A strand displacement signal amplification-assisted fluorescence assay for sensitive detection of microRNA

MicroRNA is a vital biomarker because of its abnormal expression in the emergence and development of diseases, especially in cancers. Herein, a label-free fluorescent sensing platform is proposed for detecting microRNA-21, coupled with the cascade toehold-mediated strand displacement reaction and magnetic beads. Target microRNA-21 acts as an initiator to trigger the cascade toehold-mediated strand displacement reaction and it outputs double-stranded DNA. After magnetic separation, the double-stranded DNA is intercalated by SYBR Green I, resulting in an amplified fluorescent signal. Under the optimal conditions, a wide linear range (0.5–60 nmol/L) and low limits of detection (0.19 nmol/L) are exhibited. What's more, the biosensor shows great specificity and reliability between microRNA-21 and other microRNAs involved in cancer (microRNA-34a, microRNA-155, microRNA-10b, and let-7a). Owing to the properties of fabulous sensitivity, high selectivity, and simplicity of operator, the proposed method paves a promising way for microRNA-21 detection in cancer diagnosis and biological research.     

A New Initiator Cholesteryl Chloroformate for Cupper-Based Atom Transfer Radical Polymerization of Methyl Methacrylate

The polymerization of metyl methacrylate (MMA) was studied in detail by use of CuCI/L as a catalyst and cholesteryl chloroformate (CC) as an initiator. It was found that the atom transfer radical polymerization of MMA could proceed when L equals to a multidentate aliphatic amine ligand, N,N,N‘,N“,N“-penta(methyl acrylate)diethylenetriamine (MA5-DETA), and no polymerization was occurred while L=2,2‘-bipyridine and 1,10phenanthroline. The linear proportionality of the molecular weights to the conversions and straight lines observed in ln[M]0/[M] versus time plots indicated that the present polymerization system had the typical controlled polymerization characteristics.     

海洛因滥用者唾液中毒品代谢物的SPE-GC/MS-SIM分析

本文首先考查了不同类型不同厂家的固相萃取柱的萃取效果,采用提取效果较好的50 mg/mL的Clean-screen CSDAUL01混合型强阳离子固相萃取柱,对唾液中的吗啡、可待因、6-单乙酰吗啡等3种鸦片类毒品进行提取后,将提取液吹干;再经MBTFA衍生化后,进行GC/MS-SIM检测。以乙基吗啡为内标,3种毒品的线性相关系数均大于0.99,线性范围为10～1000 ng/mL,相对回收率分别为85%～110%、94%～107%和75%～92%;相对标准偏差小于10%。3种毒品的检测限分别为2 ng/mL、1 ng/mL和2 ng/mL。该方法灵敏度高、重现性好、操作简便,可用于鸦片类毒品滥用者及中毒者唾液中的毒品及其代谢物的检测。     

A new molecularly imprinted polymer for selective extraction of cotinine from urine samples by solid-phase extraction

Cotinine, the main metabolite of nicotine in human body, is widely used as a biomarker for assessment of direct or passive exposure to tobacco smoke. A method for molecularly imprinted solid-phase extraction (MISPE) of cotinine from human urine has been investigated. The molecularly imprinted polymer (MIP) with good selectivity and affinity for cotinine was synthesized using cotinine as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with aqueous standards, by comparing recovery data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from the aqueous solutions resulted in more than 80% recovery. A range of linearity for cotinine between 0.05 and 5 μg mL⁻¹ was obtained by loading 1 mL blank urine samples spiked with cotinine at different concentrations in acetate buffer of pH 9.0, and by using double basic washing and acidic elution. The intra-day coefficient of variation (CV) was below 7% and inter-day CV was below 10%. This investigation has provided a reliable MISPE–HPLC method for determination of cotinine in human urine from both active smokers and passive smokers. Figure     

Disposable amperometric magnetoimmunosensor for the sensitive detection of the cardiac biomarker amino-terminal pro-B-type natriuretic peptide in human serum

A novel amperometric magnetoimmunosensor using an indirect competitive format is developed for the sensitive detection of the amino-terminal pro-B-type natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture solution containing variable concentrations of the antigen and a fixed concentration of an HRP-labeled detection antibody. Accordingly, the target NT-proBNP in the sample and that immobilized on the MBs compete for binding to a fixed amount of the specific HRP-labeled secondary antibody. The immunoconjugate-bearing MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE). The amperometric responses measured at –0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H₂O₂ as the enzyme substrate, are used to monitor the affinity reaction. The developed magnetoimmunosensor provides attractive analytical characteristics in 10-times diluted human serum samples, exhibiting a linear range of clinical usefulness (0.12–42.9 ng mL⁻¹) and a detection limit of 0.02 ng mL⁻¹, which can be used in clinical diagnosis of chronic heart failure in the elderly and for classifying patients at risk of death after heart transplantation. The magnetoimmunosensor was successfully applied to the analysis of spiked human serum samples.     

A sensitive immunosorbent bio-barcode assay combining PCR with icELISA for detection of gonyautoxin 2/3

In the current study, we developed a nanosphere bio-barcode technology to detect trace gonyautoxin 2/3 (GTX 2/3). GTX 2/3-glucose oxidase (GOX) conjugates were first prepared as the coating antigen in a periodate reaction. Subsequently, gold nanoparticles (NP) dual-labeled with anti-GTX 2/3 monoclonal antibodies (Mab) and DNA oligonucleotides were synthesized via a one-step preparation method. Combining PCR with indirect competitive ELISA (icELISA), a novel immunosorbent bio-barcode assay was established utilizing the Mab-NP-dsDNA complex to convert enzymatic signals to DNA signals. Importantly, the limit of detection of the method was lower than 0.74 μg mL⁻¹. Thus, the immunosorbent bio-barcode assay is a rapid and high-throughput screening tool to detect GTX 2/3 in aquatic products.     

A sensitive method for the determination of tritium in urine

An analytical procedure was developed for the determination of tritium in urine. The content of contaminants in urine after treatment is reduced markedly so that no influence is expected on tritium measurements by a liquid scintillation counter. The established analytical procedure was successfully applied for urine from the general public in Japan.     

Development of a sensitive enzyme immunoassay for the detection of phenyl-beta-D-thioglucuronide in human urine

Immunoassays for the measurement of glucuronides in human urine can be a helpful tool for the assessment of human exposure to toxic chemicals. Therefore an enzyme imimunoassay (EIA) for the specific detection of phenyl-beta-D-thioglucuronide was developed. The immunoconjugate was formed by coupling p-aminophenyl-beta-D-thioglucuronide to the carrier protein thyroglobulin leaving an exposed glucuronic acid. The hapten-protein conjugate was adsorbed to gold colloids in order to enhance the immunogenic effect. Rabbits were injected with the immunogold conjugates to raise polyclonal antibodies. The resulting competitive assay showed an inhibition by phenyl-beta-D-thioglucuronide at sample concentrations of 23.0 +/- 1.3 ng/mL (50% B/B0) and a high cross-reactivity to p-aminophenyl-beta-D-thioglucuronide (120%). Little cross-reactivities (< 2%) were observed for potential urinary cross reactants. In addition human urine samples were incubated with beta-glucuronidase in order to investigate the EIA for specific matrix effects. An integration of high-performance liquid chromatography (HPLC) and EIA was developed in an attempt to decrease the matrix effects and increase the sensitivity of the overall method. The hyphenated technique HPLC-EIA may be used to monitor human exposure to toxic thiophenol which is excreted by mammals as urinary phenyl thioglucuronide.     

Molecularly imprinted polymers for highly sensitive detection of morphine using surface plasmon resonance spectroscopy

Molecular imprinting technology is applied in surface plasmon resonance spectroscopy for highly sensitive and selective detection of morphine（MO）.As SPR-based sensor of MO,the preparation of molecular imprinted polymer is as follows: methacryhc acids（MAA）,ethylene glycol dimethacrylate（EGDMA）,azodiisobutyronitrile（AIBN） were used as functional monomer,cross-linker and initiator,respectively.The experiment results showed that morphine imprinted polymer had the performance of high sensitivity and specificity,i.e.the relative signal of SPR response was proportional to the concentration of morphine in acetonitrile in the range of 10^-9mol/L to 10^-6mol/L（1 ppb-1 ppm） with LOD of 10^-10mol/L,and MO was distinguished from its analogs,such as codeine.     

A highly sensitive and specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of antibiotic olaquindox in animal feed samples

The use of olaquindox (OLA) as an additive in animal feedstuffs has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for determination of OLA in animal feed samples was developed. OLA was activated by N′N-carbonyldiimidazole and coupled with bovine serum albumin (BSA) and ovalbumin (OVA). It was found that the sensitivity and specificity of the two antisera were very similar, with the IC₅₀ values of 16 ng mL⁻¹ and 19 ng mL⁻¹, respectively. Cross-reactivity was less than 35% for four structurally related compounds and no recognition of five other antibiotics was observed. The better antiserum I was selected for further experiments, for example testing stability, solvent effect, accuracy, and precision. The IC₅₀ value for eight standard curves was in the range 12–18 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.31 ± 0.11 ng mL⁻¹. The ELISA tolerated 5% methanol without significant influence on IC₅₀ value. The recoveries of spiked OLA in five different animal feed types including auxin, pig complex feed, fish complex feed, broiler concentrated feed, and pig premix feed were in the range 88.3–119.0% and the intra-assay relative standard deviation (RSD) was within 4.7–33.5% (n = 3). The ELISA for unspiked feed samples was confirmed by high-performance liquid chromatography (HPLC), with a high correlation coefficient of 0.9862 (n = 5). The proposed ELISA could be a feasible quantitative/screening method for OLA analysis in feed samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput, and low expense. Figure Polyclonal antibody based ELISA for detection of olaquindox     

氯嘧磺隆胶体金免疫检测试纸条的制备与应用

利用胶体金标记的经亲和层析纯化的氯嘧磺隆单克隆抗体,氯嘧磺隆-牛血清白蛋白,羊抗鼠IgG制备了氯嘧磺隆胶体金免疫检测试纸条。该试纸条的最低检测极限为100μg/L,检测时间10 m in。8个土壤样品分别添加0~2000μg/L的氯嘧磺隆标样,用0.15 mol/L NaHCO3提取2 h后用气相色谱和试纸条进行检测,如果以100μg/L作为阳性与阴性样品的区分标准,气相色谱和试纸条检测结果完全一致。所制备的胶体金免疫检测试纸条可以用于土壤中氯嘧磺隆残留的筛选。     

Monoclonal antibody-based enzyme-linked immunosorbent assay for detection of total malachite green and crystal violet residues in fishery products

A rapid easy-to-use trace level direct competitive enzyme-linked immunosorbent assay (dc-ELISA) detection of total residual malachite green (MG), crystal violet (CV) and their corresponding primary metabolites leucomalachite green (LMG) and leucocrystal violet (LCV) in fishery products in a single assay was developed. The monoclonal antibodies, anti-MG and anti-CV mAbs, were prepared using carboxyl-malachite green (CMG) and cationized bovine serum albumin (cBSA) conjugates as immunogen. The linear range for the quantitative detection of total MG, CV and their primary metabolites LMG and LCV was between 0.15 to 4.5?ng?mL^?1 with a half maximal inhibitory concentration (IC₅₀) at 0.56?±?0.04?ng?mL^?1 (n?=?5). The anti-MG mAbs exhibited 98% cross-reactivity to CV, less than 0.1% cross-reactivity with LMG and LCV, and no cross-reactivity with chloramphenicol, enrofloxacin, sulfadiazine, and tetracycline. Application of the dc-ELISA in fish tissue samples gave a limit of detection (LOD) of 0.37?ng?g^?1. The improved total detection lead to a recovery of 74.60?±?8.38% at 0.5?ng?g^?1 and 87.47?±?12.83% at 2.0?ng?g^?1 that was better than existing techniques. The dc-ELISA showed total MG in 7 out of 44 field fish samples that were confirmed with LC-MS/MS. The easy-to-use, inexpensive, and rapid dc-ELISA for the detection of total MG, CV and their corresponding primary metabolites holds promise for field applications.     

One step and highly sensitive headspace solid-phase microextraction sample preparation approach for the analysis of methamphetamine and amphetamine in human urine

A fiber-stable, repeatable and highly sensitive headspace solid-phase microextraction (HS-SPME) method was developed for the analysis of methamphetamine (MA) and amphetamine (AM) in urine using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. For sample preparation, the test specimen was placed in a 7 ml vial along with the additives (KOH and NaCl) and the internal standards (d8-MA and d8-AM), a glass insert containing heptafluorobutyric anhydride (HFBA) and heptafluorobutyric chloride (HFBCl) as derivatizing reagents was inserted into the vial, the vial was then sealed tightly. A SPME device with a 100 microm polydimethylsiloxane fiber was inserted into the vial and the fiber was exposed to the headspace in the insert, then the vial was heated and stirred at 100 degrees C and 600 rpm for 20 min for evaporation/adsorption/derivatization. The vaporized analytes (AM and MA) in the vial diffused into the glass insert though the holes on the insert, they absorbed onto the fiber, and then interacted with the vapor of the derivatizing reagent. Some of the analytes in the headspace of the glass insert may react with the vapor of the derivatizing reagent first, and then adsorb onto the fiber. The needle was finally removed and inserted into the injection port to desorb the analytes with the fiber exposed to the liner of the GC-MS system for analysis. By combining HFBCl and HFBA as derivatizing reagents and placing them in an insert, the HS-SPME method achieves high sensitivity for the analysis of AM and MA. Correlation coefficients derived from typical calibration curves in the 1.0-1700 ng ml(-1) range are 0.998 for MA and 0.994 for AM. The limits of detection and the limits of quantitation using a sample size of 1 ml are 0.3 and 1.0 ng ml(-1), respectively, for both MA and AM in urine specimens. Because the water hydrolysis of derivatizing reagent is much faster than the acylation reaction of the primary and secondary amines with the derivatizing reagent, the amphetamines cannot be acylated effectively over heated aqueous solution, and therefore this study provides a new acylation design in moisture surroundings. The proposed process also simplifies the procedure for urine sample preparation, and makes the automation of SPME possible.     

中空结构的双金属有机骨架材料作为固相微萃取纤维涂层用于多环芳烃的高灵敏检测

环境样品中多环芳烃(PAHs)含量较低且样品基质复杂,直接利用仪器进行含量测定比较困难,因此在仪器分析之前需要对环境样品进行必要的前处理。大多数前处理技术的萃取效率取决于萃取材料的特性。目前,金属有机骨架材料(MOFs)作为一种由金属离子与有机配体自组装而成的多孔材料,已经被用作固相微萃取(SPME)的涂层材料应用于PAHs的萃取,但是这些MOFs涂层材料由于目标物较难达到其深层的吸附位点,使得萃取过程往往需要较长的平衡时间;此外,大多数MOFs由单金属离子配位构成,能够提供的开放金属活性位点种类比较单一,较难获得最佳的萃取性能。这些问题在一定程度上限制了MOFs材料在SPME领域的应用。该研究制备了一种中空结构的双金属有机骨架材料(H-BiMOF),并将其作为SPME的涂层材料,用于萃取环境样品中痕量的PAHs。由于中空的结构和双金属的组成,H-BiMOF涂层材料拥有比表面积利用率高、传质距离短等优点,可以使萃取过程快速地达到平衡。同时,双金属的引入提供了种类丰富的金属活性位点,提高了对PAHs这类富电子云目标物的萃取效率。与气相色谱-串联质谱(GC-MS/MS)相结合,建立了一种用于环境水样中PAHs分析的新方法。所建立的分析方法具有检出限低(0.01~0.08 ng/L)、线性范围宽(0.03~500.0 ng/L)、重复性良好(相对标准偏差≤9.8%, n=5)等优点,并成功地用于实际湖水样品中7种PAHs的检测。实验结果表明,所建立的分析方法适用于环境样品中PAHs的分析与监测。     

Optimization of a sensitive method for the determination of nitro musk fragrances in waters by solid-phase microextraction and gas chromatography with micro electron capture detection using factorial experimental design

A solid-phase microextraction method (SPME) followed by gas chromatography with micro electron capture detection for determining trace levels of nitro musk fragrances in residual waters was optimized. Four nitro musks, musk xylene, musk moskene, musk tibetene and musk ketone, were selected for the optimization of the method. Factors affecting the extraction process were studied using a multivariate approach. Two extraction modes (direct SPME and headspace SPME) were tried at different extraction temperatures using two fiber coatings [Carboxen–polydimethylsiloxane (CAR/PDMS) and polydimethylsiloxane–divinylbenzene (PDMS/DVB)] selected among five commercial tested fibers. Sample agitation and the salting-out effect were also factors studied. The main effects and interactions between the factors were studied for all the target compounds. An extraction temperature of 100 °C and sampling the headspace over the sample, using either CAR/PDMS or PDMS/DVB as fiber coatings, were found to be the experimental conditions that led to a more effective extraction. High sensitivity, with detection limits in the low nanogram per liter range, and good linearity and repeatability were achieved for all nitro musks. Since the method proposed performed well for real samples, it was applied to different water samples, including wastewater and sewage, in which some of the target compounds (musk xylene and musk ketone) were detected and quantified. Figure Stardardized Pareto charts for the main effects and interactions     

A new colorimetric test for detection of hydroxyl groups in solid-phase synthesis

A new colorimetric test (methyl red-DIC test) for monitoring the presence of hydroxyl groups on resins for use in solid-phase synthesis was developed. The resin became orange-red when the carboxyl group of methyl red formed ester linkages with the resin’s hydroxyl groups. This new test can be used not only for qualitative analysis but also quantitative analysis by using pictures of the relationship between color depth and hydroxy loading rates.     

A sensitive and selective method for the detection of diazepam and its main metabolites in urine by gas chromatography-tandem mass spectrometry

A gas chromatography-tandem mass spectrometry method for detection of diazepam, nordazepam and oxazepam is presented. The method associates electron capture ionization and multiple reaction monitoring (MRM). No derivatization is performed; oxazepam undergoes thermal degradation during chromatographic injection and is thus quantified via its decomposition product. The negative molecular ions are so stable that they do not dissociate when collision is performed under "classical" conditions (i.e. with argon as collision gas). With xenon as collision gas, the energy transfer is sufficient to provide two product ions for diazepam and nordazepam and one product ion for the decomposition product of oxazepam. The sample preparation part involves liquid/liquid extraction with TOXI-TUBES A extraction tubes; it provides recovery yields between 68 and 95%, depending of the benzodiazepine considered, with coefficients of variation below 6% for 10 samples. The applicability of the method was demonstrated on urine extracts. From 1 mL of urine, the method provides quantitation limits of 0.15 ng/mL for diazepam, 1.0 ng/mL for nordazepam and 1.5 ng/mL for oxazepam. Mechanisms of dissociation of M*(-) ions of benzodiazepines are suggested.     

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins.     

A graphene-based electrochemical sensor for sensitive detection of paracetamol

An electrochemical sensor based on the electrocatalytic activity of functionalized graphene for sensitive detection of paracetamol is presented. The electrochemical behaviors of paracetamol on graphene-modified glassy carbon electrodes (GCEs) were investigated by cyclic voltammetry and square-wave voltammetry. The results showed that the graphene-modified electrode exhibited excellent electrocatalytic activity to paracetamol. A quasi-reversible redox process of paracetamol at the modified electrode was obtained, and the over-potential of paracetamol decreased significantly compared with that at the bare GCE. Such electrocatalytic behavior of graphene is attributed to its unique physical and chemical properties, e.g., subtle electronic characteristics, attractive π-π interaction, and strong adsorptive capability. This electrochemical sensor shows an excellent performance for detecting paracetamol with a detection limit of 3.2 × 10⁻⁸ M, a reproducibility of 5.2% relative standard deviation, and a satisfied recovery from 96.4% to 103.3%. The sensor shows great promise for simple, sensitive, and quantitative detection and screening of paracetamol.     

Preparation of novel solid-phase microextraction fibers by sol-gel technology for headspace solid-phase microextraction-gas chromatographic analysis of aroma compounds in beer

3-(Trimethoxysilyl)propyl methacrylate (TMSPMA) was first used as precursor as well as selective stationary phase to prepare the sol-gel-derived TMSPMA-hydroxyl-terminated silicone oil (TMSPMA-OH-TSO) solid-phase mircroextraction (SPME) fibers for the analysis of aroma compounds in beer. TMSPMA-OH-TSO was a medium polarity coating, and was found to be very effective in carrying out simultaneous extraction of both polar alcohols and fatty acids and nonpolar esters in beer. The extraction temperature, extraction time, and ionic strength of the sample matrix were modified to allow for maximium sorption of the analytes onto the fiber. Desorption temperature and time were optimized to avoid the carryover effects. To check the matrix effects, several different matrices, including distilled water, 4% ethanol/water (v/v) solution, a concentrated synthetic beer, a "volatile-free" beer and a real beer were investigated. Matrix effects were compensated for by using 4-methyl-2-pentanol as internal standard and selecting the "volatile-free" beer as working standard. The method proposed in this study showed satisfactory linearity, precision and detection limits and accuracy. The established headspace SPME-gas chromatography (GC) method was then used for determination of volatile compounds in four beer varieties. The recoveries obtained ranged from 92.8 to 105.8%. The relative standard deviations (RSD, n = 5) for all analytes were below 10%. The major aroma contributing substances of each variety were identified via aroma indexes.