gmL(-1) for fish sperm DNA (fsDNA), 7.0x10(-9)-1.8x10(-6)gmL(-1) for calf thymus DNA (ctDNA) and 6.0x10(-9)-1.0x10(-6)gmL(-1) for yeast RNA (yRNA). The detection limits (S/N=3) of fsDNA, ctDNA and yRNA are 2.7x10(-10)gmL(-1), 4.8x10(-10)gmL(-1) and 7.2x10(-10)gmL(-1), respectively. The studies indicate that there are interactions among nanoAg, CTMAB and fsDNA through electrostatic and chemical affinity, and the nanoAg-CTMAB complex can induce the structural change of base stacking and helicity of fsDNA.     

A novel lipid droplets-targeting fluorescent probe based on dicyanisophorone and carbazole for Cu2+ and its application

A novel fluorescent probe, LCH , based on dicyanisophorone and carbazole, was prepared for the visual detection of Cu²⁺. The probe LCH could recognize Cu²⁺ by fluorescence quenching in EtOH/H₂O (1/4, v/v) solution, which could be easily identified under the 365 nm UV lamp, and the detection limit was as low as 0.785 μM. The recognition mechanism of probe LCH with Cu²⁺ was determined by combining ¹H NMR titration, MS, and theoretical calculations. Practical application experiments showed that probe LCH could be used to detect Cu²⁺ in the test strip experiments. Cell imaging experiments showed that the probe LCH owned good cell permeability and could be applied to the imaging of Cu²⁺ in HepG2 cells. In addition, fluorescence colocalization experiments showed that LCH could target lipid droplets. These results indicate that the probe LCH will have a good application prospect in environmental detection and clinical medicine.     

Construction of a novel cell-trappable fluorescent probe for hydrogen sulfide (H2S) and its bio-imaging application

Analytical and Bioanalytical Chemistry - Fluorescence detection of H2S in living organisms is greatly advantageous because it is nondestructive and can be used for in situ analysis. We have...     

A novel fluorescent probe derived from isophorone and its application in imaging in vivo hypoxia

Herein, an isophorone-derived fluorescent probe, ARP, was developed to realize the imaging of in vivo hypoxia under the catalysis of the typical enzyme AzoR. Under the excitation at 456 nm, the detecting system with ARP could exhibit a red emission signal at 638 nm. ARP indicated advantages including high sensitivity (LOD = 0.37 Eq), high selectivity, anti-interference ability and wide pH adaption (3.0–12.0). Moreover, ARP could image the aggravation of the hypoxia in both the incubating oxygen percentage scale and the time scale in living cells. The AzoR-activated mechanism was convinced by the experimental result of adding inhibitors and illustrated by the molecular docking simulation. ARP could co-locate with the commercial dyes in the endoplasmic reticulum (ER), lysosomes, and mitochondria, which meant that ARP might be sensitive to the occurrence of the oxidation-reduction process or the passage through the intracellular membranes. Further, ARP achieved the imaging of hypoxia in both mice tumor model as well as tying model. ARP could distinguish the tumor site or the tied-up position according to the hypoxia extent. Therefore, this work raised a new trial for the imaging of the hypoxia, and might be helpful in future designs of more pragmatic fluorescent implements.