Colorimetric detection of proteins based on target-induced activation of aptazyme

The detection of protein is vital to fundamental research as well as practical applications. However, most detection methods depend on antibody-based assays which are faced with many shortcomings. Herein, we propose a colorimetric method for protein assays based on target-triggered activation of aptazyme, which may offer simple, rapid and cost-effective detection of the target protein. In this method, the conformation change of aptazyme induced by target protein is designed to be associated with aptazyme activation. Consequently, in the presence of the target protein, the designed DNA linkers will be cleaved into two fragments that fail to cross-link gold nanoparticles (GNPs), thus the color of GNP solution remains red, while the color will be changed in the absence of the target. Because of the advantages of aptazyme such as economic synthesis, stable, easy modification and its ability to accomplish signal recognition and signal amplification simultaneously, the method is thermostable, simple and cost-efficient. In this work, we have taken the detection of vascular endothelial growth factor (VEGF) as an example, which can present an analytical performance with as low as 0.1 nM detection limit, spanning a detection range of 3 orders of magnitude. What is more, the principle of this proposed new method can be extended as a universal assay method not only for the detection of analytes which have an aptamer but also for those analytes that have ligands.     

A light detection cell to be used in a micro analysis system for ammonia

This paper describes the design, realization and characterization of a micromachined light detection cell. This light detection cell is designed to meet the specifications needed for a micro total analysis system in which ammonia is converted to indophenol blue. The concentration of indophenol blue is measured in a light detection cell. The light detection cell was created using KOH/IPA etching of silicon. The KOH/IPA etchant was a 31 wt.% potassium hydroxide (KOH) solution with 250 ml isopropyl alcohol (IPA) per 1000 ml H(2)O added to it. The temperature of the solution was 50 degrees C. Etching with KOH/IPA results in 45 degrees sidewalls ({110} planes) which can be used for the in- and outcoupling of the light. The internal volume of the realized light detection cell is smaller than 1 mul, enabling measurements on samples in the order of only 1 mul. Measurements were performed on indophenol blue samples in the range of 0.02 to 50 muM. In this range the measurements showed good reproducibility.     

Miniaturization of batch- and flow-type chemiluminescence detectors in capillary electrophoresis

Glass and PTFE tubes as detection cells were put in small light-tight boxes to achieve miniaturization of batch-and flow-type chemiluminescence detectors for capillary electrophoresis. These light-tight boxes which included a detection cell and a photosensor module were successfully designed. In the batch-type detector using a glass tube as a detection cell, the influences of a repeated injection of sample and a reagent volume of the detection cell on chemiluminescence intensity were examined in detail. By using 3.8 mm I.D. glass tube including 400 microl chemiluminescence reagent solution, the chemiluminescence peaks were reproducibly observed for the repeated injection experiment up to the eight injection with each run time of 3.0 min. Dansyl-Trp was determined over the range 3 x 10(-8)-1 x 10(-5) M with the detection limit of 0.43 fmol (S/N=3). In the flow-type detector using a PTFE tube as a detection cell, both ends of the PTFE tube were connected to three-way joints; a chemiluminescence reagent solution was delivered into the cell and a capillary was inserted through one of the joints while an electrode was inserted through the other one. Dansyl-Trp was determined over the range 1 x 10(-7)-1 x 10(-5) M with the detection limit of 1.3 fmol (S/N=3). By using the compact flow-type detector, a mixture of dansyl-amino acids was separated and detected in micellar electrokinetic chromatography mode.     

Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids

Capillary electrophoresis (CE) is a useful method to quantify drugs in biological fluids. However, especially for blood or plasma samples, the sensitivity is not sufficient to quantify drugs and their metabolites as they often need to be quantified in the lower microg/L range. To overcome this limitation and to increase the sensitivity, two strategies are applied: first, to increase the amount of analyte added to the capillary and, second, to increase the sensitivity on the detector site. To improve the sensitivity on the detector site, alternative detection techniques to UV detection, e.g., laser-induced fluorescence detection (LIF) or mass spectroscopy (MS), can be applied. However, LIF detection can only be used for fluorescent analytes and the current equipment for CE-MS coupling provides only small improvements in sensitivity compared to UV detection. The detection window for UV detection can be enhanced using capillaries with an extended light path (bubble cell) or Z-shaped capillaries. Sensitivity improvements up to a factor of 10 have been reported. Increasing the amount of analyte in the capillary can be done either by chromatographic or by electrokinetic methods. Chromatographic methods such as on-capillary membrane preconcentration have been used for several analytes. However, no validated application has been reported to date. In contrast, several validated examples can be found in which electrokinetic techniques like sample stacking have been applied to achieve limits of quantification in the lower microg/L range. In conclusion, to date, electrokinetic techniques such as field-amplified sample injection offer the most promising results in achieving a sufficient sensitivity to quantify drugs in biological fluids.     

流动注射分光光度法测定砷的研究

本文采用自行设计制造的氢化物发生及吸收装置,将流动注射技术同氢化物发生分光光度法结合在一起,设计了一种不使用载气的流动注射分析系统并对该系统的测定条件进行了实验研究,实验用HNO_3-AgNO_3-聚乙烯醇-乙醇混合液作吸收液,结果证明该系统具有操作简便,成本低廉,分析速度快(30次/小时),灵敏度高,重现性好及线性范围可调的优点。     

Determination of anthocyanins in wine based on flow-injection, liquid--solid extraction, continuous evaporation and high-performance liquid chromatography--photometric detection

A continuous method, easy to automate, for the determination of anthocyanins in wine based on the coupling of continuous liquid–solid extraction, evaporation, HPLC individual separation and photometric detection is proposed. The target analytes are removed from the wine in a continuous fashion using a C₁₈ minicolumn and eluted with an aqueous solution (pH 2) with 16% acetonitrile. The eluted fraction is concentrated by solvent evaporation assisted by heat and dragging off the vapour using a flow of N₂. For in-line preconcentration, a continuous evaporation module was designed and located in the manifold between the solid-phase minicolumn and the injection valve of the chromatograph. In this way, injection of the sample into the dynamic system leads the plug through it for liquid–solid extraction of the anthocyanins, partial evaporation of the eluent (with a preconcentration factor as required) and transport to the high-pressure injection valve of the chromatograph, where individual separation and subsequent photometric detection take place. The method thus developed for the determination of malvidin-3-glucoside, cyanidin-3-glucoside and peonidin-3-glucoside anthocyanins in Spanish red wines is more sensitive than the batch manual method based on the same steps, has better linearity of the calibrations curves with lower detection limits and much wider determination range for the most abundant anthocyanins in wine. In addition, the method can be fully automated with low acquisition and maintenance costs.     

Online preconcentration and two‐dimensional separation of cationic compounds via hyphenation of capillary zone electrophoresis with cyclodextrin‐modified micellar electrokinetic capillary chromatography

A novel preconcentration/separation approach, which online combined CZE with CD‐modified MEKC, was developed for simultaneous enhancing resolving power and detection sensitivity. CZE with cation‐selective exhaustive injection and transient ITP preconcentration was used as the first dimension, from which the effluent fractions were further analyzed by CD‐modified MEKC acting as the second dimension. As the key to successful integration of CZE with MEKC, a new interface was designed and electroaccumulation focusing strategy was employed to avoid analyte band diffusion at the interface. The comprehensive 2‐D system was successfully established with only one high voltage and four electrodes. The grouping of two orthogonal separation techniques, together with analytes preconcentration techniques, significantly enhanced resolution and sensitivity for 2‐D separation of cationic compounds. The resulting electrophoregram was quite different from that of either single CZE or MEKC. Up to 14 000‐ to 35 000‐fold improvement in sensitivity was obtained relative to conventional electrokinetic injection method. The limits of detection (S/N=3) were in the range of 0.03–0.1 μg/L. The number of theoretical plates was in the range of 103 000–184 000. This method was successfully applied to the analysis of trace cationic cardiovascular drugs in wastewater.     

Infection Monitoring in Wounds

Wound infection monitoring is a challenging task. It is only solvable by designing an integrable and cost-efficient sensor which measures a relevant set of parameters. One viable parameter is the formation of neutrophil extracellular traps (NETs). Their task is trapping pathogens in the wound. A wound infection results in massive release of them which can be detected with impedimetric methods. Our investigations focused on the characterization of the biological process with an in vitro model. The model environment is a cell culture with neutrophil granulocytes cultured on interdigitated electrodes which represent the sensor surface. Detected impedance changes caused by NET-formation were in the range of 35% and even higher. This implies that impedance measurements are suitable for NET detection. We derived a measurement and evaluated it by differing conditions like changing stimulation agent and varying the cell number. For both conditions the results of impedance and phase angle deviation can be confirmed. In combination with other parameters a sensor can be designed for specific detection of wound infections. These aspects are integrated in our sensor concept.     

Merging zones in flow injection analysis : Part 4. Simultaneous spectrophotometric determination of total nitrogen and phosphorus in plant material

The merging zones approach is used in a single flow injection system for the simultaneous Spectrophotometric determination of nitrogen and phosphorus in plant material based on the Berthelot and molybdophosphate reactions. A multiple proportional injector is designed to introduce samples and reagents into water carrier streams in such a way that only one analytical path with one detection unit is required. For both processes, catalysis is employed; a detailed study of the reagent composition is reported for the Berthelot reaction. The proposed system is characterized by a high degree of sample and reagent dispersion; 60 samples per hour can be analyzed with a consumption of reagents in the range of microliters per determination. Total recoveries of 98.3% N and 99.1% P were estimated from 20 runs of a typical plant sample containing around 3% N and 0.3% P in dry matter. The results of the present method agree with those obtained by Technicon AutoAnalyzer procedure.     

电感耦合等离子体发射光谱仪高电离能元素进样系统的研制

设计开发了一套电感耦合等离子体发射光谱仪（ICP-OES）高电离能元素进样系统.系统既保留了ICP-OES原有的检测功能，又利用超声波振动空化作用的物理方法与氢化反应的化学方法有机结合，有效增加单位体积内待测元素检测浓度，使高电离能元素的检测灵敏度在原有基础上提高3~5倍.系统的研制扩展了ICP-OES关于高电离能元素的检测功能.     

Low-molecular-mass anion screening for forensic environmental analyses by capillary zone electrophoresis with indirect UV detection

A method for low-molecular-mass anion screening is described using a buffer composed of 5-sulfosalicylate (SS) as a visualizing ion, hexadimethrine bromide as an electroosmotic flow modifier and Tris as a pH buffer component, at pH 8.6. All ions with effective mobility higher than 2610⁻⁹ m² s⁻¹ V⁻¹ can be separated within 7.5 min under −30 kV. By using the moderately mobile SS (5410⁻⁹ m² s⁻¹ V⁻¹), not only the sensitivity of the detection is improved due to its high UV absorptivity, but also a smaller overall overloading effect is achieved. Meanwhile, the resolution of the high mobility ions, which is normally critical, remains almost the same as compared to a chromate buffer. With an electrokinetic injection, the limit of detection (LOD) of the common ions is 2–13 nM and the detection range is linear up to 0.5–3 μM. With a hydrostatic injection the LOD is 0.15–1 μM and the detection range is linear up to 25–200 μM. The identification of ions is performed by comparing the mobility of the ions with that of standards, taking the apparent and effective mobility of HCO₃⁻, which is normally present in the sample solution, as a reference.     

An ultrasensitive aptasensor for prostate specific antigen assay based on Exonuclease T-aided cyclic cleavage

An ultrasensitive electrochemical aptasensor is presented for prostate specific antigen(PSA) detection. DNA tetrahedronaptamer is designed, which not only facilitates the molecular self-assembly events,but also improves the recognition efficiency between PSA and aptamer sequence on the electrode interface. The DNA conformation on top of DNA tetrahedron changes accordingly, which can be further digested by Exonuclease T(Exo T), a type of single-strand specific nuclease. Electrochemical species are removed synchronously and the initial PSA level can thus be determined. A linear range from 0.5 pg mL~(-1) to50 ng mL~(-1) is achieved with the limit of detection(LOD) as low as 0.15 pg mL~(-1). Moreover, this proposed method is highly selective and is successfully applied to determine PSA in human serum samples.     

A novel surface ionization source for ion mobility spectrometer

A surface ionization (SI) source is designed and prepared for ion mobility spectrometer (IMS). The source acts not only as an emitter but also an ion injector which can inject ions periodically into the drift region of drift tube. Using the dual-role source, the dimension of the drift tube can be decreased and the circuit for high voltage can be simplified efficiently. The IMS with the SI source has a response range of ∼4 orders of magnitude and a good reproducibility to tri-ethylamine. Compared with radioactive ionization (RI), the ultra-short time for ion injection and the zero level base line of ion mobility spectrum are characteristics of the surface ionization.     

Electrochemically assisted injection – a new approach for hyphenation of electrochemistry with capillary-based separation systems

A new concept based on the electrochemical conversion of analyte species during the injection into capillary flow systems is presented. This approach is termed electrochemically assisted injection (EAI). In a specially designed injection cell containing the analyte solution a conversion efficiency of about 83% can be achieved. Potassium octacyanotungstate(IV) served as a model compound for the analytical characterisation of EAI applying capillary flow injection analysis with double-pulse amperometric detection. Capillary electrophoresis experiments were performed using EAI to study the electrochemical oxidation of various ferrocene derivatives in acetonitrile solution. The electropherograms recorded with UV detection show separated signals for the ferrocene compounds and their oxidation products. The migration behaviour and the stability of ferrocenium cations and other reaction products were investigated.     

Solid phase extraction for the determination of ibuprofen by means of HPLC and UV detection in the lower ng range in plasma and urine

Summary Ibuprofen in the lower ng range could, up to now, only be determined in biological samples by means of specific detectors. In the following a time-saving HPLC method with UV detection after solid phase extraction is described. The lower detection limit is 0.5 ng/20 l of sample volume. The linear range is between 0 and 10.0 ng of ibuprofen per injection. The mean standard deviation and mean coefficient of variation are 0.12 ng and 2.6%. The selectivity of the method is assured.     

流动注射-分光光度法在线全自动测定正磷酸盐

通过将GB/T6913-2008分光光度法在线化,设计研制了正磷酸盐流动注射自动在线监测仪;优化了显色剂浓度、载流液浓度、采样环长度等实验参数,建立了水中正磷酸盐流动注射全自动在线分析方法,并考察了其实际使用效果.结果表明,当待测水样中PO34浓度在0～15.0 mg/L范围内时,吸光度和浓度呈良好的线性关系,检出限为...     

Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (lambda(ex) = 330 nm, lambda(em) = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r(2) > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 microL injection volume.     

Simultaneous determination of hydroxylamine and cyanide in formulations containing pralidoxime salts by flow injection

A flow injection method is described for the simultaneous determination of cyanide and hydroxylamine which are known decomposition products of formulations containing pralidoxime salts used in the treatment of anticholinesterase poisoning. By using the diffusion of HCN from the carrier stream followed by amperometric detection, high selectivity and sensitivity and a wide dynamic range can be achieved. Hydroxylamine is determined by its oxidation with iodine to nitrite which can then be determined colorimetrically. The gas diffusion unit effectively acts as a stream splitter for the two analytes allowing their simultaneous determination from a single sample injection. The performance of the system and its applicability to thermally stressed pralidoxime solutions are described.     

Analysis of pesticide residues in fruits, vegetables, and milk by gas chromatography/tandem mass spectrometry.

A method for detection, quantitation, and confirmation of more than 100 pesticides by gas chromatography (GC) with ion trap mass spectrometry (MS/MS) has been developed. The sensitivity of this method for many analytes is equal to or lower than those of selective GC detectors such as flame photometric detectors and electrolytic conductivity detectors. Using MS/MS, very low detection limits and good confirmation (1 precursor ion and 2 or more product ions) are achieved simultaneously. The entire list of pesticides is screened with 2 injections per sample. Samples are introduced onto the column by a temperature-programmed cold injection to maximize response. Each pesticide is run with its own unique set of parameters, which fragment the compound, retaining only the precursor ion. This ion is then refragmented to create a product spectrum. The selectivity of MS/MS gives a very clean spectrum, making compound identification and confirmation clear, even with a relatively dirty food matrix. If care is taken to maintain the injection port and guard column, this method can reliably identify and confirm more than 100 pesticides at the low parts-per-billion range.