Fluorimetric study of the interaction between human serum albumin and quinolones-terbium complex and its application

A highly sensitive spectrofluorimetric method is proposed for determination of human serum albumin (HSA) and some quinolone drugs. Using quinolones-terbium (Tb3+) complex as a fluorescent probe, in the buffer solution of pH 7.8, HSA can remarkably enhance the fluorescence intensity of the quinolones-Tb3+ complex at 545 nm and the enhanced fluorescence intensity of Tb3+ ion is in proportion to the concentration of HSA and quinolone drugs. Optimum conditions for the determination of HSA were also investigated. The linear ranges and limits of detection are 8.0 x 10(-9) to 8.0 x 10(-8) mol L(-1), 4.20 x 10(-9) mol L(-1) (for HSA); 1.0 x 10(-6) to 4.0 x 10(-6) mol L(-1), 1.87 x 10(-8) mol L(-1) (for norfloxacin) and 1.0 x 10(-7) to 1.0 x 10(-6) mol L(-1), 4.82 x 10(-8) mol L(-1) (for enoxacine), respectively. This method is simple, practical and relatively free interference from coexisting substances, as well as much more sensitive than most of the existing assays.     

High selective determination iron(II) by its enhancement effect on the fluorescence of pyrene-tetramethylpiperidinyl (TEMPO) as a spin fluorescence probe

A novel fluorescence method determination for iron(II) with a high selectivity and sensitivity has been proposed, based on the enhancement of fluorescence signals resulting from specific redox reaction between synthesized spin fluorescence probe pyrene-tetramethylpiperidinyl (TEMPO) and iron(II). Under the experimental conditions, fluorescent probe displayed a rapid and linear response for iron(II) over the concentration range from 2.4 x 10(-7) to 3.6 x 10(-6) mol/L. The limit of detection was 4.0 x 10(-8) mol/L. The relative standard deviation of six replicate measurements was 1.90% for 3.0 x 10(-7) mol/L iron(II). Because of the specific redox reaction between developed spin fluorescence probe and iron(II), there are few interference by other ions, especially in the presence of relative high concentration iron(III). The method has been successfully applied for iron(II) determinations in two different kinds of real samples. Results determined by the proposed method agree favorably with those determined UV-vis spectrometry method with 1,10-phenanthroline.     

Spectrofluorimetric study on the inclusion interaction between vitamin K3 with p-(p-sulfonated benzeneazo)calix[6]arene and determination of VK3

The characteristics of host-guest complexation between p-(p-sulfonated benzeneazo) calix[6]arene (SBC6A) and vitamin K3 (VK3) were investigated by fluorescence spectrometry. A 1:1 stoichiometry for the complexation was established and was verified by Job's plot. An association constant of 4.95 x 10(3)L mol(-1) at 20 degrees C was calculated by applying a deduced equation. The interaction mechanism of the inclusion complex was discussed. It was found that the fluorescence of SBC6A could be remarkably quenched by an appropriate amount of VK3 especially when non-ionic surfactant Triton X-100 existed. According to the obtained results, a novel sensitive spectrofluorimetric method for the determination of VK3 based on supramolecular complex was developed with a linear range of 5.0 x 10(-7) -3.0 x 10(-5)mol L(-1) and a detection limit of 2.0 x 10(-7)mol L(-1). The proposed method was used to determine VK3 in commercial preparations with satisfactory results.     

A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1

A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1 (AFB1) was achieved. AFB1-binding induced formation of a hairpin structure and closeness of fluorophore label and quencher probe, causing fluorescence decrease.     

Self-locked aptamer probe mediated cascade amplification strategy for highly sensitive and selective detection of protein and small molecule

In this work, a novel self-locked aptamer probe mediated cascade amplification strategy has been constructed for highly sensitive and specific detection of protein. First, the self-locked aptamer probe was designed with three functions: one was specific molecular recognition attributed to the aptamer sequence, the second was signal transduction owing to the transduction sequence, and the third was self-locking through the hybridization of the transduction sequence and part of the aptamer sequence. Then, the aptamer sequence specific recognized the target and folded into a three-way helix junction, leading to the release of the transduction sequence. Next, the 3’-end of this three-way junction acted as primer to trigger the strand displacement amplification (SDA), yielding a large amount of primers. Finally, the primers initiated the dual-exponential rolling circle amplification (DE-RCA) and generated numerous G-quadruples sequences. By inserting the fluorescent dye N-methyl mesoporphyrin IX (NMM), enhanced fluorescence signal was achieved. In this strategy, the self-locked aptamer probe was more stable to reduce the interference signals generated by the uncontrollable folding in unbounded state. Through the cascade amplification of SDA and DE-RCA, the sensitivity was further improved with a detection limit of 3.8 × 10⁻¹⁶ mol/L for protein detection. Furthermore, by changing the aptamer sequence of the probe, sensitive and selective detection of adenosine has been also achieved, suggesting that the proposed strategy has good versatility and can be widely used in sensitive and selective detection of biomolecules.     

基于无标记的核酸适配体荧光生物传感器检测凝血酶

设计了一个简单、通用、基于核酸适配体无标记的高敏感、高专一检测凝血酶的荧光方法。以无标记凝血酶核酸适配体单链DNA为识别元素,Pico Green染料传导互补双链的荧光信号。Pico Green是一种不对称菁,当其单独存在时不产生荧光信号,而当其被吸附到互补的双链DNA上时,可产生很强的荧光信号,但被吸附到单链DNA上时,却无明显的信号改变。基于该性质,将其用于凝血酶的检测。该方法对凝血酶的响应线性范围为1.0×10~(-14)~1.0×10~(-7)mol/L,相关系数(r~2)为0.99,检出限为1.0×10~(-14)mol/L。1.0×10~(-8)mol/L两种干扰物质(牛血清蛋白和细胞色素C)的存在不影响凝血酶的检测,表明该方法对凝血酶具有非常好的专一性。该方法成功应用于对人血清样品的检测,其平均回收率为97%~102%。方法可简单、灵敏、特异性地检测凝血酶,有望用于医学临床诊断等领域。     

用于检测细胞内谷胱甘肽的比率型荧光探针

设计合成了1种用于检测生物巯基的比率型荧光探针(4),并考察了其对谷胱甘肽的识别作用.在4-羟乙基哌嗪乙磺酸(HEPES)缓冲液中,探针4可与谷胱甘肽快速反应,溶液颜色由淡黄色变为粉红色,从而实现"裸眼"检测,且在608 nm处的荧光信号增强.在1.6×10-5~2×10-4mol/L范围内,探针4能够定量检测谷胱甘肽,检出限为8.9×10-7mol/L.此外,探针4还可用于MCF-7细胞中谷胱甘肽的成像.     

Study on the electrochemiluminescence behavior of ABEI and its application in DNA hybridization analysis

The electrogenerated chemiluminescence (ECL) behavior of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was studied and it was found that ABEI could produce emission light when oxidized at a +1.0 V (vs. Ag/AgCl) potential in alkaline solution. The addition of H2O2 markedly improved the ECL sensitivity. The pH value of the solution as well as the H2O2 concentration and working potential all have influences on the ECL response. Under optimal conditions, ABEI can be detected in the range 1.3 x 10(-6)-6.5 x 10(-12) mol L(-1). A detection limit of 2.2 x 10(-12) mol L(-1) for ABEI was obtained at a signal-to-noise ratio of 3. ABEI was then used as a marker to label a known sequence oligonucleotide, which was used as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were evaluated by the ECL measurements. The results showed that only a complementary sequence could form a double-stranded DNA with the DNA probe and give a strong ECL response. A three-base mismatch sequence and non-complementary sequence have no response. The intensity of the ECL was linearly related to the concentration of the complementary sequence in the range 9.6 x 10(-11)-9.6 x 10(-8) mol L(-1), the detection limit was 3.0 x 10(-11) mol L(-1).     

基于主客体竞争模式的凝血酶适配体传感器的研究

基于β-环糊精(β-CD)主客体竞争模式,构建了开关型凝血酶适配体电化学传感器.将末端修饰了二茂铁(Fc)的核酸适配体通过与β-CD的主客体识别固定在金电极表面,当凝血酶存在时,适配体由原来的直立线状构型变为"G-四链体",远离电极表面,适配体探针的氧化还原电流强度减小,即"Signal-off".利用此效应对凝血酶进行了灵敏检测,结果表明,在5.0×10-13~5.0×10-9 mol/L浓度范围内,凝血酶的浓度与电化学响应信号呈良好的线性关系,检出限为2.0×10-13 mol/L(3σ).与其它蛋白分子相比,本方法对凝血酶蛋白的检测具有高特异性.本传感器构建简单,再生性好,为生物血清样本中凝血酶的实时高效检测提供了方法.     

Electrochemical aptamer sensor for highly sensitive detection of mercury ion with Au/Pt@carbon nanofiber‐modified electrode

In this paper, an electrochemical aptamer sensor was proposed for the highly sensitive detection of mercury ion (Hg²⁺). Carbon nanofiber (CNF) was prepared by electrospinning and high‐temperature carbonization, which was used for the loading of platinum nanoparticles (PtNPs) by the hydrothermal method. The Pt@CNF nanocomposite was modified on the surface of carbon ionic liquid electrode (CILE) to obtain Pt@CNF/CILE, which was further decorated by gold nanoparticles (AuNPs) through electrodeposition to get Au/Pt@CNF/CILE. Self‐assembling of the thiol‐based aptamer was further realized by the formation of Au‐S bond to get an electrochemical aptamer sensor (Aptamer/Au/Pt@CNF/CILE). Due to the specific binding of aptamer probe to Hg²⁺ with the formation of T‐Hg²⁺‐T structure, a highly sensitive quantitative detection of Hg²⁺ could be achieved by recording the changes of current signal after reacting with Hg²⁺ within the concentration range from 1.0 × 10^?15 mol/L to 1.0 × 10^?6 mol/L and the detection limit of 3.33 × 10^?16 mol/L (3σ). Real water samples were successfully analyzed by this method.     

Determination of adenosine disodium triphosphate (ATP) using oxytetracycline-Eu3+ as a fluorescence probe by spectrofluorimetry

A new spectrofluorimetric method was developed for determination of adenosine disodium triphosphate (ATP). We studied the interactions between oxytetracycline (OTC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-vis absorption and fluorescence spectra. Using oxytetracycline (OTC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the OTC-Eu3+ complex at lambda = 612 nm and the enhanced fluorescence intensity of Eu3+ ion is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP are 8.00 x 10(-8)-1.50 x 10(-6) mol L(-1) with detection limits of 2.67 x 10(-9) mol L(-1). This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in samples. The mechanism of fluorescence enhancement between oxytetracycline (OTC)-Eu3+ complex and ATP was also studied.     

Spectrofluorimetric determination of thiols by use of N[P-(2-benzoxazolyl)phenyl]maleimide

The weak fluorescence of N-[P-(2-benzoxazolyl)phenyl]maleimide (BOPM) can be greatly enhanced by thiol-containing compounds. A sensitive and simple spectrofluorimetric method based on the use of BOPM has been developed for the determination of thiols such as cysteine (Cys) and reduced glutathione (GSH). Calibration plots were linear in the concentration range from 0 to 1.6 x 10(-7) mol L(-1) for Cys and 0 to 1.7 x 10(-7) mol L(-1) for GSH. The detection limits (3a) were 2.36 x 10(-10) mol L(-1) for Cys and 1.49 x 10(-10) mol L(-1) for GSH. Many other amino acids (present at 100-fold greater concentrations) did not interfere with the determination. The proposed method has been used for the determination of Cys in protein hydrolysate and cystine electrolyte or GSH in serum, with recoveries of 95.4-103.7%.     

A fluorescent probe for zinc ions based on N-methyltetraphenylporphine with high selectivity

N-methyl-alpha,beta,gamma,delta-tetraphenylporphine (NMTPPH) has been used to detect trace amount of zinc ions in ethanol-water solution by fluorescence spectroscopy. The fluorescent probe undergoes a fluorescent emission intensity enhancement upon binding to zinc ions in EtOH/H(2)O (1:1, v/v) solution. The fluorescence enhancement of NMTPPH is attributed to the 1:1 complex formation between NMTPPH and Zn(II) which has been utilized as the basis for the selective detection of Zn(II). The linear response range covers a concentration range of Zn(II) from 5.0x10(-7) to 1.0x10(-5)mol/L and the detection limit is 1.5x10(-7)mol/L. The fluorescent probe exhibits high selectivity over other common metal ions except for Cu(II).     

基于水溶性共轭聚合物荧光增强测定精氨酸

在水溶液中,精氨酸(Arg)能使水溶性共轭聚合物P1的荧光高灵敏、特异性地增强,从而建立了一种免标记的荧光增强型Arg检测新方法.该方法Arg检测的线性范围为6.0×10-8～3.06×10-6 mol/L和3.06×10-6～7.11×10-5 mol/L;检出限为2.0×10-8 mol/L.方法简单、快捷、选择性好、灵敏度高,可用于盐酸精氨酸注射液样品的检测.     

Determination of ATP as a fluorescence probe with europium(III)-doxycycline.

A new spectrofluorimetric method has been developed for the determination of adenosine disodium triphosphate (ATP). We studied the interactions between the doxycycline (DC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-visible absorption and fluorescence spectra. Using doxycycline (DC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP could remarkably enhance the fluorescence intensity of the DC-Eu3+ complex at lambda = 612 nm. The enhanced fluorescence intensity of the Eu3+ ion was in proportion to the concentration of ATP. The optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP were 1.00 x 10(-7) - 2.00 x 10(-6) mol L(-1) with detection limits of 4.07 x 10(-8) mol L(-1). This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to the determination of ATP in samples. The mechanism of fluorescence enhancement between the doxycycline (DC)-Eu3+ complex and ATP was also studied.     

基于苯并吲哚的荧光探针对Al3+的识别及应用

设计合成了一种结构简单且基于苯并吲哚衍生物的荧光探针(H),通过紫外-可见吸收光谱(UV-Vis)和荧光发射光谱研究了荧光探针H对金属阳离子的识别性能。 结果表明,在乙醇(EtOH)和4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(pH=7.4) (体积比为1:1)的混合溶液体系中,荧光探针H对Al³⁺有专一性识别且不受其它金属阳离子干扰。 探针H与Al³⁺的配合比为1:1,配合常数K_a为0.52×10² mol/L,检测限为1.75 μmol/L。 探针通过荧光发射光谱对水样及食品中的Al³⁺进行了定量检测,具有一定实际应用价值。     

A new spectrofluorometric probe for the determination of trace amounts of CoA in injection, human serum and pig livers.

A new spectrofluorometric method has been developed for the determination of trace amounts of coenzyme A (CoA). Using europium (Eu3+)-tetracycline (TC) complex as a fluorescent probe in the buffer solution of pH 6.80, CoA could remarkably enhance the fluorescence intensity of the Eu3+-TC complex at lambda = 612 nm after adding H(5)IO(6) and the enhanced fluorescence intensity is in proportion to the concentration of CoA. Optimum conditions for the determination of CoA were also investigated. The dynamic range for the determination of CoA is 6.08 x 10(-8) - 1.84 x 10(-5) mol L(-1) with detection limit of 4.62 x 10(-8) mol L(-1). This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to determination of CoA in injection, human serum and pig liver samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Eu3+-TC system and the CoA-Eu3+-TC system have been also discussed.     

Optochemical sensor for an ornidazole assay using 1-amino-4-allyloxyanthraquinone as a fluorescent indicator.

To improve the stability of optochemical sensors (optodes), the fluorescence indicator 1-amino-4-allyloxyanthraquinone (AAA), which was synthesized by reacting allyl bromide with 1-amino-4-hydroxyanthraquinone, was covalently immobilized on surface-modified glass slides. The resulting sensor was used to determine the content of ornidazole based on fluorescence quenching. It showed a linear response toward ornidazole in the concentration range of 9.0 x 10(-6) to 1.0 x 10(-3) mol L(-1) with a detection limit of 8 x 10(-6) mol L(-1) at pH 7.5. This AAA-immobilized sensor has a rapid response, high stability and good selectivity to ornidazole.     

Aptamer Biosensor for Selective and Rapid Determination of Insulin

Insulin plays an important role in glucose metabolism and its detection in biological fluids is of interest. In this study, a triple-helix molecular switch was employed for the simple, sensitive, and rapid determination of insulin. The triple-helix molecular switch was composed of a target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide acting as a signal transduction probe. This approach takes advantage of unique properties of aptamers and triple-helix molecular switches such as high affinity, selectivity, and stability. In the absence of insulin, the fluorescence of triple-helix molecular switch is on. Upon addition of insulin, the aptamer binds to its target, leading to the release of the signal transduction probe, folding of the signal transduction probe to a stem loop structure, and the quenching of the fluorescence. This sensor showed a high selectivity toward insulin and a limit of detection as low as 9.97 nM. The sensor was employed for the determination of insulin in biological samples. This platform may be generalizable for a variety of molecules.     

Amperometric detection studies of Nafion/indium hexacyanoferrate film for the determination of electroinactive cations in ion chromatography

An amperometric detector based on the chemical modification of Nafion and indium (III) hexacyanoferrate (II, III) thin film (Nafion/In-CN-Fe) onto a glassy carbon (GC) electrode, was first successfully used for the determination of electroinactive cations (Li+, Na+, K+, Rb+, Cs+, NH4+) in single column ion chromatography (IC). A set of well-defined peaks of electroinactive cations was obtained. The detection limits of the cations are 8.9 x 10(-6) mol/L for Li+, 2.3 x 10(-6) mol/L for Na+, 5.2 x 10(-6) mol/L for K+, 4.8 x 10(-6) mol/L for Rb+, 4.0 x 10(-6) mol/L for Cs+ and 5.3 x 10(-6) mol/L for NH4+ at a single-to-noise ratio of 3. The proposed method was quick, sensitive and simple. The cations in rainwater and mineral water were successfully analyzed by this method.