Near-infrared fluorimetric determination of nucleic acids by shifting the ion-association equilibrium between tetracarboxy aluminum phthalocyanine and tetra-N-hexadecylpyridiniumyl porphyrin

A new method was developed for determination of micro amounts of nucleic acids based on near-infrared (near-IR) fluorescence recovery, employing a two-reagent system which is composed of an anionic tetracarboxy aluminum phthalocyanine (AlC₄Pc) and a cationic tetra-N-hexadecylpyridiniumyl porphyrin (TC₁₆PyP). The fluorescence of the AlC₄Pc, with the maximum emission wavelength at 701 nm, could be quenched by TC₁₆PyP at its proper concentration, but recovered by adding nucleic acids. Under optimal conditions, the recovered fluorescence is proportional to the concentration of nucleic acids. The calibration graphs are linear over the range of 1-200 ng mL⁻¹ for fish sperm DNA (FS DNA) and 2-400 ng mL⁻¹ for calf thymus DNA (CT DNA). The corresponding detection limits are 0.59 ng mL⁻¹ for FS DNA and 0.82 ng mL⁻¹ for CT DNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.     

Determination of nucleic acids based on shifting the association equilibrium between tetracarboxy aluminum phthalocyanine and poly-lysine

A new method based on near-infrared (near-IR) fluorescence recovery was presented for the determination of nucleic acids. This method employed a two-reagent system composed of anionic tetracarboxy aluminum phthalocyanine (AlC4Pc) and polycationic poly-lysine. The fluorescence of AlC4Pc, with the maximum excitation and emission wavelengths at 620 and 701 nm, respectively, was quenched by poly-lysine with a proper concentration, but recovered by adding nucleic acids. Under optimal conditions, the recovered fluorescence was in proportional to the concentration of nucleic acids. The linear ranges of the calibration curves were 5-200 ng mL(-1) for both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) with the detection limit of 2.6 ng mL(-1) for ctDNA and 2.1 ng mL(-1) for fsDNA. The relative standard deviation (n = 6) was 1.9 and 1.3% for 50 ng mL(-1) ctDNA and fsDNA, respectively. The proposed method was applied to the determination of nucleic acids in synthetic samples with satisfactory results.     

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 μg/mL for CT (calf thymus) DNA or SM (salmon sperm) DNA, and 0.10–¶1.6 μg/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.     

Near-infrared fluorimetric determination of nucleic acids by shifting the ion-association equilibrium between heptamethylene cyanine and Alcian blue 8GX

A new method based on near-infrared (near-IR) fluorescence recovery, employing a two-reagent system which is composed of an anionic heptamethylene cyanine (HMC) and a polycationic phthalocyanine dye, Alcian blue 8GX, is presented for the determination of nucleic acids. With a maximum excitation wavelength at 766 nm and a maximum emission wavelength at 796 nm, the fluorescence recovery is linear with the concentration of nucleic acids added. Factors including the acidity of the medium, the reaction time, the optimal ratio of the two reagents, as well as the influence of foreign substance were all investigated. Meanwhile, the mechanism of fluorescence recovery was also studied. Under the optimal conditions, the linear ranges of the calibration curves were 10-250 ng ml⁻¹ for calf thymus DNA (CT DNA) and 10-200 ng ml⁻¹ for yeast RNA. The detection limits were 6.8 ng ml⁻¹ for CT DNA and 6.3 ng ml⁻¹ for yeast RNA, respectively. The method has been applied to the analysis of practical samples and the recovery results were satisfactory.     

A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins

An amplified colorimetric method has been developed for the detection of protein and cancer cells based on the assembly of nucleic acids and proteins for the first time. In this process, the assembly of nucleic acids was triggered by a biotinylated DNA strand after a sandwich immunoreaction. The biotinylated DNA strand and sandwich immunocomplex were connected by streptavidin. Then, the assembly of biotinylated bovine serum albumin (Biotin-BSA) and streptavidin-horseradish peroxidase (SA-HRP) occurred at a node of the assembled products of nucleic acids through the biotin-streptavidin reaction. Under the catalysis of horseradish peroxidase, 3,3′,5,5′-tetramethylbenzidine (TMB) was oxidized by H₂O₂ and the oxidized product was analyzed by its UV–vis absorbance signal and sensitive colorimetric detection. This colorimetric sensor could not only achieve the quantitative determination of protein by UV–vis absorbance but could also be applied for semiquantitative determination by digital visualization. Using alpha-fetoprotein (AFP) as the model target, this proposed colorimetric method showed a wide linear range from 5 pg/mL to 1 ng/mL with a detection limit of 1.95 pg/mL by the instrument, and even 5 pg/mL target protein could be distinguished simply by the naked eye. This approach was then expanded to detect cancer cells based on the recognition of folic acid receptors that were over-expressed on the cancer cells by folic acid-tethered DNA. More importantly, this strategy can be further used as a universal colorimetric method for the determination of viruses or other proteins by changing the corresponding antibodies.     

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.     

Determination of nucleic acids by near-infrared fluorescence quenching of hydrophobic thiacyanine dye in the presence of Triton X-100.

A near-infrared (near-IR) fluorescence quenching method was developed for the determination of nucleic acids in aqueous solution by using a cationic heptamethylene thiacyanine as a probe. The near-IR cationic cyanine showed maximum excitation and emission wavelengths at 800 and 825 nm, respectively, in the presence of Triton X-100; the fluorescence of the cyanine could be greatly quenched by DNA. The calibration graphs were linear over the range of 10-400 ng/mL for CT (calf thymus) DNA and over the range 5-400 ng/mL for FS (fish sperm) DNA under optimal conditions. The corresponding detection limits were 5.2 ng/mL for CT DNA and 2.5 ng/mL for FS DNA. The relative standard deviation (n = 8) was 3.1% for 75 ng/mL CT DNA and 2.2% for 75 ng/mL FS DNA, respectively. Preliminary research showed that the fluorescence quenching might be ascribed to the formation of dye aggregate facilitated by DNA.     

Resonance Rayleigh scattering study of the interaction of bleomycinA<Subscript>5</Subscript> 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 bathochromic 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 conditions 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.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Novel fluorescent colloids as a DNA fluorescence probe

Fluorescent perylene colloids in the 80–90 nm size range have been prepared by the reprecipitation method. These nanoparticles were modified by cetyltrimethylammonium bromide (CTAB) which inhibited their growth. The nanoparticles also readily interacted with DNA. The fluorescence emission was measured at _ex/_em=400/565 nm. The fluorescence decrease of colloid–CTAB in aqueous solution was measured in the presence of nucleic acids. Under the optimum 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 0.02–5.1 µg mL^–1 for FS (fish sperm) DNA or CT (calf thymus) DNA. The detection limits were 0.01 µg mL^–1 for FS DNA and 0.012 µg mL^–1 for CT DNA, respectively. Based on this approach, a new quantitative method for DNA assay is presented in this paper.     

Spectrofluorimetric determination of nucleic acids as 8-hydroxyquinoline/ yttrium ternary complexes

The formation of nucleic acids/8-hydroxyquinoline/yttrium(III) ternary complexes and their fluorescent properties have been studied. The nucleic acids studied include native and thermally denatured calf thymus DNA, fish sperm DNA and yeast RNA. In the range of pH 7.6–8.5, controlled by NH₃-NH₄C1 buffer, ternary complexes are formed that fluoresce at different wavelengths with different nucleic acids. Based on the fluorescence reactions, sensitive spectrofluorimetric methods for nucleic acids are proposed. In optimal conditions, the calibration curves were linear in the range 0.5–4.0 gml^–1 for calf thymus DNA, 0.5–2.5 g ml^–1 for fish sperm DNA and 0.5–4.0 g ml^–1 for yeast RNA. The limits of determination (3 ) were 0.030 g ml^–1 for calf thymus DNA, 0.020 g ml^–1 for fish sperm DNA and 0.090 g ml^–1 for yeast RNA. Corresponding to the interferences of coexisting substances, six synthetic samples were constructed and the results of determination were satisfactory.     

Fluorometric method for the determination of hydrogen peroxide and glucose with Fe3O4 as catalyst

In this paper, we utilized the instinct peroxidase-like property of Fe₃O₄ magnetic nanoparticles (MNPs) to establish a new fluorometric method for determination of hydrogen peroxide and glucose. In the presence of Fe₃O₄ MNPs as peroxidase mimetic catalyst, H₂O₂ was decomposed into radical that could quench the fluorescence of CdTe QDs more efficiently and rapidly. Then the oxidization of glucose by glucose oxidase was coupled with the fluorescence quenching of CdTe QDs by H₂O₂ producer with Fe₃O₄ MNPs catalyst, which can be used to detect glucose. Under the optimal reaction conditions, a linear correlation was established between fluorescence intensity ratio I₀/I and concentration of H₂O₂ from 1.8 × 10⁻⁷ to 9 × 10⁻⁴ mol/L with a detection limit of 1.8 × 10⁻⁸ mol/L. And a linear correlation was established between fluorescence intensity ratio I₀/I and concentration of glucose from 1.6 × 10⁻⁶ to 1.6 × 10⁻⁴ mol/L with a detection limit of 1.0 × 10⁻⁶ mol/L. The proposed method was applied to the determination of glucose in human serum samples with satisfactory results.     

Determination of nucleic acids with a near infrared cyanine dye using resonance light scattering technique

A new method for the determination of nucleic acids has been developed based on the enhancement effect of resonance light scattering (RLS) with a cationic near infrared (NIR) cyanine dye. Under the optimal conditions, the enhanced RLS intensity at 823 nm is proportional to the concentration of nucleic acids in the range of 0-400 ng mL-1 for both calf thymus DNA (CT DNA) and fish sperm DNA (FS DNA), 0-600 ng mL-1 for snake ovum RNA (SO RNA). The detection limits are 3.5 ng mL-1, 3.4 ng mL-1 and 2.9 ng mL-1 for CT DNA, FS DNA and SO RNA, respectively. Owing to performing in near infrared region, this method not only has high sensitivity endowed by RLS technique but also avoids possible spectral interference from background. It has been applied to the determination of nucleic acids in synthetic and real samples and satisfactory results were obtained.     

Determination of nucleic acids based on the fluorescence quenching of Hoechst 33258 at pH 4.5

It was found the strong fluorescence emitted by the bis-benzimidazole derivative Hoechst 33258 at 490 nm could be efficiently quenched in pH 4.5 buffer when nucleic acids were added. Analysis of fluorescence intensity showed that the procedure was a static quenching dominated one, which was also demonstrated by the electron absorption spectra and lifetime of the excited state. The binding constant and numbers of binding sites were obtained from the Scatchard plot. The decreased fluorescence intensity was in proportion to the concentration of nucleic acids in the range 40-1800 ng ml⁻¹ for dsDNA and 26-1700 ng ml⁻¹ for ssDNA. The limits of detection were 12 and 8 ng ml⁻¹, respectively. The sensitivity of the method was about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the widely used fluorescence enhancement method using the same dye. Application results to synthetic samples showed simplicity, rapidity and satisfactory reproducibility of the presented method. Measurement of real samples extracted from leaves of Crassula argentea and E. coli genome also gave satisfactory results, which were in good agreement with those obtained using spectrophotometric method.     

Flow-injection determination of hydrogen peroxide based on fluorescence quenching of chromotropic acid catalyzed with Fe(II)

Flow-injection analysis system (FIA system), which was based on Fe(II)-catalyzed oxidation of chromotropic acid with hydrogen peroxide, was developed for the determination of hydrogen peroxide. The chromotropic acid has a fluorescence measured at λ_em = 440 nm (emission wavelength) with λ_ex = 235 nm (excitation wavelength), and the fluorescence intensity at λ_em = 440 nm quietly decreased in the presence of hydrogen peroxide and Fe(II), which was caused by Fe(II)-catalyzed oxidation of chromotropic acid with hydrogen peroxide. By measuring the difference of fluorescence intensity, hydrogen peroxide (1.0 × 10⁻⁸-1.0 × 10⁻³ mol L⁻¹) could be determined by the proposed FIA system, whose analytical throughput was 40 samples h⁻¹. The relative standard deviation (RSD) was 1.03% (n = 10) for 4.0 × 10⁻⁸ mol L⁻¹ hydrogen peroxide. The proposed FIA technique could be applied to the determination of hydrogen peroxide in rain water samples.     

A Novel Method to Determine DNA by Use of Molecular “Light Switch” of Ru(phen)2(dppz)

A novel fluorimetric method was developed for selective determination of DNA with the molecular “light switch” complex of Ru(phen)₂(dppz)²⁺. The maximum fluorescence intensity was produced in the pH range 9.3–11.5, with the maximum excitation and emission wavelength of 453.0 and 598.0 nm, respectively. Under the optimum conditions, the fluorescence intensity was in proportion to the concentration of DNA. The linear range for calfthymus DNA, salmon sperm DNA, and herring sperm DNA are 0–0.9 μg/mL. The limits of detection for calfthymus DNA, salmon sperm DNA, and herring sperm DNA are 2.0, 1.8, and 5.4 ng/mL, separately. When the proposed method was used to determine DNA in the presence of some coexisting substances, a satisfactory result was obtained.     

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.     

Determination of nucleic acids using fluorescence probe sensitized by microemulsion

Because the fluorescence of azur A can be quenched by adding nucleic acid, a sensitive fluorometric method for determination of nucleic acids at nanogram levels was established. Using optimal conditions, the calibration curves were linear in the range of 0-6.0 microg/mL for calf thymus deoxyribonucleic acid (ct DNA) and 0-7.0 microg/mL for herring sperm DNA (hs DNA). The limits of determination were 3.5 and 3.8 ng/mL, respectively, which shows the high sensitivity of this method. Triton X-100 microemulsion was applied as a sensitive media to enhance the sensitivity. The binding mode concerning the interactions of azur A with nucleic acids was also studied and the association constant with different binding numbers was obtained. The method has been applied to the determination of nucleic acid in both synthetic and real samples, such as cauliflower and pork liver, with satisfactory results.     

基于酶催化反应的核酸定量新方法

近年来 ,将染料自缔合或诱导缔合用于核酸定量测定备受关注 [1～ 3 ] .但是将酶与染料的缔合用于核酸定量测定尚未见报道 .氯化血红素 (hemin)可作为辣根过氧化物酶 (HRP)的模拟酶 ,能催化 H2 O2氧化对 -羟基苯乙酸 (p- HPA)生成荧光产物——联二对 -羟基苯乙酸的反应 [4 ,5] .由于 hemin在碱性介质中是阴离子化合物 ,能与阳离子化合物如阿尔新蓝 (Alcian Blue 8GX)发生缔合作用 ,从而使自身的催化性质被抑制 .当加入带负电荷的脱氧核糖核酸 (DNA)时 ,由于阿尔新蓝与 DNA的强烈作用使hemin与阿尔新蓝的缔合物被破坏 ,hemin的催化活…     

LC determination of biopterin reduced forms by UV-photogeneration of biopterin and fluorimetric detection

An off-line photoirradiation LC fluorimetric method to determine tetrahydrobiopterin (BH₄), by photogeneration of biopterin (BIO), is described, as an alternative way to the chemical oxidation procedure. To minimize the uncontrolled BH₄ oxidation, due to environmental oxygen, an antioxidant, dithiothreitol (DTT), was used. The acidity of the medium, as well as the presence of hydrogen peroxide, affects the rate of the photoreaction and the nature of the obtained fluorescent photoproducts. The best conditions were achieved by irradiation in hydrochloric acid (0.2 M) medium, in presence of 100 mM hydrogen peroxide, and using an irradiation time of 20 min. The method was tested in the analysis of serum samples containing BH₄, and recoveries between 89 and 105% were found. Also, the proposed method allows the resolution of BH₄ and BIO, in the same sample, by injection of non-irradiated and irradiated sample aliquots in the chromatographic system.