Voltammetric Determination of Lead Labelled Albumin and of Albumin Antiserum by Immunoassay

Abstract

Differential pulse polarography was utilized in voltammetric immunoassay to determine human albumin and albumin antiserum. Albumin was labelled with lead, which exhibited a reduction peak with E_1/2 at ?680 mV vs SCE. The current is proportional to the concentration of lead labelled albumin, and hence the albumin at a fixed lead concentration, and it decreases in proportion to the amount of albumin antiserum added. Albumin concentrations of 2.0 × 10^?6–10 × 10^?6 M and 10–50 μl of goat albumin antiserum (titer 1:16 = maximum dilution causing precipitation with equal volume of 1 mg/ml human albumin) were measured in the presence of 2 × 10^?6 M lead.     

A Chemiluminescence Reaction Between Hydroxyl Radical and Rhodamine 6G and its Applications

Abstract

A novel chemiluminescence (CL) reaction between hydroxyl radical and ascorbic acid is described in this paper. Hydroxyl radical generated on line by the reaction between Fe³⁺ solution and H₂O₂ solution in HCl medium could oxidize rhodamine 6G to produce weak chemiluminescence. It was found that ascorbic acid could enhance the chemiluminescence and the excited rhodamine 6G was the emitter of the chemiluminescence reaction. The possible mechanism of the CL system was also discussed. Ascorbic acid can be determined in the range of 2.0×10^?6?8.0×10^?4 mg/ml with a detection limit of 1×10^?6 mg/ml (3σ). A complete analysis could be done in 1 minute with the relative standard deviation of 3.1% for 5.0×10^?5 mg/ml (n=11). In order to study the chemiluminescence reaction further, the application to the determination of ascorbic acid in food using the chemiluminescence reaction combined with flow injection is investigated.     

A Selective Fluorimetric Method for the Determination of Some β-Lactamic AntibioticsSupported by Natural Science Foundations of China and Shandong province

Abstract

Based on the reaction between acetylacetone-formaldehyde and β-lactamic antibiotics at pH=4.0, in which product can emit strong fluorescence, a selective simple fluorimetric method is described for the determination of both α-aminocephalosporins (namely cepharadine and cefalexin) and 6-aminopenicillanic acid (6-APA) in pure form and in pharmaceutical form. Other β-lactamic antibiotics free from α-amino group do not interfere with the assay. The linear ranges are 1.0×10^?4-8.0×10^?2 mg/ml, 2.0×10^?4-3.0×10^?2 mg/ml and 3.0×10^?4-2.0×10^?2mg/ml for cepharadine, cefalexin and 6-APA, respectively. Their detection limits (S/N=3) are 3.0×10^?5mg/ml, 4.0×10^?5mg/ml and 6.0×10^?5mg/ml.     

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.     

Piezoelectric Crystal Biosensor System for Detection of Escherichia Coli

Abstract

A piezoelectric crystal biosensor system was applied to the detection of Escherichia coli. the system consists of an oscillator, a frequency counter, a flow cell and a modified piezoelectric crystal. Anti-E. coli antibody is immobilized on the surface of the crystal. It is used as an E. coli detection by measuring its resonant frequency shift due to a mass change caused by specific binding of the micro organisms to the surface. the frequency shift correlates with an E. coli concentration in the range of 10⁶?10⁸ cells·cm^?3. the resonant frequency shift is increased by further treatment to bind micro-particles modified with anti-E. coli antibody. This method allows us to improve the determination limit to 10⁵ cells · cm^?3.     

Determination of Albumin in Urine by a Quartz Crystal Microbalance Label-Free Assay

Albumin is a vital plasma protein in the control of osmotic pressure of human blood and is used as a screening marker for renal disease and Type I and Type II diabetic mellitus. A mass-sensitive immunosensor based on quartz crystal microbalance was used to determine microalbumin in urine on the antihuman albumin layer which was bonded to the protein A layer on the surface of a silver electrode. The analytical conditions were optimized for protein A and antihuman albumin on the electrode, and a calibration curve and the correlation between the microalbumin measured by the immunosensor and by a conventional method were evaluated. The results demonstrated that the optimal conditions for protein A coating on the silver electrode were 1?mg/ml and 2?h incubation at room temperature. The optimal conditions for antihuman albumin on the protein A-modified surface were 0.5?mg/ml at 2?h. The dose-response curve was linear from 0.0001 to 0.1?mg/ml. The comparison for the determination of microalbumin in urine by this method and a conventional approach showed good correlation with R²?=?0.999 (P?<?0.01). The receiver operating characteristic curve analysis showed 98.7% sensitivity, 100% specificity, and 1.00 for the area under the curve for the 0.02?mg/ml cutoff value. This system was label free and easy to use with high sensitivity and specificity. Therefore, the system may be used in place of commercial methods in the clinical laboratory.     

Use of A Novel Acetone-H2O2−C10− Chemiluminescence System for the Determination of Iodide Ion

Abstract

The present paper reports a new chemiluminescence system, i.e, acetone-H₂O₂?C10^?, which can be catalyzed by iodide ion (I^?). Based on this catalysis, a new chemiluminescence method for the determination of trace iodide ion is proposed. the optimum conditions are reported in this note. the detection limit is 2 × 10^?11 g/ml I^?, the linear dynamic range is 4 × 10^?10 g/ml to 3 × 10^?7 g/ml I^?, and the variation coefficient at an iodide concentration of 5 × 10^?9 g/ml I^? (n=10) is 4.6%. the method has been satisfactorily applied to the determination of trace iodide ion in water.     

Study on a Fluorometric Method for the Determination of Protein in Serum Using Quercetin‐Lanthanum (III)‐Sodium Dodecyl Benzene Sulfonate‐Protein System

Abstract

It was found that the fluorescence intensity of lanthanum (III) (La³⁺)‐quercetin (Qu) complex is greatly enhanced by proteins in the presence of sodium dodecyl benzene sulfonate (SDBS). Based on this finding, a new fluorimetric method for the determination of proteins was developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins in the range of 2.5×10^?8 to 1.0×10^?5 g/mL for bovine serum albumin (BSA), 5.0×10^?8 to 1.5×10^?5 g/mL for human serum albumin (HSA), and 1.0×10^?7 to 1.5×10^?5 g/mL for egg albumin (EA). Their detection limits (S/N=3) are 5.0×10^?9 g/mL, 7.0×10^?9 g/mL, and 2.1×10^?8 g/mL, respectively. The interaction mechanism was also studied.     

Preconcentration of Volatile Hydrocarbons as Air Pollutants by C-18 Bonded Silica used for Solid Phase Extraction

ABSTRACT

C₁₈-Silica used for Solid-Phase Extraction exhibits the same degree of adsorption of volatile hydrocarbons as compared to conventional Tenax adsorbent. The vapor pressure of the hydrocarbons and the velocity of the air sample through the sorbent are dominants of the preconcentration. Recovery of 80% for n-hexane and 98% for p-xylene at a concentration of 10 mg.m^?3 was obtained at 10 ml.g^?.min^? velocity.

C₁₈Bond Elut cartridges have been successfully used for quantitative determination of hydrocarbons as air pollutants by gas chromatography. The detection limit for p-xylene using preconcentration from 1 L air sample and a S/N ratio of 5 was 0.1 mg.m^?3. After regeneration of C₁₈Bond Elut cartridges by washing with acetonitrile and diethyl ether and drying at 85°C/15 min, their preconcentration potential remain acceptable upon reusing at least three times.     

Spectrophotometric Method for the Estimation of Vitamin C in Drug Formulations

Abstract

The use of 1-chloro-2, 4-dinitrobenzene is described for spectrophotometric estimation of ascorbic acid. The procedure is based on the interaction of ascorbic acid with 1-chloro-2, 4-dinitrobenzene in alkaline medium. The product absorbs maximally at 380 nm and has the molar absorptivity 0.14 × 10⁴1 mole^?1cm^?1. Beer's law is obeyed in the concentration range 0.12–0.6 mg/10ml of ascorbic acid.     

1-[(2-Hydroxy-phenylimino)-methyl]-naphthalen-2-ol: application in detection and adsorption of aluminum ions

A novel “on–off” Al³⁺ ions fluorescence-enhanced sensor (E)-1-(((2-hydroxyphenyl) imino)methyl)naphthalen-2-ol (AH-2) and its hydrogel hybrid (PAMN) were synthesized. AH-2 showed excellent selectivity and ultrasensitive to Al³⁺ ions; the detection limit was 2.36?×?10^–9 M. The most plausible complexation mechanism was studied by ¹H NMR, FT-IR, HR-MS, Job’s plot and theoretical calculation. And, it was interesting that PAMN could adsorb Al³⁺ ions with a removal rate of over 99%, which also could easily be distinguished by the naked eye in UV lamp (365 nm). Before and after adsorption of Al³⁺ ions, the microstructures of PAMN were analyzed by scanning electron microscope and X-ray energy spectrometer. The silica gel detect plates prepared in this work could rapidly and conveniently detect Al³⁺ ions with a concentration greater than 5?×?10^–6 M (0.13 mg/L) in aqueous solution, and the detection concentration (0.13 mg/L) was lower than the national standard concentration of Al³⁺ ions (0.2 mg/L) in city tap water of china.

     

A Label Free Electrochemical Nanobiosensor Study

Abstract

Nano-porous silicon (PS) is an attractive material for incorporation into biosensors, because it has a large surface area combined with the ability to generate both optical and electrical signals. In this paper, we describe a label-free nanobiosensor for bovine serum albumin (BSA). Nano-porous silicon produced in our laboratory was functionalized prior to immobilization of anti-BSA antibody on the surface. Reaction with BSA in phosphate buffered saline (PBS) buffer resulted in an impedance change which was inversely proportional to the concentration of the analyte. The system PBS buffer/antigen-antibody/PS constitutes an electrolyte-insulator-semiconductor (EIS) structure, thus furnishing an impedance EIS nanobiosensor. The linear range of the sensor was 0–0.27 mg mL^?1 and the sensitivity was less than 10 µg mL^?1.     

Zinc-Selective Membrane Electrode Based On 5, 6, 14, 15-Dibenzo-l, 4-Dioxa-8, 12-Diazacyclopentadecane-5, 14-Diene

ABSTRACT

A new PVC membrane for zinc ions based on DBDA15C4 as membrane carrier was prepared. The electrode shows a linear stable response over a wide concentration range (5.0 × 10^?5-1.0 × 10^?1 M) with a slope of 22 mV/decade and a limit of detection of 3.0 × 10^?5 M (2.0 μg/ml). It has a very short response time of less than 5 s and can be use for at least 11 months without any divergence in potential. The zinc ion-selective electrode exhibited very good selectivities with respect to alkali, alkaline earth and some transition and heavy metal ions and could be used in a pH range 1.5-7.0. It was successfully applied for the direct determination of zinc in a pharmaceutical sample and, as an indicator electrode, in potentiometric titration of Zn²⁺ ions.     

Acid Phosphatase Assay with a Wireless Magnetoelastic Biosensor

Abstract

A wireless remote‐query disposable magnetoelastic (ME) biosensor was developed for the assay of acid phosphatase (ACP). The sensor was fabricated by applying a magnetoelastic ribbon with a layer of pH‐sensitive polymer and upon it a sensing film containing bovine serum albumin (BSA) and adenosine‐5′‐monophosphate (5′‐AMP). The ACP‐catalyzed hydrolysis of 5′‐AMP decreases the solution pH, resulting in the polymer shrinking and consequently the resonance frequency of the magnetoelastic sensor increasing. The kinetic parameters were measured to be 1.64×10^?3 M (Michaelis constant) and 130 Hz/min (maximum initial rate). The proposed sensor can determine 0.2 to 1.2 U/ml of ACP.     

Determination of Diethylstilbestrol by Time-resolve Fluoroimmunoassay

Abstract

A time-resolved fluoroimmunoassay (TRFIA) was developed for the determination of diethylstilbestrol (DES). The method was based on a competitive immunoassay using europium-labeled anti-DES antibody and DES-bovine serum albumin (DES-BSA) as coated antigen. The TRFIA exhibited a typical response for DES at concentrations of 0.001–100 ng · mL^?1, the linear correlation coefficient is 0.9933, and the detection limit (LOD) is 0.595 pg · mL^?1. Some serum and water samples have been analyzed by using this method with satisfactory results. Compared with the routine fluorescence immunoassay (FIA), this method was more sensitive. The TRFIA may offer a valuable alternative method for the DES detection and could be applied to routine analysis.     

Construction and Characterization of an Anti‐Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Abstract

A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrP^c). After five rounds of panning against recombinant bovine prion protein (rb‐PrP^c) and ELISA test, two positive clones with high affinity to rb‐PrP^c, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrP^c were 3.24×10^?8 M, 8.82×10^?8M, and 8.10×10^?9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrP^c with detection limit of 1 pg/ml.     

Calorimetric Determination of Phosphorylated Serum Cholinesterase and Its Reactivation by an Antidote

Abstract

Serum cholinesterase, inhibited in vitro up to 92% by 0, 0 dimethyl-2, 2-dichlorovinyl phosphate (4.52 × 10^?9 mole/ml of 40% serum), was reactivated up to 35% with 2-pyridine aldoxime methiodide (3.5 × 10^?6 mole/ml of 40% serum). Evaluation was based on the reaction rate between cholinesterase and acetylcholine chloride (2.2 × 10^?6 mole) measured with a microcalorimeter. It was thus possible to approximate the “normal” or pre-exposure level of serum cholinesterase, while simultaneously determining the extent of poisoning.     

Umbelliferone Phosphate as a Substrate for Acid and Alkaline Phosphatase

Abstract

The results of a complete study of 8 substrates for acid and alkaline phosphatase indicated 7-hydroxycoumarin (umbelliferone) phosphate to be the best substrate for the analysis of these enzymes. Using this ester, from 10^?6 to 2 × 10^?2 units per ml. of alkaline phosphatase and 10^?5 to 0.06 units per ml. of acid phosphatase can be determined with an accuracy and precision of about 1.5%. Samples of serum as small as 1 μ;1. can be assayed. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Determination of 19-nortestosterone residues in aquaculture tissues by enzyme-linked immunosorbent assay and comparison with liquid chromatography and tandem mass spectrometry

19-Nortestosterone (17β-NT) was oximated by carboxymethoxylamine and then coupled with bovine serum albumin (BSA) in a mixed-anhydride reaction in order to produce an antibody. The conjugate rate of 17β-NT and BSA was estimated to be 24 by ultraviolet spectrophotometry. Polyclonal antibody of 17β-NT was acquired from the animal immunized with the conjugate. Through an indirect enzyme-linked immunosorbent assay (ELISA), which demonstrated that the synthesis of immunogen was successful, the titre of antiserum was found to be 6.4?×?10⁵. Based on the purified antibody, a competitive indirect ELISA was developed. ELISA revealed that the limit of detection (LOD) was 0.07?ng?g^?1, the recovery (in edible tissues) was 71–89%, and the working range was 0.05–31.25?ng?g^?1. The preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy revealed that this ELISA method could be used in the practical detection of 17β-NT in tissue samples. Moreover, this method was compared with high-performance liquid chromatography tandem mass spectrometry, for which the transition for quantification of 17β-NT was 275.4/109.1.     

Development of a Flow Immunoassay System for the Detection of Salmonella Typhimurium

Abstract

A flow immunoassay system has been developed for the detection of Salmonella typhimurium by immobilization of antibody to Salmonella onto tygon tubing by adsorption and onto polyethylene tubing, by covalent binding. After liquid containing Salmonella cells or Salmonella antigen was allowed to interact with the immobilized. antibody, glucose oxidase labeled antibody to Salmonella was injected to the system. The bound antigen was quantified by electrochemical detection of H₂O₂ produced from the enzymic oxidation of glucose to gluconic acid. In view of the low non-specific binding, sensitivity and reusability, the polyethylene immunoreactor provided a better performance. The system was capable of detecting as low as 10⁵ ? 10⁶ Salmonella cells/ml in less than 10 minutes. The immunoreactor could be regenerated for at least 50 times during one month of the experiment. The detection limit of the tygon immunoreactor was somewhat higher (10⁶ ? 10⁹ cells/ml). Even though the anti-Salmonella antibody was easily immobilized on the tygon tubing, the system was not reusabl and exhibited a very high non-specific binding.