Fluorimetric determination of nucleic acids with terbium and lutetium

In this paper, fluorescence-enhancement of Tb-nucleic acids [fish sperm DNA (fsDNA) and yeast RNA (yRNA)] by Lu³⁺ is studied in detail and is applied to determine nucleic acids. The experiments indicated that under the optimum conditions, a linear relationship was obtained between the fluorescence intensity (I_f) and the concentration of nucleic acids. The linear range is 1.2×10⁻⁸-1.0×10⁻⁴ g/ml for DNA and 3.0×10⁻⁸-8.0×10⁻⁴ g/ml for RNA. The detection limits (signal/noise=3) for DNA and RNA were 4.8×10⁻⁹ and 7.0×10⁻⁹ g/ml, respectively. The mechanism of the co-luminescence effect is also discussed.     

Study on the interaction between nucleic acid and Eu3+-oxolinic acid and the determination of nucleic acid using the resonance light scattering technique

At pH 9.75, the resonance light scattering (RLS) intensity of OA–Eu³⁺ system is greatly enhanced by nucleic acid. Based on this phenomenon, a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS is proportional to the concentration of nucleic acid in the range of 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g/ml for herring sperm DNA, 8.0 × 10⁻¹⁰ to 1.0 × 10⁻⁶ g/ml for calf thymus DNA and 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and 0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between nucleic acid and OA–Eu³⁺ is also investigated.     

Fluorescence enhancement effect for the determination of DNA with calcein-cetyl trimethyl ammonium bromide system

A new spectrofluorimetric method was developed for the determination of trace amounts of DNA using the calcein as a fluorescent probe. In the presence of appropriate amounts of the cationic surfactant cetyl trimethyl ammonium bromide (CTAB), the anionic dye calcein dimerizes. The weak fluorescence intensity of the dimer was enhanced by adding DNA at pH 6–7. The interaction between calcein–CTAB and DNA was studied on the basis of this behavior and a new method was developed for determining DNA. Under the optimal conditions, the enhanced fluorescence intensity was in proportion to the concentration of DNA in the range of 4.0 × 10⁻⁶ to 8.0 × 10⁻⁵ g L⁻¹ for fsDNA and thermally denatured ctDNA (4.5 × 10⁻⁶ to 9.0 × 10⁻⁵ g L⁻¹). The detection limits (S/N = 3) were 2.0 × 10⁻⁶ and 2.2 × 10⁻⁶ g L⁻¹, respectively. This method was used for determining the concentration of DNA in synthetic samples with satisfactory results.     

Simple and sensitive assay for nucleic acids by use of the resonance light-scattering technique with copper phthalocyanine tetrasulfonic acid in the presence of cetyltrimethylammonium bromide

On the basis of enhancement of resonance light scattering (RLS) of copper phthalocyanine tetrasulfonic acid (CuTSPc) by nucleic acids and cetyltrimethylammonium bromide (CTMAB) under suitable conditions, a new RLS method for determination of nucleic acids in aqueous solutions has been developed. At pH 9.80–10.95 and ionic strength 0.01 mol L^–1 (NaCl), the interaction of copper phthalocyanine tetrasulfonic acid with nucleic acids in the presence of cetyltrimethylammonium bromide results in enhanced RLS signals at 282.0 nm, 383.6 nm, and 616.2 nm in the enhanced regions. It was found that the enhanced RLS intensity at 383.6 nm was proportional to the concentration of nucleic acids within suitable ranges. The limits of detection were 10.6 ng mL^–1 for fish sperm DNA and 32.4 ng mL^–1 for calf thymus DNA when the concentration of copper phthalocyanine tetrasulfonic acid was 2.0×10^–6 mol L^–1. This method is rapid, simple and sensitive. In addition, the reagents used are relatively inexpensive, stable, and easily synthesised. The method can be applied to the determination of nucleic acids in the presence of coexisting substances, and we have applied it to the determination of DNA in synthetic samples, with satisfactory results.     

四磺化铁酞菁-阿尔新蓝8GX-对-羟基苯乙酸-H2O2催化荧光反应体系在核酸定量中的应用

阳离子荧光染料阿尔新蓝8GX与阴离子荧光染料四磺化铁酞菁发生缔合作用，使四磺化铁酞菁的荧光猝灭，当核酸存在时，削弱了离子缔合作用使四磺化铁酞菁的荧光恢复。荧光增强的程度与核酸的量成线性关系，据此实现了对核酸的定量测定。     

Fluorimetric determination of phytic acid in urine based on replacement reaction

A sensitive fluorimetric method for determination of phytic acid in human urine samples was described. The method was based on a fluorimetric replacement reaction, in which the added phytic acid replaced the Cu²⁺ ion from Cu²⁺-gelatin complex, liberating the fluorescent gelatin molecule. The fluorescence of the solution was accordingly recovered proportionally to the amount of the foreign phytic acid. The excitation wavelength was 273.5 nm and the characteristic emission wavelength was 305.0 nm, respectively. The calibration graph was obtained by plotting the recovered fluorescent intensity at maximum 305.0 nm against the added standard phytic acid, and was divided into two sections. One section was linear over the range of 0.40-2.40 mg L⁻¹ with a linear regression equation of I_f = −0.895 + 15.146c (R² > 0.9993), and the other over the range of 2.40-9.20 mg L⁻¹ with a linear regression equation of I_f = −29.526 + 26.113c (R² > 0.9996), respectively. The relative standard deviation (R.S.D.) at 95% confidence degree for a 2.0 mg L⁻¹ of standard phytic acid within 1 month was less than 1.26% (n = 5), indicating the procedure is reproducible. The detection and the quantification limits of phytic acid were estimated to be 0.23 and 0.40 mg L⁻¹, respectively. The proposed method was applied to the determination of phytic acid in urine samples and the found concentrations of phytic acid in urine were in the range of 0.49-0.75 mg L⁻¹ with recoveries of 96.2-108.8%. Comparison of the obtained results with the reported HPLC was performed, indicating the proposed method was reliable.     

Fluorescence-based nucleic acid detection and microarrays

Recent analytical innovations for nucleic acid detection have revolutionized the biological sciences. Single nucleic acid sequence detection methods have been expanded to incorporate multiplexed detection strategies. A variety of nucleic acid detection formats are now available that can address high throughput genomic interrogation. Many of these parallel detection platforms or arrays, employ fluorescence as the signaling method. Fluorescence-based assays offer many advantages, including increased sensitivity, safety and multiplexing capabilities, as well as the ability to measure multiple fluorescence properties. Multiplexed microarray platforms provide parallel detection capabilities capable of measuring thousands of simultaneous responses. This review will discuss both single target detection and microarray applications with a focus on gene expression and pathogenic microorganism (PM) detection.     

Silver nanoparticles fluorescence enhancement effect for determination of nucleic acids with kaempferol-Al(III)

Nucleic acids can greatly enhance fluorescence intensity of the kaempferol (Km)-Al(III) system in the presence of silver nanoparticles (AgNPs). Based on this, a novel method for the determination of nucleic acids is proposed. Under studied conditions, there are linear relationships between the extent of fluorescence enhancement and the concentration of nucleic acids in the range of 5.0 × 10⁻⁹ to 2.0 × 10⁻⁶ g mL⁻¹ for fish sperm DNA (fsDNA), 7.0 × 10⁻⁹ to 2.0 × 10⁻⁶ g mL⁻¹ for salmon sperm DNA (smDNA) and 2.0 × 10⁻⁸ to 3.0 × 10⁻⁶ g mL⁻¹ for yeast RNA (yRNA), and their detection limits are 2.5 × 10⁻⁹ g mL⁻¹, 3.2 × 10⁻⁹ g mL⁻¹ and 7.3 × 10⁻⁹ g mL⁻¹, respectively. Samples were satisfactorily determined. And the system of Km-Al(III)-AgNPs was used as a fluorescence staining reagent for sensitive DNA detection by DNA pattern of agarose gel electrophoresis analysis. The results indicate that the fluorescence enhancement should be attributed to the formation of Km-Al(III)-AgNPs-nucleic acids aggregations through electrostatic attraction and adsorption bridging action of Al(III) and the surface-enhanced fluorescence effect of AgNPs.     

Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acid analysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concentration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the approach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies. Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementation of planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescent field associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity are discussed.In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible with established industrial standard formats allowing for integration into industrial high throughput environments, are presented, as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparison with a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguide platforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, when using conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acids requires massive sample preparation and signal or target amplification steps.     

Voltammetric study of the interaction of ciprofloxacin-copper with nucleic acids and the determination of nucleic acids

The behavior of the ciprofloxacin (CPFX) complex with copper, Cu(II)L₂, at a mercury electrode has been investigated in borax–boric acid buffer. The adsorption phenomena were observed by linear sweep voltammetry. The mechanism of the electrode reaction was found to be reduction of Cu(II)L₂ adsorbed on the surface of the electrode by an irreversible charge transfer to metal amalgam, Cu(0)(Hg). In the presence of DNA, the formation of the electrochemically non-active complexes Cu(II)L₂–DNA results in the decrease of the equilibrium concentration of Cu(II)L₂ and its peak current. Under the optimum conditions, the decrease of the peak current is proportional to DNA concentration. The linear ranges are 6.67×10⁻⁸ to 1.20×10⁻⁵ g ml⁻¹ for calf thymus DNA (ctDNA), 3.30×10⁻⁸ to 2.33×10⁻⁶ g ml⁻¹ for fish sperm DNA (fsDNA) and 1.0×10⁻⁸ to 1.2×10⁻⁶ g ml⁻¹ for yeast RNA. The detection limits are 5.00×10⁻⁹, 3.00×10⁻⁹ and 2.50×10⁻⁹ g ml⁻¹, respectively. This method exhibits good recovery and high sensitivity.     

Spectrophotometric determination of bromide

A Spectrophotometric method for bromide determination has been developed. Bromide is converted to tribromide which absorbs at 267 nm. The detection limit is 0.01 g/ml bromide. As little as 0.4 g of bromide can be determined in an excess of chloride and sulfate with a coefficient of variation of 4%.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

Bioanalytical applications of isothermal nucleic acid amplification techniques

The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.     

Fluorimetric determination of aminocaproic acid in pharmaceutical formulations using a sequential injection analysis system

A sequential injection analysis (SIA) methodology for the fluorimetric determination of aminocaproic acid in pharmaceutical formulations is proposed. The developed analytical procedure is based on the derivatisation reaction of the aminocaproic primary amine with o-phthalaldehyde (OPA) and N-acetylcysteine (NAC) and fluorimetric detection of the formed product (λ_ex = 350 nm; λ_em = 450 nm). The implementation of a SIA flow system allowed for the development of a simple, fast and versatile automated methodology, which exhibits evident advantages regarding the US Pharmacopoeia 24 (USP 24) reference procedure. By combining the SIA time-based sample insertion with a subsequent zone sampling approach, which permitted to select for detection of a well-defined sample zone, it was possible to implement an on-line dilution strategy that enabled the expansion of the analytical working range of the methodology, and thus its application in dissolution studies, without manifold re-configuration.Linear calibration plots were obtained for aminocaproic acid concentrations up to 6 × 10⁻⁵ mol l⁻¹. The developed methodology exhibit a good precision, with a R.S.D. < 2.0% (n = 15) and the detection limit was 2.5 × 10⁻⁷ mol l⁻¹. The obtained results complied with those furnished by the reference procedure with a relative deviation lower than 1.2%. No interference was found.     

Rapid,single-step nucleic acid detection

A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay. An erratum to this article can be found at     

姜黄素-La3+-CTMAB-核酸体系的荧光增强效应及其应用

研究了镧离子(La3+)-姜黄素(CU)-十六烷基三甲基溴化铵(CTMAB)-核酸荧光增强体系.建立了测定核酸的新方法.体系的最优条件为:六次甲基四胺(HMTA)-HCl缓冲溶液(pH 5.80)中,1.00× 10-3 mol/L阳离子表面活性剂CTMAB存在下,姜黄素浓度为2.00×10-5 mol/L,La3+的浓度为1.40×10-4 mol/L时,核酸能增强La3+ -CU络合物的荧光强度,而且体系荧光的增强程度与核酸的加入量在一定范围内呈线性关系.fsDNA,ctDNA和yRNA线性范围分别为7.00×10-4～10.00 mg/L,4.00×10-4～10.00 mg/L和7.00×10-4～10.00 mg/L;检出限分别为0.17,0.02和0.14 μg/L.与已报道的核酸的分析方法相比,本方法具有较宽的线性范围和较高的灵敏度.研究表明,核酸对体系荧光的增强源于DNA主链上PO3-4与CU之间的静电结合,以及通过氢键和疏水力进行的沟槽式结合,为探针分子提供了疏水性的微环境,降低了体系的非辐射能量损失,使体系的荧光强度增加.     

Analysis of salicylic acid based on the fluorescence enhancement of the As(III)-salicylic acid system

A new, simple and sensitive spectrofluorimetric method for the determination of salicylic acid (λ_ex = 315 nm, λ_em = 408 nm) using As(III) as a sensitizing reagent has been investigated by measuring the increase of fluorescence intensity of salicylic acid due to the complexation of As(III)-salicylic acid in presence of sodium dodecyl sulfate (SDS) 10⁻³ M. Under optimum conditions, a significant relationship was obtained between the fluorescence intensity and salicylic acid concentration. A linear calibration curve was obtained in the range 13.8-13812 μg l⁻¹ with product-moment correlation coefficient (R) 0.99985 and detection limit 4.2 μg l⁻¹. The R.S.D. is 2.35% (n = 5).The method was applied successfully to the determination of salicylic acid in human serum.     

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.     

Peptide nucleic acid stabilized perovskite nanoparticles for nucleic acid sensing

Nanostructural hybrid organic-inorganic metal halide perovskites offer a wide range of potential applications including photovoltaics, solar cells, and light emitting diodes. Up to now the surface stabilizing ligands were used solely to obtain the optimal properties of nanoparticles in terms of dimensionality and stability, however their possible additional functionality was rarely considered. In the present work, hybrid lead bromide perovskite nanoparticles (PNP) were prepared using a unique approach where a peptide nucleic acid is used as a surface ligand. Methylammonium lead bromide perovskite colloidal nanoparticles stabilized by thymine-based peptide nucleic acid monomer (PNA-M) and relevant trimer (PNA-T) were prepared exhibiting the size below 10 nm. Perovskite structure and crystallinity were verified by X-ray powder diffraction spectroscopy and high resolution transmission electron microscopy. PNP-PNA-M and PNP-PNA-T colloidal dispersions in chloroform and toluene possessed green-blue fluorescence, while Fourier-transform infrared spectroscopy (FT-IR) and quantum chemical calculations showed that the PNA coordinates to the PNP surface through the primary amine group. Additionally, the sensing ability of the PNA ligand for adenine nucleic acid was demonstrated by photoluminescence quenching via charge transfer. Furthermore, PNP thin films were effectively produced by the centrifugal casting. We envision that combining the unique, tailored structure of peptide nucleic acids and the prospective optical features of lead halide perovskite nanoparticles could expand the field of applications of such hybrids exploiting analogous ligand chemistry.     

多巴胺和抗坏血酸在十六烷基三甲基溴化铵/碳纳米管电极上的检测

研究了十六烷基三甲基溴化铵(CTMAB)/多壁碳纳米管修饰玻碳电极的制备以及多巴胺和抗坏血酸在该修饰电极上的电化学行为。在CTMAB和多壁碳纳米管的协同作用下,该修饰电极对多巴胺和抗坏血酸均具有显著的催化氧化作用,多巴胺和抗坏血酸的氧化峰电位分别为223mV和15mV,实现了在抗坏血酸共存时测定多巴胺。在pH7.0的磷酸盐缓冲溶液中,多巴胺和抗坏血酸的线性范围分别为2.0×10-6～2.0×10-3mol/L和4.0×10-5～1.0×10-2mol/L,检出限分别为6.0×10-7mol/L和1.0×10-5mol/L。