No abstract

An automated, disk-based, enzyme-linked immunosorbent assay (ELISA) system is presented in this work. Magnetic beads were used as the antibody carriers to improve the assay sensitivity and shorten the reaction time. The magnetic module integrated on the system is capable of controlling the magnetic beads to either move in the incubation stage or immobilize at a specific location during washing stage. This controlling mechanism utilizes a passive controlling approach so that it can be performed through disk spinning without the need of active control from external devices. The movement of the magnetic beads was investigated and the optimal rotational speed was found to be related to the ratio of the processing time to the cycle time of the magnetic beads. Comparing to ELISA conducted on microtiter plates, similar test results could be achieved by the disk-based ELISA but the entire protocol can be finished automatically within 45 min with much less reagent consumption. V C 2014 AIP Publishing LLC. [http://dx

We developed a procedure for the resolution of esters of amino acids in 95% t-butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t-butanol(95%) and water (5%) at 25"C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L-amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to sapomfy the unreacted ester. The D-antipode precipitated at pH 6.2-6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. o 1994 Wiey-Liss, Inc. KEY WORDS: resolution, D,L-&Oacid esters, alkaline protease, simple separation, high enantiomeric excess and yieldsThe search for proteases for peptide synthesis that are stable in organic solvents is the subject of extensive investigation. 's2 Several studies have demonstrated the possibility of using proteases to catalyze peptide synthesis in organic solvents, but a drawback of those reactions is the poor stability of the enzyme in organic solvents. Many immobilization processes have been developed to overcome this. Here we describe the use of an inexpensive enzyme, alcalase, as a catalyst for resolution of amino acids, some of which have uncommon side chains and are found in many biologically and pharmaceutically important peptides. 35 Most of these unnatural amino acids cannot be obtained by fermentation or recombinant DNA technology. Resolution is an effective way to produce optically pure unnatural amino acids.Alcalase is a proteolytic enzyme prepared from a selective strain of Bacillus lichenifomis. The major enzyme component in alcalase is subtilisin Carlsberg (alkaline protease A), which is a serine protease and which is widely used as additives in detergents as a digesting enzyme. This alcalase can maintain enzymatic activity and stability in organic solvents.6v7 Application of alcalase in organic synthesis has been limited, although it is a potent and inexpensive catalyst.'*' Scheme 1 shows the reaction sequence of the resolution. The hydrolysis catalyzed by alkaline protease was conducted in a mixture of t-butanol (95%) and water (5%) at 25°C with a pH controller at pH 8.5. The hydrolyzed amino acid was insoluble under these conditions and precipitated during the course of hydrolysis. The precipitate was separated by filtration, and the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester of the D-&O acid.