A highly sensitive,dual-signal assay based on rhodamine B covered silver nanoparticles for carbamate pesticides

A highly sensitive sensor for determination of carbamate pesticides based rhodamine B (RB) modified silver nanoparticle (RB-AgNPs) was developed. Compared with the classical method, it combined colorimetric with fluorescence for detecting carbamate pesticides in complex solutions. Carbamate pesticides can inhibit the activity of acetylcholinesterase (AChE), thus preventing the generation of thiocholine. On the other hand, thioncholine can transform the yellow RB-AgNPs solutions gray color and unquenches the fluorescence of RB simultaneously. Once the activity of AChE was inhibited by the pesticide, the color of the RB-AgNPs solution remains yellow and the fluorescence of RB molecules remains quenched. Under optimized experimental conditions, carbaryl was detected in a concentration range from 0.1 ng/L to 8.0 ng/L with a detection limit of 0.023 ng/L (it was detected by fluorescence spectra). This simple method is suitable for determination of carbamate pesticides in complex samples, such as tomato, apple and river water.     

基于石墨烯/纳米金/壳聚糖复合膜固定化酶的有机磷生物传感器

将乙酰胆碱酯酶(AChE)固定到还原态氧化石墨烯(rGO)/纳米金(Au NPs)/壳聚糖(CS)复合膜修饰的玻碳电极表面,制备了高灵敏度的电化学生物传感器,用于有机磷农药的检测。由于rGO具有较大的比表面积和良好的导电性,复合材料不仅为保持AChE的生物活性提供了良好的生物相容环境,而且rGO/Au NPs的协同作用也提高了传感器的灵敏度。CS/SiO2复合溶胶-凝胶网格状的结构为酶的固定化提供了良好的载体。农药抑制率与乐果浓度的负对数在0.1~10.0ng/mL范围内呈线性关系,检出限为0.02ng/mL(S/N=3)。     

尼罗红在离子表面活性剂水溶液中的荧光特性

尼罗红(NR)分子具有大的芳香环和基态时可与水分子形成氢键的吸电子基, 它对增溶在表面活性剂胶束栅栏层的环境尤其敏感, 在十二烷基三甲基溴化铵(C12TABr)胶束水溶液中表现为双重荧光, 最大发射波长分别位于578和630 nm. 十二烷基硫酸钠(SDS)胶束的反离子解离度大于C12TABr胶束, 这不仅增大了NR周边环境的极性, 也增多了溶剂化水, 导致与NR氢键作用增强, 荧光强度低于C12TABr, 但有效促进了分子内扭转电荷转移(TICT)激发态形成, 其布居甚至可达到98%以上, 表观上仅出现了在634 nm的单重荧光峰. NR对环境的敏感特性很好地反映了Gemini表面活性剂初始形成胶束的残缺结构信息, 是检测这类具有强烈相互作用两亲分子聚集行为的良好探针.     

Electrochemical Assay of Effects of Organophosphate Poisoning on Acetylcholinesterase from Pheretima via 2,6-Dimethyl-p-benzoquinone

Electroanalytical techniques could be a reliable and promising alternative to classical and sophisticated methods because of their simplicity(small and portable), easy use, the ability to deliver fast response with high sensitivity and selectivity. A square wave voltammetric method was developed for the assessment of organophosphorus(OPs) compound impact on acetylcholinesterase(AChE) of Pheretima with 2,6-dimethyl-p-benzoquinone(2,6- DMBQ) as a redox indicator. The substrate of acetylthiocholine is hydrolyzed by AChE and the produced thiocholine reacts with 2,6-DMBQ to give an obvious shift of electrochemical signal. The reduction peak of 2,6-DMBQ is located at around -0.18 V which is far away from the oxidation potential of possible interference components often present in biosample. The decreased rate of reduction current was related with the activity of AChE. The inhibition of parathion-methyl on AChE was assessed. The inhibiton rate of OPs on AChE activity increased quickly during the first 10 min inhibition, and after that the value of inhibition rate approached to be constant. AChE lost almost 29.3% of acti- vity after 10 min incubation with 1 mg/mL parathion-methyl and 67.5% of activity with 10 mg/mL parathion-methyl, while the activity that corresponds to 40 mg/mL parathion-methyl was nearly completely inhibited(94.9%). Compared to cyclic voltammetry and amperometry, Square wave voltammetry(SWV) method is a high sensitive electroanalysis with fast scan-rate(only several seconds for one signal value) which is useful to prevent the electrodes from possible fouling or passivation. This method can be employed to assess the inhibition of organophosphate on AChE and investigate OPs impact on environmental animals.     

层层自组装纳米金与乙酰胆碱酯酶电化学生物传感器检测有机磷农药

采用层层自组装技术制备了快速检测有机磷农药的生物传感器,利用带正电荷的高分子聚电解质聚二烯丙基二甲基氯化铵(PDDA)将乙酰胆碱酯酶(AChE)和金纳米粒子(AuNPs)通过静电力逐层固定到玻碳电极(GCE)表面,并采用交流阻抗和微分脉冲伏安法研究了此生物传感器的电化学行为。由于金纳米粒子优异的电催化性能和良好的生物相容性,使固定化的乙酰胆碱酯酶对其底物具有更高的亲和力和更快的响应速度。实验结果表明:修饰金纳米粒子后,传感器的氧化电流明显增大,在4.6×10-5～5.3×10-3mol/L范围内,固定化酶的抑制率与甲基对硫磷浓度的对数成正比,检出限为7.6×10-6mol/L。该生物传感器具有制备方法简便、成本低、灵敏度高等优点,已成功用于蔬菜样品中甲基对硫磷含量的测定。     

纳米SnSe2空心球上乙酰胆碱酯酶的固定及辛硫磷传感器的构建

描述了一种将乙酰胆碱酯酶固定在SnSe2空心球上检测辛硫磷的简单方法,用水热法合成了SnSe2空心球,并用透射电镜对其表征.固定的乙酰胆碱酯酶能保持其生物学活性,催化乙酰胆碱为胆碱,胆碱被氧化产生可检测的信号.基于辛硫磷对乙酰胆碱酯酶活性有抑制作用这个机理,在理想条件下,这种传感器对辛硫磷检测的线性范围是0.008～56μg/mL,检测限为0.004μg/mL.这种新型的传感器有很好的稳定性和重现性.这项工作表明SnSe2空心球可以作为固定乙酰胆碱酯酶的理想载体并用于构建相应的传感器.     

Amperometric Biosensor Based on Immobilization Acetylcholinesterase on Manganese Porphyrin Nanoparticles for Detection of Trichlorfon with Flow‐Injection Analysis System

A highly sensitive amperometric biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso‐tetraphenylporphyrin (MnTPP) nanoparticles (NPs)‐modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE‐CHIT/MnTPPNP/GC biosensor integrated with a flow‐injection analysis (FIA) system was used to monitor trichlorfon (typical OP). A wide linear inhibition response for trichlorfon is observed in the range of 1.0 nM–1.0 mM, corresponding to 10–83% inhibition for AChE with a detection limit of 0.5 nM.     

Surface-enhanced Raman scattering detection of cholinesterase inhibitors

A new sensitive surface-enhanced Raman scattering (SERS) assay for detection of cholinesterase inhibitors such as organophosphorous pesticides using silver colloidal nanoparticles was developed and optimized. Acetylcholinesterase (AChE) mediated the hydrolysis of acetylthiocholine to produce thiocholine, which interacted with the silver nanoparticles to give a specific SERS spectrum. Variation in enzyme activity due to inhibition was measured from changes in intensity of a characteristic peak (772 cm⁻¹) of the SERS spectrum that was directly correlated with the concentration of produced thiocholine. The method was demonstrated for the detection of paraoxon as reference AChE inhibitor. Limit of detection of paraoxon for 5 min incubation at 25 °C was 1.8 × 10⁻⁸ M. This assay can be utilized for the detection of trace amounts of any AChE inhibitor.     

Evidence for lipids-calcium ions interactions using fluorescent probing in paediatric nutrition admixtures

The aim of this work was to envisage a new analytical fluorescent method to study the molecular interactions between cations and negatively charged lipid droplets contained in total parenteral nutrition (TPN) admixtures. For this purpose, two fluorescent probes were tested: 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, commonly named nile red (NR), and 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS). NR, a neutral molecule, and TNS, an anionic one, are both polarity probes. Their fluorescence emission was enhanced in an apolar environment. They were used at 1 and 2.5 muM, respectively. Results showed that scattered light was very intense in weak aqueous dilution (1/10 vv(-1)) of fat emulsion and appeared as an experimental constraint. The sensitivity of fluorescence measurement in fat emulsion samples was constantly higher for NR than for TNS. When calcium addition occurs, as in pharmaceutical practice, a dramatic increase of fluorescence emission signal was showed for TNS, but no effect was observed for NR. As a conclusion, it was pointed out that the interactions between lipid droplets and calcium ions were likely to take place at the interface of the droplet and that TNS was a more appropriate probe than NR to prove it. Thus, fluorescent probing appeared to be a convenient new analytical tool for the investigation of lipid-cations interactions in TPN mixtures.     

Homogeneous fluorescence-based immunoassay via inner filter effect of gold nanoparticles on fluorescence of CdTe quantum dots

Homogeneous immunoassays are becoming more and more attractive for modern medical diagnosis because they are superior to heterogeneous immunoassays in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin (IgG) as a model analyte, has been developed. This assay relies upon the inner filter effect (IFE) of gold nanoparticles (AuNPs) on CdTe QDs fluorescence. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the IFE of AuNPs on CdTe QDs fluorescence is greatly enhanced, resulting in a decrease of fluorescence intensity in the system. Based on this phenomenon, a wide dynamic range of 1-100 pg mL(-1) for determination of IgG can be obtained. The proposed method shows a detection limit of 0.3 pg mL(-1) for human IgG, which is much lower than the corresponding absorbance-based approach and compares favorably with other reported fluorescent methods. This immunoassay method is simple, rapid, cheap, and sensitive. The proposed method has been successfully applied to measuring IgG in serum samples, and the obtained results agreed well with those of the enzyme-linked immunosorbent assay (ELISA).     

Ultrasensitive endonuclease activity and inhibition detection using gold nanoparticle-enhanced fluorescence polarization

We have developed a highly sensitive and selective fluorescence polarization assay method based on the specificity of the DNA cleavage reaction with the enhancement of gold nanoparticles (AuNPs) for assaying endonuclease activity and inhibition. This assay can detect EcoRI endonuclease down to 5.0×10(-4) U mL(-1) with a detection range from 5.0×10(-4) to 10 U mL(-1).     

Binding of acetylcholinesterase to multiwall carbon nanotube-cross-linked chitosan composite for flow-injection amperometric detection of an organophosphorous insecticide

A novel method for immobilization of acetylcholinesterase (AChE) by binding covalently to a cross-linked chitosan-multiwall carbon nanotube (MWNT) composite is described. In addition a sensitive, fast, cheap and automatizable flow injection detection of an organophosphorous insecticide was developed. The MWNTs were homogeneously distributed in the chitosan membrane which showed a homogeneous porous structure. The immobilized AChE could catalyze the hydrolysis of acetylthiocholine with a K(M)app value of 177 microM to form thiocholine, which was then oxidized to produce detectable signal in a linear range of 1.0-500 microM and fast response. MWNTs could catalyze the electrooxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of an organophosphorous insecticide on the enzymatic activity of AChE, using Sulfotep as a model compound, the conditions for the flow-injection detection of the insecticide were optimized. Both biocompatibility of chitosan and inherent conductive properties of MWNTs favored the detection of the insecticide from 1.5 to 80 microM along with good stability and reproducibility. 95 % reactivation from inhibited AChE could be regenerated by using 2-pyridinealdoxime methiodide within 15 min for 15 times. The detection of Sulfotep samples exhibited satisfactory results. The proposed flow-injection analysis device can be applied to automated determination and characterization of enzyme inhibitors.     

Aptamer‐Assisted Gold Nanoparticles/PEDOT Platform for Ultrasensitive Detection of LPS

Neatly arranged gold nanoparticles (AuNPs) were directly electrodeposited on an electrochemically polymerized self‐assembled monolayer (SAM) of thiol‐functionalized 3,4‐ethylenedioxythiophene (EDOT) derivative, EDTMSHA. A thiolated single‐stranded DNA (ssDNA) aptamer with high specificity to LPS was immobilized on the AuNPs/conducting polymer composite film, serving as sensing platform for LPS detection. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscope (SEM), and atomic force microscopy (AFM) were utilized to characterize the modification and detection processes. The electron transfer resistance was found to have a linear relationship with LPS concentration from 0.1 pg/mL to 1 ng/mL.     

Aptasensor for visual and fluorometric determination of lysozyme based on the inner filter effect of gold nanoparticles on CdTe quantum dots

The authors describe an aptasensor for visual and fluorescent detection of lysozyme via an inner filter effect (IFE). The assay is based on the fact that red gold nanoparticles (AuNPs) act as powerful absorbers of the green fluorescence of CdTe because of spectral overlap. If the lysozyme-binding aptamer is adsorbed onto the surface of the AuNPs, the salt-induced aggregation of AuNPs (that leads to a color change from red to blue) does not occur and the IFE remains efficient. If lysozyme is present, it will bind the aptamer and thereby prevent its adsorption on the AuNPs. As a result, the salt-triggered aggregation of the AuNPs will occur. Consequently, color will change from red to blue, and green fluorescence will pop up because the IFE is suppressed. Under optimum conditions, fluorescence is linearly related to lysozyme concentration in the 1.0 nM to 20 nM concentration range, with a 0.55 nM limit of detection. The method is perceived to be of wider applicability in that it may be used to design other visual and fluorescent assays if appropriate aptamers are available.

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Graphical abstract The fluorescence intensity of QDs is quenched by gold nanoparticles (AuNPs) due to an inner filter effect. Aptamers can adsorb on AuNPs to prevent the salt-induced aggregation. AuNPs serve a dual function as fluorescence quencher and colorimetric reporter.

     

A sensitive and selective fluorescent probe for acetylcholinesterase: Synthesis,performance, mechanism and application

The detection of acetylcholinesterase (AChE) activity is of great significance for studying the physiological functions of AChE and clinical diagnosis of pesticide poisoning. Herein, a small-molecule fluorescent probe BDFA was rationally designed and readily synthesized via a one-step reaction, which enables qualitative and quantitative detection of AChE. BDFA emits a slight fluorescence in an aqueous medium, while the fluorescence is significantly enhanced under the catalysis of AChE. Mechanism studies reveal that BDFA eliminates the N, N-dimethyl carbamate protective group in the presence of AChE and then spontaneously undergoes intramolecular cyclization conversion to generate an intense fluorescent product. Based on the above mechanism, BDFA exhibits a sensitive, selective, rapid and stable “turn-on” fluorescence response to AChE, without interference from pH, ions, thiols, amino acids and other enzymes. The fluorescence intensity of BDFA at 525 nm has a linear relationship with the AChE concentration in the range of 0.0045–1.0 U/mL, and the detection limit is 4.5 mU/mL. Moreover, BDFA is suitable for rapidly diagnosing AChE activity in blood samples, thus providing an efficient and convenient tool for diagnosing organophosphorus and carbamate pesticide poisoning. Compared with the reported AChE fluorescent probes, BDFA exhibits apparent advantages including simple synthesis, low detection limit and fast response speed.     

基于聚苯胺纳米纤维复合膜界面构建电化学细胞传感器及其对癌细胞的识别检测

合成了聚苯胺纳米纤维,直径在50～70 nm之间;基于静电作用构建聚苯胺纳米纤维-纳米金复合膜界面,并在此界面上层层组装修饰叶酸分子,构建叶酸功能化传感界面,基于叶酸分子与癌细胞表面过量表达的叶酸受体之间的特异性识别作用,将此传感界面应用于对癌细胞的识别和捕获。结果表明:叶酸功能化传感界面能够特异性识别和捕获叶酸受体过量表达的癌细胞。采用电化学阻抗技术,以HeLa细胞为模型,应用于对癌细胞的识别和检测,细胞在1.0×104～6.4×106cells/mL浓度范围内与阻抗变化值ΔRct呈良好的线性关系;检出限为2000 cells/mL。本方法简单、快速灵敏、重现性和稳定性良好;制备的传感器可以再生使用。     

A Disposable Biosensor for Organophosphorus Nerve Agents Based on Carbon Nanotubes Modified Thick Film Strip Electrode

A disposable biosensor based on acetylcholinesterase‐functionalized acid purified multi‐wall carbon nanotubes (CNTs) modified thick film strip electrode for organophosphorus (OP) insecticides was developed. The degree of inhibition of the enzyme acteylcholinesterase (AChE) by OP compounds was determined by measuring the electrooxidation current of the thiocholine generated by the AChE catalyzed hydrolysis of acteylthiocholine (ATCh). The large surface area and electro‐catalytic activity of carbon nanotubes lowered the overpotential for thiocholine oxidation to 200 mV (vs. Ag/AgCl) without the use of mediating redox species and enzyme immobilization by physical adsorption. The biosensor detected as low as 0.5 nM (0.145 ppb) of the model organophosphate nerve agent paraoxon with good precision, electrode to electrode reproducibility and stability. Analysis of real water sample using the sensor demonstrated the feasibility of the application of the sensor for on site monitoring of OP compounds.     

A Uniquely Configured Acetylcholinesterase‐Lecithin Biomimetic Sensing Structure at Cyclohexane/Water Interface for Electrochemical Determination of Fenthion in Organic Solvent

An acetylcholinesterase (AChE)‐lecithin biomimetic structure was constructed at the oil/water interface for the direct determination of fenthion in cyclohexane. Indophenol acetate in oil phase was hydrolyzed by AChE at the two‐phase interface to produce indophenol. Square wave voltammetry was used to monitor the current decrease of substrate to assess AChE activity. The AChE incorporated in the lecithin‐based biomimetic layer possessed higher enzymatic activity and was more sensitive to fenthion inhibition than freestanding AChE. A linear relationship between the inhibition percentage and logarithm of fenthion concentration was obtained in a concentration range from 1 ng/mL to 1 mg/mL.     

Ethylenediaminetetraacetic Acid Functionalized Gold Nanoparticles for Sensitive Colorimetric Detection of Chromium(III)

A cost‐effective and sensitive colorimetric method was described for the determination of chromium(III) ion (Cr³⁺) by using ethylenediaminetetraacetic acid functionalized gold nanoparticles (EDTA‐AuNPs) as a probe. The stable and dispersed EDTA‐AuNPs were prepared by reducing HAuCl4 with sodium borohydride in presence of EDTA as a capping agent. Upon the addition of Cr³⁺, the colour of EDTA‐AuNPs solution changed from red to violet, which was in response to the surface plasmon absorption of dispersed and aggregated EDTA‐AuNPs. The procedure allowed the determination of Cr³⁺ in the range of 0.1–1.0 mol/L. The limit of detection for Cr³⁺ was 0.08 mol/L. The relative standard deviation was 2.5 % for eight repeated measurements of 0.6 mol/L Cr³⁺ solution. The method was applied to the determination of Cr³⁺ in water samples.     

Dual Amplified Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Using Hemin/G‐Quadruplex Immobilized onto Fe3O4‐AuNPs or (Hemin‐Amino‐rGO‐Au) Nanohybrid

A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe₃O₄‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe₃O₄‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H₂O₂ in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe₃O₄‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.