An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

用生物素-中性抗生物素蛋白结合导致的位阻来控制连接生物素的缓激肽生物活性(英)

缓激肽是一含有9个氨基酸残基的多肽,其残基序列为Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹-OH,在激肽释放酶的作用下, 从其大的前体多肽——激肽原而形成的.许多发病机理,如发炎、疼痛、哮喘等都与缓激肽有关. 它能与PC12细胞表面的受体作用,引起细胞器内的钙离子释放,在共焦显微镜下,通过观察钙指示剂Fluo-3荧光增加来监测缓激肽的生物活性.在这项研究中,利用固相肽合成方法合成了连接生物素的缓激肽,Biotin-Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹-OH,通过对其生物活性的研究发现：a.它能保持象天然的缓激肽那样的生物活性;b.由于中性抗生物素蛋白与连接的生物素的结合引起的空间位阻阻碍它与细胞表面受体的相互作用,从而抑制了它的生物活性;c.在有自由的生物素存在的条件下,自由生物素与连接生物素与中性抗生物素蛋白的竞争结合,能够使得与中性抗生物素蛋白结合的连接生物素的缓激肽从抗生物素蛋白上脱离,因而恢复其生物活性.因此,可利用生物素和抗生物素蛋白来控制连接生物素的缓激肽的生物活性.这对于研究生物体系中生物活性的结构相关性具有重要的意义.     

Cu@Au alloy nanoparticle as oligonucleotides labels for electrochemical stripping detection of DNA hybridization

Synthesis of the novel Cu@Au alloy nanoparticle and its application in an electrochemical DNA hybridization detection assay is described in this article. We report a low-temperature method for generating core-shell particles consisting of a core of Cu and a thin layer of Au shell that can be readily functionalized with oligonucleotides. Core-shell Cu@Au particles were successfully labeled to a 5'-alkanethiol capped oligonucleotides probe that is related to the colitoxin gene. The DNA genetic sensing assay relies on the electrostatic adsorption of target oligonucleotides onto conducting polypyrrole (PPy) surface at the glassy carbon electrode (GCE), and its hybridization to the alloy particle-oligonucleotides DNA probe. Hybridization events between probe and target were monitored by the release of the copper metal atoms anchored on the hybrids by oxidative metal dissolution and the indirectly determination of the solubilized Cu2+ ions by sensitive anodic stripping voltammetry (ASV). The detection limit is 5.0 pmol l(-1) of target oligonucleotides. The Cu@Au core-shell nanoparticles combining the surface modification properties of Au with the good electrochemical activity of Cu core shows their perspective application in the electrochemical DNA hybridization analysis assay.     

Successively amplified electrochemical immunoassay based on biocatalytic deposition of silver nanoparticles and silver enhancement

A successively signal-amplified electrochemical immunoassay has been reported on the basis of the biocatalytic deposition of silver nanoparticles with their subsequent enlargement by nanoparticle-promoted catalytic precipitation of silver from the silver-enhancer solution. The immunoassay was carried out based on a heterogeneous sandwich procedure using polystyrene microwells to immobilize antibody. After all the processes comprising the formation of immunocomplex, biocatalytic deposition of silver nanoparticles and following silver enhancement were completed, the silver on polystyrene microwells was dissolved and quantified by anodic stripping voltammetry (ASV). The effect of relevant experimental conditions, including the concentration of ascorbic acid 2-phosphate (AA-p) substrate and Ag(I) ions, the biocatalytic deposition time, and of crucial importance, the silver enhancement time, were investigated and optimized. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1-10 ng ml(-1) with a detection limit of 0.03 ng ml(-1). Scanning electron microscope (SEM) was applied to characterize the silver nanoparticles before and after silver enhancement on the surface of polystyrene microplates. By coupling the highly catalytic effect of enzyme and nanoparticles to successively amplify the analytical signal, the sensitivity of immunoassay was enhanced so dramatically that this approach would be a promising strategy to achieve a lower detection limit for bioassays.     

Acidithiobacillus ferrooxidans fixation on mercuric surfaces and its application in stripping voltammetry

The adsorption (fixation) of bacteria Acidithiobacillus ferrooxidans on Hg and Cu metallic surfaces was qualitatively studied owing to two independent methods: frequency measurement using a quartz crystal microbalance and light absorption measuring at the Hg/bacterial culture interface. A method using a dropping mercury electrode (DME) allowed quantifying this bacterial fixation. Determining the superficial tension at Hg/bacterial culture interface led to determine bacteria adsorption on Hg through the Gibbs isotherm. A modified stripping voltammetry was proposed taking benefit of both bacterial adsorption on Hg surface and metal fixation capacity on biomass. Metal preconcentration on the biologically modified Hg electrode appeared to improve the measurement sensitivity of differential pulse anodic stripping voltammetry (DPASV). The height of the detected peaks was thus increased of 17.6% for copper, 48.4% for lead, and 132% for cadmium determinations compared to those obtained with an unmodified mercury electrode. Such augmentation depended on bulk bacteria concentration and bacteria preconcentration.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.     

Ultra-trace analysis of soluble zinc, cadmium, copper and lead in Windermere lake water using anodic stripping voltammetry and atomic absorption spectroscopy

SUMMARY. The concentrations of trace metals in filtered and unfiltered lake water were measured using anodic stripping voltammetry, before and after digestion by ultra-violet irradiation, and by atomic absorption spectroscopy with electrothermal atomization. Total soluble components were estimated to be: zinc, 2.1 μg 1⁻¹; cadmium, <0.05 μg l⁻¹; lead, <(0.1 μg l⁻¹ and copper, 0.3 μg 1⁻¹. Atomic absorption spectrophotometric results and u.v.-digested, anodic stripping voltammetric results were in good agreement. All measurable zinc was electrochemically labile whereas copper above the detection limit of 0.09 μg l⁻¹ was electrochemically inert.     

ZnO quantum dot labeled immunosensor for carbohydrate antigen 19-9

Using ZnO quantum dots as electrochemical and fluorescent labels, a sandwich-type sensitive immunoassay was developed to detect carbohydrate antigen 19-9 (CA 19-9) which is a preferred label for pancreatic cancer. The immobilization process was mainly carried out through the electrostatic adsorption based on the high isoelectric point of ZnO, and the sandwich structure was built through the immunoreaction of CA 19-9 antibodies and antigens. The immunological recognition of CA 19-9 was converted into detection of the amplified signals of the square wave stripping voltammetry (SWV) and intrinsic photoluminescence of the labeled quantum dots. The electrochemical assay demonstrated a dynamic range of 0.1-180 U/ml with detection limit of 0.04 U/ml, while the optical spectral detection revealed 1-180 U/ml with detection limit of 0.25 U/ml.     

Simultaneous determination of Zn,Cd, Pb,and Cu in urine of patients with blackfoot disease using anodic stripping voltammetry

Blackfoot disease (BFD) is an endemic peripheral vascular disorder resulting in gangrene of the lower extremities, especially the feet, among residents in a limited area on the southwest coast of Taiwan. In the present study, the concentrations of zinc, cadmium, lead, and copper in urine of BFD patients with matched normal controls are investigated by differential pulse anodic stripping voltammetry (DPASV) on a hanging mercury drop electrode (HMDE). The analytical results indicate that urinary copper, cadmium, and lead of the BFD patients are significantly higher than those of the controls. In addition, the patients showed a significantly lower concentration of zinc in the urine than the normal controls. The possible connection of these elements with the etiology of the disease is discussed.     

Determination of zinc, copper, lead and cadmium in some medicinally important leaves by differential pulse anodic stripping analysis

Levels of zinc, copper, lead and cadmium have been determined in some medicinally important leaves by differential pulse anodic stripping voltammetry (DPASV). High pressure digestion with nitric acid (HPA) was used for sample digestion. The accuracy of the method was verified by the parallel analysis of leaves with inductively coupled plasma atomic emission spectroscopy (ICP-AES) and recovery studies by the analysis of standard reference materials. Based on elemental levels the utility of these leaves in medicine are discussed. Statistical treatment has been used in order to understand the correlation between elements in these leaves.     

Graphene/quantum dot bionanoconjugates as signal amplifiers in stripping voltammetric detection of EpCAM biomarkers

A sensitive electrochemical immunosensor for the detection of epithelial cell adhesion molecule (EpCAM) antigen, a common marker for tumors of epithelial origin, employing bionanoconjugates as signal-transduction labels has been developed. The bionanoconjugates were fabricated by carboxylation of the two-dimensional graphene oxide nanosheets (GRs) and immobilizing streptavidin and amine-functionalized CdSe quantum dots (QDs) on carboxylated GRs via carbodiimide coupling chemistry, followed by the immunoreaction with the biotinylated secondary antibodies. Since carboxylated GRs have a higher density of active sites, it allows a large number of CdSe QDs to be immobilized onto the surface of the bionanoconjugates, and hence, enhance the sensitivity of the immunosensor. The method enabled detection limits of 100 fg/mL and 1 pg/mL (based on the S/N=3) in PBS buffer and serum samples, respectively, using anodic stripping voltammetric readout. The immunosensor showed a good selectivity, reproducibility, and long-storage stability, and may become a promising technique for the early detection of tumor biomarker in clinical/biological samples.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using nanoCuS tags

A novel and sensitive biosensor for the determination of short sequence of DNA based on flow injection (FI)-chemiluminescence (CL) system of luminol-H2O2-Cu2+ was developed in the present work. The DNA probe labeled with copper sulfide nanoparticles (CuS NPs) could hybridize with target DNA immobilized on glass-carbon electrode (GCE). The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu2+ after the cupric ions was dissolved from the hybrids. A preconcentration process of cupric ions was performed by anodic stripping voltammetry (ASV) technology to improve the sensitivity of the biosensor. Under the optimum conditions, the CL intensity was proportional to the concentration of target DNA in the range of 2.0 x 10(-12)-1.0 x 10(-10)M. A detection limit of 5.5 x 10(-13)M of target DNA was achieved. The CL intensity of two-base mismatched sequences and noncomplementary sequences were also detected. The experiments indicated that two-base mismatched sequences showed weaker CL intensity and noncomplementary sequences gave no response at all.     

An electrochemical aptasensor based on hybridization chain reaction with enzyme-signal amplification for interferon-gamma detection

A novel electrochemical aptasensor based on hybridization chain reaction (HCR) with enzyme-signal amplification was constructed for the detection of interferon-gamma (IFN-γ). In this aptasensor, the recognition probes which contained the sequence of IFN-γ aptamer were initially binded to IFN-γ, and the unbound recognition probes were captured on the electrode as an initiator to trigger the HCR. The two DNA hairpins bio-H1 and bio-H2 were opened by the recognition probe, and bound one by one on the electrode. The biotin was used as a tracer in the hairpins and streptavidin-alkaline phosphatase (SA-ALP) as a reporter molecule. Then, SA-ALP converted its electro-inactive substrate 1-naphthyl phosphate into an electroactive derivative 1-naphthol generating amplified electrochemical signal by differential pulse voltammetry (DPV). The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated for enzymatic dephosphorylation of 1-naphthyl phosphate. The electrochemical signal observed was inversely related to the concentration of IFN-γ. The proposed approach showed a high sensitivity for IFN-γ in a concentration range of 0.5-300 nM with a detection limit of 0.3 nM. The sensing system also provided satisfactory results for the detection of IFN-γ in the cell media.     

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy.

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein-streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 microg/ml.     

Advantages and disadvantages of voltammetric method in studying cadmium-metallothionein interactions.

A sensitive and chemical species-selective technique of differential pulse anodic stripping voltammetry (DPASV) was applied in studying the cadmium-metallothionein (Cd-MT) interaction. The amperometric titrations of the purified MT20 and MT10 fractions, isolated by verified biochemical procedures from the digestive gland of cadmium-exposed mussels Mytilus galloprovincialis, with Cd2+ ions were performed in the buffered sodium chloride solution of 0.59 M ionic strength, pH 7.9 and 25 degrees C. Applying the DPASV method at various cadmium to metallothionein ratio several groups of chemical species were recorded. The data on the available ligand concentration to complex cadmium ions (CL), the apparent concentration stability constants (K,) of the respective complexes and the reliability of the determined complexing parameters are discussed. In quantifying the Cd-MT interaction the interference of dithiotreitol (DTT), which is used as the reducing agent in isolation and purification of MTs, is documented.     

Toxicity of CdTe QDs with different sizes targeted to HSA investigated by two electrochemical methods

QDs have large scale application in many important areas with potential of unintentional exposure to the environment or organism during processing of a nanotechnology containing product’s life cycle. In this paper, two classical electrochemical methods, cyclic voltammetry and electrochemical impedance spectroscopy were applied to investigate the influence of particle sizes of CdTe QDs on their toxicity targeted to human serum albumin (HSA) under simulative physiological conditions. The results show that the toxicity of yellow emitting QDs (YQDs) on HSA is slightly stronger than that of the green-emitting (GQDs) and red-emitting QDs (RQDs). We also compared these two classical electrochemical methods with the traditional fluorescence spectroscopy through the above results. The electrochemical methods may be more accurate and comprehensive to investigate the toxicity of QDs at the biomacromolecular level under certain conditions, though fluorescence spectroscopy is simpler and more sensitive.     

An electrochemical approach to quantitation and characterization of metallothioneins

An analysis was made of the potential of the electrochemical approach to quantitation and characterization of metallothioneins. The Brdi ka procedure for determination of sulfhydryl groups was shown to be of utility as a standard determination method while anodic stripping voltametry was found to offer advantage in determination of protein-bound zinc and cadmium. Cyclic voltametry was observed to differentiate metallothioneins from other sulfhydryl- or disulfide-containing compounds, suggesting considerable potential as a characterizing method for metallothioneins.     

Determination of lead in white lupin by anodic stripping voltammetry

An anodic stripping voltammetry method for the simultaneous determination of trace heavy metals in nutrient solutions, soils and plants has been developed at a hydrodynamic electrochemical sensor. Several parameters were optimized in order to enhance sensitivity. Calibration curves in different media are presented. The study of the uptake of lead by white lupin (Lupinus albus L.) was carried out. Toxicity symptoms were observed and compared with levels of lead measured in roots and leaves.     

Simultaneous detection of dual proteins using quantum dots coated silica nanoparticles as labels

Simultaneous detection of multianalytes associated with a particular cancer is beneficial for disease diagnosis. Here, a facile immunosensing strategy was designed to allow simultaneous electrochemical detection of dual proteins, in a single run. CdSe and PbS water-soluble quantum dots (QDs) were prepared and coated on monodisperse silica nanoparticles as labels for proteins detection. Rabbit immunoglobulin G antigen (IgG) and carcinoembryonic antigen (CEA) were chosen as model proteins for analysis. After a typical sandwich immunoassay, CdSe and PbS QDs labels were introduced onto the Au substrates' surface, which were then dissolved and could be simultaneously monitored by square-wave-voltammetric (SWV) stripping measurements. Under selected conditions, IgG and CEA could be assayed in the ranges of 0.05-40 ng mL(-1) and 0.05-25 ng mL(-1), respectively. The proposed method possessed high sensitivity, good precision, and satisfactory reproducibility and regeneration.