A new ITO-based Aß42 biosensor for early detection of Alzheimer's disease.

In this study, a novel label-free impedimetric immunosensor was fabricated for rapid, selective, and sensitive quantitative analysis of Aß42 protein for use in the diagnosis of Alzheimer's disease. The immunosensor was fabricated using inexpensive and disposable indium tin oxide polyethylene terephthalate (ITO-PET) electrodes. After the electrodes were modified with 3-glycidoxypropyldimethoxymethylsilane (GPDMMS), the antibody specific to the Aß42 protein (anti-Aß42) was immobilized. The affinity interaction between anti-Aß42 and Aß42 in the immobilization steps in immunosensor fabrication and in the quantitation of Aß42 were analyzed using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) techniques. Additionally, the morphological changes occurring on the electrode surface during each immobilization step were imaged using scanning electron microscopy (SEM). The linear detection range of the immunosensor was determined as 1-100 pg/mL with the limit of detection value of 0.37 pg/mL. Analytical properties of the biosensor, including reproducibility, repeatability, storage stability, selectivity, and regeneration were investigated. The kinetic behavior of antibody-antigen complex formation was determined for the first time using single frequency impedance (SFI) analysis on an Aß42 biosensor. The potential for use of the immunosensor in clinical studies was demonstrated by analysis of Aß42 in commercially purchased human serum.

A new electrochemical impedance biosensor based on aromatic thiol for alpha-1 antitrypsin determination.

Alpha-1 antitrypsin (A1AT) is one of the acute phase proteins which are synthesized in the liver. A1AT inhibits the activity of many proteases, but its main task is to protect the lungs from the attack of neutrophil elastase. In an autosomal hereditary disease known as alpha-1 antitrypsin deficiency, the A1AT level in blood serum decreases, increasing the risk of developing emphysema, liver apoptosis, and liver cancer. Thus, the detection of A1AT concentration in blood serum is very important. In this study, an impedimetric biosensor was developed, forming an SAM (self-assembled monolayer) with 4-mercaptophenylacetic acid (4MPA) on the surface of the gold electrode. An A1AT biosensor was constructed using immobilization of an A1AT-specific antibody (anti-A1AT) after activating the carboxyl groups of 4MPA with EDC/NHS. Each immobilization stage was characterized by using electrochemical impedance spectroscopy, cyclic voltammetry, and scanning electron microscopy with energy dispersive X-ray spectroscopy. With the designed biosensor, precise and highly reproducible results were obtained for A1AT concentrations in the range of 100-600 µg/mL. A1AT detection was also successfully carried out in artificial serum solutions spiked with A1AT.

A new biosensor for osteoporosis detection.

Osteoporosis is a disease that is characterized by deterioration of bone tissue and increased risk of fracture as it leads to a decrease in bone mineral density, which is an important public health problem. Today, bone mineral density is measured by radiological techniques. Alternative techniques are needed because of the disadvantages such as excessive radiation intake, the cost of radiological techniques, and the necessity for specialist personnel for the devices. The quantitative determination of biochemical markers that play a role in bone mineralization may be a good alternative for the osteoporosis diagnosis and especially in the follow-up of treatment. In this study, a specific and sensitive immunological biosensor, which quantitatively determines the osteocalcin molecule, has been developed to be used in the early osteoporosis diagnosis and to evaluate the response to the drug treatment. Anti-osteocalcin antibody was immobilized onto gold electrode surface via covalent immobilization method by using 6-mercaptohexanol, 1,4-butanedioldiglycidyl ether, ethanolamine, and glutaraldehyde. Immobilization steps and biosensor characterization were specified by cyclic voltammetry and electrochemical impedance spectroscopy. The detection time and range of Ocn biosensor were determined as 45 min and 10-60 pg µL-1 Ocn concentration, respectively. The Ocn biosensor was successfully applied in artificial serum samples spiked with Ocn.

Entrapment of laurel lipase in chitosan hydrogel beads.

Laurel seed lipase was entrapped within chitosan beads with ionotropic gelatin method using tripolyphosphate (TPP) as multivalent covalent counter ion. Immobilization yield was 78%. First, optimum immobilization conditions were determined, and morphology of chitosan beads was characterized by scanning electron microscopy. Optimum pH and temperature were evaluated as 6.0 and 40 °C, respectively. The immobilized beads saved about 55% of its activities at 60° while saved about 32% at 70 °C for 30 min. Vmax/Km values were determined as 31.75 and 2.87 using olive oil as substrate for immobilized beads and free enzyme, respectively. Immobilized beads showed the activities during 30 days at +4 °C.

Comparison of some biochemical properties of artichoke polyphenol oxidase entrapped in alginate-carrageenan and alginate gels.

Polyphenol oxidase (PPO, EC.1.14.18.1) isolated from artichoke (Cynara scolymus) was entrapped within alginate and alginate+ carrageenan beads, and the catecholase and cresolase activities of both entrapped enzymes were determined. Some properties of these immobilized enzymes such as optimum pH and temperature, kinetic parameters (Km and Vmax), thermal, and storage stability were determined and compared to each other. The highest catecholase activity was observed in alginate gel (370 U/g bead) while the highest cresolase activity was in alginate+ carrageenan gel (90 U/g bead). For catecholase and cresolase activities, optimum pHs of alginate and alginate+ carrageenan beads were determined to be 7.0 and 4.0, respectively. Optimum temperatures for catecholase activity were determined to be 40°C for both entrapped enzymes. These values for cresolase activity were 30°C and 20°C, respectively. Immobilized artichoke PPOs greatly preserved their thermal stability which exists anyway. The catalytic efficiency value (Vmax/Km) of the alginate beads is approximately high as two-and-a-half folds of that of alginate+κ-carrageenan beads for cresolase activity. These values were very close for catecholase activity. Immobilized beads saved their both activities after 30 days of storage at 4°C.

Optimization of polyphenol oxidase immobilization in copper alginate beads.

Polyphenol oxidase (PPO, EC 1.14.18.1) was isolated from artichoke head (Cynara scolymus L.) by using 0.1 M Tris-HCl buffer (pH 7.0), concentrated by (NH4)2SO4 precipitation, and immobilized in copper-alginate beads. Immobilization yield was determined to be 70%. The cresolase and catecholase activities of enzyme immobilized at optimum immobilization conditions were found to be 13.3 and 670 U g beads min(-1), respectively. Effects of immobilization conditions such as alginate concentration, CaCl2 concentration, amount of loading enzyme, bead size, and amount of beads on enzymatic activity were investigated. Optimum alginate and CuCl2 concentration were found to be 2 % and 3 % (w/v), respectively. Using bead (diameter 3 mm) amount of 0.25 g maximum enzyme activities were observed for both polyphenol activities. The initial concentrations of loading free enzyme were 6.5 U mL(-1) and 5815 U mL(-1) for cresolase activity and catecholase activities, respectively. Beads prepared at optimum immobilization conditions were suitable for up to 8 repeated uses.

Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads.

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.

Optimization of alpha-amylase immobilization in calcium alginate beads.

alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C.

Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation.

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.

Some biochemical properties of polyphenol oxidase from celery.

Polyphenol oxidase (PPO, EC 1.14.18.1) was extracted from celery roots (Apium graveolens L.) with 0.1 M phosphate buffer, pH 7.0. The PPO was partially purified by (NH4)2SO4 and dialysis. Substrate specificity experiments were carried out with catechol, pyrogallol, L-DOPA, p-cresol, resorcinol, and tyrosine. The Km for pyrogallol, catechol, and L-DOPA were 4.5, 8.3, and 6.2mM, respectively, at 25 degrees C. Data for Vmax/Km values, which represent catalytic efficiency, show that pyrogallol has the highest value. The optimum pH and temperature were determined with catechol, pyrogallol, and L-DOPA. Optimum pH was 7.0 for catechol and L-DOPA, and 7.5 for pyrogallol. Optimum temperatures for maximum PPO activity were 25 degrees C for pyrogallol, 40 degrees C for catechol, and 45 degrees C for L-DOPA. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 60 degrees C. The order of inhibitor effectiveness was: L-cysteine > ascorbic acid > glycine > resorcinol > NaCl.