Ultrasensitive enzyme-free immunoassay for squamous cell carcinoma antigen using carbon supported Pd–Au as electrocatalytic labels

A novel nonenzymatic sandwich-type electrochemical immunosensor has been developed to detect squamous cell carcinoma antigen (SCCA). Nitrogen-doped graphene sheet (N-GS) was used to increase capacity of capturing primary antibodies (Ab₁). Carbon-supported Pd–Au binary nanoparticles (Pd–Au/C) were synthesized and used to label secondary antibodies (Ab₂). The specific binding of SCCA and antibodies enabled a quantitative attachment of Pd–Au/C on the electrode surface. Electrocatalytic analysis showed that the prepared Pd–Au/C exhibit excellent electrocatalytic activity towards hydrogen peroxide (H₂O₂). We use current response of electrocatalytic labels Pd–Au/C to detect the concentration of SCCA. The unique nonenzymatic immunosensor exhibits a relatively wide linear range from 0.005 to 2 ng mL⁻¹ and high sensitivity with a low detection limit of 1.7 pg mL⁻¹. The immunsensor also shows good reproducibility (4.2%) and stability (5.8%), which makes it an enormous application prospect in clinical research.     

N-(β-Carboxypropionyl)luminol as a new chemiluminescence label in immunoassay

A new chemilurninescence label N-(β-carboxypropionyl)luminol (CPL) was used to label sheep anti-human IgG (SaHIgG).The labeled antibody was stable and could be detected at least down to 10-17~10-16 mol.The molar incorporation ratio was estimated to be 0.26 mol of CPL per mol of SaHIgG.There were no apparent changes in the immunoreactivity of the labeled SaHIgG and in the quantum efficiency of the CPL after labeling.     

A paper-based competitive lateral flow immunoassay for multi β-agonist residues by using a single monoclonal antibody labelled with red fluorescent nanoparticles

An ultrasensitive paper based lateral flow assay is described for rapid and simultaneous fluorometric detection of several β-agonists including clenbuterol and its chemical analogues (mabuterol, brombuterol, cimaterol, cimbuterol, bromchlorbuterol and banbuterol). A nonspecific monoclonal antibody (mAb) against clenbuterol and its analogues was prepared and employed in a competitive immunoassay where mAb conjugated to fluorescent nanoparticles and free β-agonists compete for the binding sites. This enables rapid screening for the 7 β-agonists in a single run that takes about 8 min. Detection limits for the seven β-agonists are <50 pg g^?1 of pork. Recoveries ranged from 69.5% to 102.4%, and relative standard deviations were ±15%. The assay was applied to the analysis of both using spiked and unspiked pork for β-agonists, and the results compare well to those obtained by HPLC-MS.

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Graphical abstract Schematic presentation of an ultra sensitive fluorescent nanoparticle based paper based assay for rapid detection of multi β-agonists in pork tissue.

     

Preparation of magnetic molecularly imprinted polymers based on a deep eutectic solvent as the functional monomer for specific recognition of lysozyme

A magnetized molecularly imprinted polymer (MIP) was prepared via a surface-imprinting technique. An allyl-based deep eutectic solvent was chosen as the functional monomer to obtain the polymer for specific recognition of lysozyme. It was deposited on silica-coated magnetite nanoparticles. The structure of the polymer was confirmed by X-ray diffraction, Fourier transform infrared spectrometry, transmission electron microscopy, thermogravimetric analysis and vibrating sample magnetometry. The maximum binding capacity of the imprinted polymer is found to be 108 mg·g^?1, which is higher than that of non-imprinted polymer. Compared to reference proteins such as cytochrome C, bovine hemoglobin and bovine serum albumin, the MIP shows favorable selectivity for lysozyme. Besides, the imprinted polymer can be further used to specifically recognize lysozyme from the protein mixture and chicken egg white. Reusability studies demonstrate that the polymer can be recycled four times without significant loss of adsorption capacity. The LOD of the method is 12.8 μg·mL^?1. The relative standard deviations (for n = 3) are 1.38% for precision and 2.76% for repeatability. Its facile synthesis, high adsorption performance and excellent selectivity to capture lysozyme make this polymer an attractive candidate to be applied in biomacromolecular purification.

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Graphical abstract Magnetic molecularly imprinted polymer (MIP) based on deep eutectic solvent as functional monomer was fabricated and applied for the specific recognition of lysozyme. The MIP exhibits high adsorption capacity and excellent selectivity for lysozyme.

     

Determination of carcinoembryonic antigen using a novel amperometric enzyme-electrode based on layer-by-layer assembly of gold nanoparticles and thionine

Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and electrostatic interactions onto a self-assembled monolayer configuration is de- scribed in this paper.The enzyme,horseradish peroxidase(HRP),was employed to block the possible remaining active sites of the nano-Au monolayer,avoid the non-specific adsorption,instead of bovine serum albumin(BSA),and amplify the response of the antigen-antibody reaction.Electrochemical ex- periments indicated highly efficient electron transfer by the imbedded Thi mediator and adsorbed nano-Au.The HRP kept its activity after immobilization,and the studied electrode showed sensitive response to CEA and high stability during a long period of storage.The working range for the system was 2.5 to 80.0 ng/mL with a detection limit of 0.90 ng/mL.The model membrane system in this work is a potential biosensor for mimicking the other immunosensor and enzyme sensor.     

Prussian blue–gold nanoparticles-ionic liquid functionalized reduced graphene oxide nanocomposite as label for ultrasensitive electrochemical immunoassay of alpha-fetoprotein

In this work, poly(diallyldimethylammonium chloride) (PDDA) protected Prussian blue/gold nanoparticles/ionic liquid functionalized reduced graphene oxide (IL-rGO-Au-PDDA-PB) nanocomposite was fabricated. The resulting nanocomposite exhibited high biocompatibility, conductivity and catalytic activity. To assess the performance of the nanocomposite, a sensitive sandwich-type immunosensor was constructed for detecting alpha-fetoprotein (AFP). Greatly enhanced sensitivity for this immunosensor was based on triple signal amplification strategies. Firstly, IL-rGO modified electrode was used as biosensor platform to capture a large amount of antibody due to its increased surface area, thus amplifying the detection response. Secondly, a large number of Au-PDDA-PB was conjugated on the surface of IL-rGO, which meant the enrichment of the signal and the more immobilization of label antibody. Finally, the catalytic reaction between H₂O₂ and the IL-rGO-Au-PDDA-PB nanocomposite further enhanced the signal response. The signals increased linearly with AFP concentrations in the range of 0.01–100 ng mL⁻¹. The detection limit for AFP was 4.6 pg mL⁻¹. The immunosensor showed high sensitivity, excellent selectivity and good stability. Moreover, the immunosensor was applied to the analysis of AFP in serum sample with satisfactory result.     

Adsorption of CO2 as a method for the characterisation of the structure of alumina-pillared clay catalysts

The aim of this work was to study the effect of the metal loading on the structure of two series of cobalt and manganese pillared clay-supported catalysts. For this purpose, equilibrium data for CO₂ adsorption at 273 K were analysed using Freundlich, Langmuir and Toth isotherm models, in order to estimate the adsorption parameters and to relate them with the metal loading of the catalysts. The metal distributions on the porous structure of the catalysts were studied using the temperature programmed reduction results combined with information from the nitrogen physisorption data. Comparison of all results reveals that up to a certain metal loading, about 0.5 wt% of Co and 2 wt% of Mn, the metal oxide is well dispersed into the porous structure of the pillared clay. At higher metal loadings, bulk-metal oxide particles are formed on the external surface.     

Asymmetric photodeconjugation of ammonium ene-carboxylates: temperature effects and evidence for the α-carbon of the dienolic species as a latent trigonal centre

The diastereoselective photodeconjugation of CH₂Cl₂ solutions of enantiopure ammonium ene-carboxylates was studied and the results compared with those of the enantioselective photodeconjugation of ene-esters in the presence of catalytic amounts of chiral aminoalcohols. We have observed a significant entropic effect and shown that a C-β control of the selectivity is operative in the asymmetric protonation of the photodienolic species.     

Development of a one-step immunoassay for triazophos using camel single-domain antibody–alkaline phosphatase fusion protein

Triazophos is mainly used in Asian and African countries for the control of insects in agricultural production. Camelid variable domains of heavy-chain antibodies (VHHs) show great promise in monitoring environmental chemicals such as pesticides. To improve the rate of success in the generation of VHHs against triazophos, genes specifically encoding VHH fragments from the unique allotype IgG3a of an immunized Camelus bactrianus were amplified by using a pair of novel primers and introduced to construct a diverse VHH library. Five out of seven isolated positive clones, including the VHH T1 with the highest affinity to triazophos, were derived from the allotype IgG3a. A one-step enzyme-linked immunosorbent assay (ELISA) using VHH T1 genetically fused with alkaline phosphatase (AP) had a half-maximum inhibition concentration of 6.6 ng/mL for triazophos. This assay showed negligible cross-reactivity with a list of important organophosphate pesticides (< 0.1%). The average recoveries of triazophos from water, soil, and apple samples determined by the one-step ELISA ranged from 83 to 108%, having a good correlation with those by a gas chromatography mass spectrometry (R² = 0.99). The VHH-AP fusion protein shows potential for the analysis of triazophos in various matrices.

     

Ultrasensitive electrochemical immunosensor for CA 15-3 using thionine-nanoporous gold-graphene as a platform and horseradish peroxidase-encapsulated liposomes as signal amplification

This paper describes a novel electrochemical immunosensor using a nanoporous gold (NPG)/graphene (GN) hybrid platform combined with horseradish peroxidase (HRP)-encapsulated liposomes as labels for the sensitive detection of cancer antigen 15-3 (CA?15-3). The electrochemical detection was based on the released HRP from HRP-encapsulated liposomes toward the reduction of H(2)O(2) with the help of the thionine (TH) electron mediator. In the presence of CA?15-3, HRP@liposomes and TH-NPG-GN formed a sandwich-type immunocomplex, and the immunocomplex increased with the increment of the CA?15-3 concentration in the sample. The more CA?15-3 antigen in the sample there was, the more HRP@liposomes/anti-CA?15-3 in the immunocomplex there were. Thus, the catalytic current increased. Under optimized conditions, the linear range of the immunoassay is 2 × 10(-5) to 40 U mL(-1) with a detection limit of 5 × 10(-6) U mL(-1) CA?15-3. The CA?15-3 concentrations of the clinical serum specimens assayed by the developed immunoassay showed consistent results in comparison with those obtained by a commercially available electrochemiluminescence assay. This proposed immunoassay system had many desirable merits including sensitivity, accuracy, and minimal instrumentation required. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.     

Establishing a basis for nuclear archaeometry in Australia using the 20 MW OPAL research reactor

The k ₀-method of standardisation for instrumental neutron activation analysis (INAA) has been used at the OPAL research reactor to determine the elemental composition of three certified reference materials: coal fly ash (SRM 1633b), brick clay (SRM 679) and Montana soil (SRM 2711). Of the 41 certified elements in the three materials, 88 percent were within five percent of the certified values and all determinations were within 15 percent of the certified values. The average difference between the measured and certified values was 0.1 percent, with a standard deviation of 4.1 percent. Since these reference materials are widely used as standards in the analysis of archaeological ceramics by INAA, it has been concluded that the INAA facility in Australia is particularly well-suited for nuclear archaeometry.     

Formation of Tc metal in 12 M HCl using Zn as a reductant

Amorphous TcO₂ and NH₄TcO₄ solubilized into 12 M HCl will spontaneously convert to hexachlorotechnetate (TcCl₆ ^2?). This process is accelerated upon heating but species lower than Tc(IV) are not generated by this action. TcCl₆ ^2? is kinetically unstable with regards to formation in solutions of low concentrations of HCl and will spontaneously convert back to soluble and insoluble forms of Tc(IV) in water. TcCl₆ ^2? in 12 M HCl placed in contact with the reducing metal Zn at elevated temperatures (90 °C) forms a black precipitate that contains amorphous Tc metal, TcO₂, and oxy-chlorides of Tc. Powder X-ray diffraction indicates the presence of Tc metal after thermal treatment where X-ray absorption fine structure spectroscopy indicates the presence of hexagonal Tc metal and amorphous TcO₂ in the precipitate after rinsing with 12 HCl but before thermal treatment. The resulting solution contains a mixture of Tc chlorides and oxy-chlorides following reduction where TcCl₆ ^2? is completely consumed resulting primarily in Tc₂OCl₁₀ ^4? dominating the UV–visible spectra. Reducing the solution volume and reconstituting the products into 12 M HCl while boiling the mixed solution (>24 h) will slowly convert all soluble Tc back to TcCl₆ ^2?. Expanding on previous efforts made in this laboratory to recover Tc metal from aqueous solution, we investigate its synthesis when Tc(IV) and Tc(VII) in 12 M HCl is placed in contact with the reducing metal (i.e., Zn) at elevated temperatures.     

Ultrasensitive competitive electrochemiluminescence immunoassay for the β-adrenergic agonist phenylethanolamine A using quantum dots and enzymatic amplification

Microchimica Acta - We have designed an ultrasensitive electrochemiluminescence (ECL) immunoassay for the determination of the β-adrenergic agonist phenylethanolamine A (PA). It is based on...     

Immunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.     

Low-generation carbosilane dendrimers as core for starpolymers using a Ru–ROMP catalyst

Synthesis of new metathesis initiators by coupling Ru-complexes to low generation carbosilane dendrimers (G0–Ru, G1–Ru). These initiators show a very high activity for the ROMP of NBE. Using these dendrimer complexes, multi-arm starpolymers can be developed in a controlled manner.     

Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL⁻¹ with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics.     

Commiphora pedunculata gum as a green inhibitor for the corrosion of aluminium alloy in 0.1 M HCl

The effect of Commiphora pedunculata (CP) gum on the inhibition of the corrosion of aluminum alloy AA 3001) in solutions of HCl was investigated using gravimetric and thermometric methods of monitoring corrosion. The results obtained indicated that CP gum is a good adsorption inhibitor for the corrosion of aluminum in solutions of HCl. The inhibition efficiency of CP gum was found to increase with an increase in concentration but to decrease with increasing temperature. The adsorption of CP gum on the surface of aluminum was found to be endothermic, spontaneous and to support the mechanism of physical adsorption. The Langmuir adsorption model has been used to describe the adsorption characteristics of CP gum on aluminum surface.