Detection of complement C1-inhibitor with a piezoelectric immunosensor

A novel piezoelectric immunosensor has been developed for the detection of human complement C1-inhibitor. Anti-C1-inhibitor antibody was immobilized onto the gold electrodes of a 9 MHz AT-cut piezoelectric crystal. Coating the crystal with polyethyleneimine adhesion, followed by a glutaraldehyde cross-linking method to immobilize antibody showed better results than the physical adsorption method with respect to sensitivity and reproducibility. Under the optimized experimental conditions, the sensor showed good response to the C1-inhibitor in the range from 2.0 × 10^–8 to 1.2 × 10^–6 g. Other proteins in human serum did not remarkably interfere with the detection. The crystals could be regenerated 5 times, when bound materials on the crystal surface were eluted by strong acid and strong alkali solution and subsequently cleaned in an ultrasonic cleaner.     

A New Approach to the Development of a Reusable Piezoelectric Crystal Biosensor

Abstract

A reusable piezoelectric crystal immunosensor for human albumin has been developed. The crystal was coated with protein a and then reacted with anti-human albumin antibody. Human albumin in the range 10^?4 mg/ml to 10^?1 mg/ml could be detected by the system. Crystals were regenerated by saturation with albumin and subsequent binding of a new anti-albumin antibody layer. The albumin assay could be repeated up to 5 times using the same crystal.     

A Sandwich-type Electrochemical Immunosensor for the Sensitive Determination of Salmonella Typhimurium in Food

A sandwich-type electrochemical immunosensor was developed for sensitive detection of Salmonella Typhimurium (S. Typhimurium) in milk and eggs. According to the antigen-antibody specific binding, Anti-S. Typhimurium antibody was used to recognize the S. Typhimurium, while the HRP-Anti-S. Typhimurium antibody as signal probe was used to catalyze H₂O₂ with the assistance of thionine. The immunosensor showed wide linear range from 5.6×10¹ to 5.6×10⁸ CFU/mL and low detection limit of 35 CFU/mL. The immunosensor also exhibited excellent specificity, reproducibility and stability, indicating that it had practicability in quality control of complex food samples and potential in clinical applications.     

Direct and Rapid Detection of Diphtherotoxin via Potentiometric Immunosensor Based on Nanoparticles Mixture and Polyvinyl Butyral as Matrixes

In this paper a novel potentiometric immunosensor for direct and rapid detection of diphtherotoxin (D‐Ag) has been developed by means of self‐assembly of monoclonal diphtheria antibody (D‐Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes. At first, D‐Ab was absorbed onto the surface of nanoparticles mixture, and then they were entrapped into polyvinyl butyral sol‐gel network on a platinum electrode. The detection is based on the change in the potentiometric response before and after the antigen‐antibody reaction in a phosphate buffer solution (pH 7.0). The immobilized D‐Ab exhibited direct potentiometric response toward D‐Ag. In comparison to the conventional applied methods, this strategy could allow antibodies immobilized with higher loading amount and better retained immunoactivity, as demonstrated by potentiometric response, cyclic voltammetry and electrochemical impedance spectroscopy of the immunosensor. The immunosensor with nanoparticles mixture exhibited much higher sensitivity, better reproducibility, and long‐term stability than that with gold nanoparticles or silica nanoparticles alone. The linear range was from 5.0×10^?3 to 1.2 μg?mL^?1 with a detection limit of 1.1×10^?3 μg?mL^?1. Up to 16 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with in 0.2 mol?L^?1 glycine‐hydrochloric acid (Gly‐HCl) buffer solution and 0.25 mol?L^?1 NaCl. Moreover, the immunosensor with nanoparticles mixture was applied to evaluate a number of practical specimens with potentiometric results in acceptable agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtherotoxin in the clinical diagnosis.     

Reusable Amperometric Immunosensor for the Determination of Cortisol

Abstract

A novel reusable amperometric immunosensor was developed for the determination of cortisol by co-immobilizing horseradish peroxidase (POD) and cortisol-antibody on a chemically activated affinity membrane which is mounted over the tip of an oxygen electrode. The enzymatic electrocatalytic current response to the respective substrate is inhibited by the association of the antigen to the co-immobilized antibody. The sensor can be regenerated to facilitate several assays by washing with dilute hydrochloric acid solution. The advantages of the sensor include rapid-response, simple analysis methodology and high selectivity. The calibration curve for cortisol is linear in the concentration range of 1 × 10^?7 – 1 × 10^?5M. The optimal conditions of immobilization and pH have been studied. The inhibition of enzymatic activity was confirmed by luminescence measurement utilizing the luminol- H₂O₂-POD system.     

Disposable Immunosensor for Escherichia Coli O157:H7 Based on a Multi-Walled Carbon Nanotube-Sodium Alginate Nanocomposite Film Modified Screen-Printed Carbon Electrode

A disposable immunosensor for the detection of Escherichia coli O157:H7 based on a multiwalled carbon nanotube–sodium alginate nanocomposite film was constructed. The nanocomposite was placed on a screen-printed carbon electrode, and horseradish peroxidase-labeled antibodies were immobilized to E. coli O157:H7 on the modified electrode to construct the immunosensor. The modification procedure was characterized by atomic force microscopy and cyclic voltammetry. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to E. coli O157:H7 in a concentration range of 10³–10¹⁰ cfu/mL, with a relatively low detection limit of 2.94 × 10² cfu/mL (S/N = 3). This immunosensor exhibited satisfactory specificity, reproducibility, stability, and accuracy, making it a potential alternative tool for early assessment of E. coli O157:H7.     

A MIP-based flow-through fluoroimmunosensor as an alternative to immunosensors for the determination of digoxin in serum samples

This work reports a comparative study of two automated flow-through fluorosensors for the determination of digoxin in serum samples: an immunosensor with an anti-digoxin polyclonal antibody as the reactive phase permanently immobilised on controlled-pore glass and a sensor with a selective reaction system based on a methacrylic molecularly imprinted polymer (MIP) synthesised by bulk polymerisation. The variables affecting the sensitivity and dynamic range of the sensors (e.g. the carrier and elution solutions, flow rates, pH and reagent concentrations) were optimized, and the binding characteristics of their reactive phases were compared in a competitive fluorescent assay. Digoxin was reproducibly determined by both sensors at the milligram per litre level (detection limit = 1.20 × 10⁻³ mg L⁻¹ and RSD = 4–7% for the immunosensor; detection limit = 1.7 × 10⁻⁵ mg L⁻¹ and RSD = 1–2% for the MIP sensor). No cross-reactivity with digoxin-related compounds was seen for either sensor at a digoxin/interferent ratio of 1:100. The lifetime of the immunosensor was about 50 immunoassays; its shelf life, when unused, is about 3 months. The lifetime of the MIP sensor was over 18 months. Both sensors were used to determine the digoxin concentration of human serum samples with satisfactory results.     

Label-free Microcantilever Immunosensor Based on a Competitive Immunoassay for the Determination of Clenbuterol

A label-free microcantilever immunosensor based on a competitive immunoassay is reported for the determination of clenbuterol. The immunosensor was fabricated by modifying clenbuterol–ovalbumin on the gold surface of the microcantilever with crossing linkage by L-cysteine and glutaraldehyde. Atomic force microscopy was utilized to characterize the construction of immunosensor and to measure the deflection of the microcantilever. The deflection response of the microcantilever was in negatively proportional to the concentration of clenbuterol from 1.0?×?10^?2 to 20?µg/L with a limit of detection of 1.0?×?10^?2?µg/L. The fabricated immunosensor was used to determine clenbuterol in pork samples with satisfactory results. In addition, the results were in accordance with those obtained by high-performance liquid chromatography. The reported immunosensor displayed high sensitivity and specificity together with excellent repeatability and reliability.     

Parabens determination with a hybrid FIA/HPLC system with ultra-short monolithic column

The combination of an ultra-short C18 monolithic column (5 mm long) with a flow injection analysis (FIA) scheme results in a versatile and efficient system that has been used for the chromatographic determination of four preservatives — methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP). The separation is carried out by using two carriers, A and B, consisting of a mixture of ACN: water in different proportions. The described procedure is able to separate the analytes in only 150 s. The applicable concentration range, detection limit and the relative standard deviation were the following: for MP from 1.6 × 10^-5 to 1.1 × 10^-3 M; 4.8 × 10^-4 M; 0.65%; for EP between 3.7 × 10^-5 and 2.0 × 10^-3 M; 1.2 × 10^-5 M; 1.2%; for PP between 3.9 × 10^-5 and 2.0 × 10^-3 M; 1.2 × 10^-5 M; 1.2%; and for BT between 6.0 × 10^-5 and 2.0 × 10^-3 M; 1.8 × 10^-5 M; 1.8%. The method was applied and validated satisfactorily for the determination of these parabens in commercial cosmetics samples, comparing the results with those obtained by HPLC reference method.     

Fabrication of Immunosensor Based on Polyaniline,Fullerene‐C60 and Palladium Nanoparticles Nanocomposite: An Electrochemical Detection Tool for Prostate Cancer

Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C₆₀ nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C₆₀/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C₆₀ was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C₆₀ was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C₆₀/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H₂O₂ reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10^?4 ng.mL^?1 to 38 ng.mL^?1 with a limit of detection (LOD) of 1.95×10^?5 ng.mL^?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.     

Determination Of Amino Acid Based On A Series Piezoelectric Quartz Crystal Sensor

ABSTRACT

A series piezoelectric quartz crystal (SPQC) sensor has been applied to detect L-glutamic acid and L-lysine acid. The effect of formaldehyde solution on the frequency shift was studied. Two methods were discussed. For the calibration curve method, in a neutralized formaldehyde medium, the linear ranges for determining L-glutamic acid and L-lysine acid were from 7.1×10^?6M to 6.5×10^?4M and from 6.9×10^?6 M to 7.4×10^?4 M, respectively, with the detection limit being 7.1×10^?6 M and 6.9×10^?6 M, the recoveries were 99.2% and 100.1%, the R.S.D were 1.63% (n=6) and 1.83%(n=6), respectively. Frequencimetric tiration method was also described and the lowest titratable concentrations were 8.3×10^?5M and 5.5×10^?5 M, respectively.     

A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode

Recent outbreaks of foodborne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A disposable electrochemical immunosensor for detection of Vibrio parahaemolyticus (VP) based on the screen-printed electrode (SPE) coated with agarose/Nano-Au membrane and horseradish peroxidase (HRP) labeled VP antibody (HRP-anti-VP) has been developed in this paper. Then, the immunosensor was characterized by cyclic voltammetry (CV) and laser scanning confocal microscope (LSCM). The immunosensor was incubated with the one-step immunoassay format involving VP for 30 min at room temperature (25 ± 0.5 °C). The access of the active center of HRP catalyzing the oxidation reaction of thionine by H₂O₂ was partly inhibited by VP, which connected on the surface of the immunosensor by immunoreaction. VP could be quantificationally detected according to the shift of reduction current while CV was used as electrochemical means to detect the products of the enzymatic reaction. Under the optimum conditions of immunoreaction and electrochemical detection, VP was rapidly detectable by sigmoidal curve method and form a linear calibration between 10⁵ and 10⁹ cfu/ml with an associated detection limit of 7.374 × 10⁴ cfu/ml (S/N = 3). The immunosensor had acceptable specificity, reproducibility, stability and accuracy, indicating that the immunosensor could satisfy the need of practical sample detection.     

Amperometric Immunosensor for Rapid Detection of Honeybee Pathogen Melissococcus Plutonius

European foulbrood (EFB) is a honeybee larvae disease caused by a bacterium Melissococcus plutonius. An amperometric immunosensor based on a sandwich assay was developed for rapid point‐of‐care detection of this pathogen. An in‐house made anti‐Melissococcus antibody was immobilized to a gold surface of a screen‐printed sensor via self‐assembled monolayer of cysteamine activated with glutaraldehyde. The direct impedimetric detection of captured microbial cells was tested, however, a better performance was obtained after the formation of sandwich with the peroxidase‐labeled antibody in the amperometric mode. The label‐free assay was limited by higher non‐specific binding. The limit of detection of the immunosensor was 6.6×10⁴ CFU mL^?1 (colony‐forming units) with wide linear range between 10⁵ CFU mL^?1 and 10⁹ CFU mL^?1. The whole analysis was completed within 2 h, which is shorter compared to common laboratory diagnostic tools, such as enzyme‐linked immunosorbent assay or polymerase chain reaction. Furthermore, atomic force microscopy was used for confirmation of the bacteria presence on the electrode surface. The developed immunosensor was successfully employed in the analysis of real samples of honeybees and larvae. The achieved results demonstrate the potential of the amperometric immunosensor for practical in‐field diagnosis of EFB, which can prevent infection spreading and connected losses of honeybee colonies.     

A self-assembled monolayer based electrochemical immunosensor for detection of leukemia K562A cells

A novel strategy to quantify the cell number of leukemia K562A cells using electrochemical immunosensor was developed by effective surface immunoreaction between P-glycoprotein (P-gp) on cell membrane and P-gp mouse monoclonal antibody bound on an epoxysilane monolayer modified glassy carbon electrode. The surface morphologies of the epoxysilane monolayer and the bound antibodies were studied with atomic force microscopy. The binding of target K562A cells onto the immobilized antibodies increased the electron-transfer impedance of electrochemical probe, which depended linearly on the cell concentration in the range of 5.0 × 10⁴–1.0 × 10⁷ cells mL⁻¹. The detection limit of the immunosensor was 7.1 × 10³ cells mL⁻¹. The proposed strategy showed acceptable reproducibility with an RSD of 3.4% for the linear slope and good precision with the RSD of 3.7% and 3.0% examined at the cell concentrations of 2.0 × 10⁶ and 1.0 × 10⁷ cells mL⁻¹.     

Flow Injection and Batch Spectrophotometric Determination of Ibuprofen Based on its Competitive Complexation Reaction with Phenolphthalein‐β‐Cyclodextrin Inclusion Complex

Abstract

Rapid, simple, and accurate spectrophotometric method is presented for the determination of ibuprofen by batch and flow injection analysis methods. The method is based on ibuprofen competitive complexation reaction with phenolphthalein‐β‐cyclodextrin (PHP‐β‐CD) inclusion complex. The increase in the absorbance of the solution at 554 nm by the addition of ibuprofen was measured. Ibuprofen can be determined in the range 8.0×10^?6 ?3.2×10^?4 and 2.0×10^?5?5.0×10^?3 mol l^?1 by batch and flow methods, respectively. The limit of detection and limit of quantification were 6.19×10^?6 and 2.06×10^?5 mol l^?1 for batch and 1.77×10^?5 and 5.92×10^?5 mol l^?1 for flow method, respectively. The sampling rate in flow injection analysis method was 120±5 samples h^?1. The method was applied to the determination of pharmaceutical formulations.     

A SERS-based immunoassay for porcine circovirus type 2 using multi-branched gold nanoparticles

We report on a facile immunoassay for porcine circovirus type 2 (PCV2) based on surface enhanced Raman scattering (SERS) using multi-branched gold nanoparticles (mb-AuNPs) as substrates. The mb-AuNPs in the immunosensor act as Raman reporters and were prepared via Tris base-induced reduction and subsequent reaction with p-mercaptobenzoic acid (pMBA). They possess good stability and high SERS activity. Subsequently, the modified mb-AuNPs were covalently conjugated to the monoclonal antibody (McAb) against the PCV2 cap protein to form SERS immuno nanoprobes. These were captured in a microtiterplate via a immunoreaction in the presence of target antigens. The effects of antibody concentration, reaction time and temperature on the sensitivity of the immunoassay were investigated. Under optimized assay conditions, the Raman signal intensity at 1,076 cm⁻¹ increases logarithmically with the concentrations of PCV2 in the concentration ranging from 8 × 10² to 8 × 10⁶ copies per mL. The limit of detection is 8 × 10² copies per mL. Compared to conventional detecting methods such as those based on PCR, the method presented here is rapid, facile and very sensitive.

     

Metal Enhanced Electrochemical Cyclooxygenase‐2 (COX‐2) Sensor for Biological Applications

Pain measurement is commonly required in biomedical and other emergency situations, yet there has been no pain biosensor reported in literature. Conventional approaches for pain measurement relies on Wong‐Baker face diagrams, which are grossly inadequate for situations involving children or unconscious people. We report a label‐free immunosensor for monitoring the pain biomarker cylooxygenase‐2 (COX‐2) in blood. The sensor is based on the concept of metal‐enhanced detection (MED). MED relies on the idea that the immobilization of underpotential deposition (upd) metallic films deposited either as a monolayer or electrostatically held onto a solid gold substrate could significantly amplify bimolecular recognition such as involving antigen‐antibody (Ab‐Ag) interactions. The surface bound Ab‐Ag complex insulates the electrode; causing a decrease in concentration‐dependent redox signals. A linear detection range of (3.64–3640.00)×10^?4 ng/mL was recorded with a detection limit of 0.25×10^?4 ng/mL, which was 4 orders of magnitude lower than that reported for ELISA for the same biomarker. The immunosensor exhibited selectivity of less than 6 % for potential interferents.     

Spectrophotometric Determination of Sulfate by Solvent Extraction with Nonionic Surfactant Span 20 and Crystal Violet

Abstract

The sulfate ion is extracted into toluene with nonionic surfactant span 20 and crystal violet. A linear calibration curve was obtained between the absorbance due to crystal violet in the extract and sulfate concentrations over the range 2.5 × 10^?5 to 2.5 × 10^?4 mol 1^?1 (2.4 to 24 ppm) in the aqueous phase.     

A Sensitive Chemiluminescence Method for Cinchona Alkaloids

Abstract

Chemiluminescence (CL) was achieved by oxidation of sulphide with cerium(IV) in the presence of cinchona alkaloids (quinine, quinidine or cinchonine). The CL intensity was correlated with the concentration of each cinchona alkaloid. Based on this phenomenon, sensitive CL methods for these alkaloids were described. Quinine (4×10^?8～1×10^?4 g/ml), quinidine (1×10^?7 ～ 1×10^?3 g/ml) and cinchonine (1×10^?6 ～ 8×10^?4 g/ml) could be determined with detection limits of 1×10^?8 g/ml, 4×10^?8 g/ml and 6×10^?7 g/ml, respectively.