Detection system for reaction-rate analysis in a low-volume proteinase-inhibition assay.

High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-microL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-microL level and detected in 2-microL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore (N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 microL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 microL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC50) for actinonin of 73 +/- 16 and 100 +/- 14 nM were obtained in the 2- and 200-microL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.

Quantitative Evaluation of SERS-Active Ag Film Nanostructure by Atomic Force Microscopy.

The reliability of an image analysis algorithm for atomic force microscopy (AFM) of thin metal films was evaluated by comparison with manual analysis of images and transmission electron micrographs of Ag films deposited on Formvar-coated Cu grids. In order to extract quantitative nanostructural information using the algorithm discussed herein, the optimal fitting parameters were found to be low-pass filtering to reject high-frequency noise, a 5 × 5 point grid for identification of particle maxima, and a linear least-squares fit to a hemispheroidal model of particle shape. Metal particle dimensions were defined from the height and radius of the hemispheroid fit. Due to the close spacing of particles in these Ag films, tip geometry causes the greatest error in the height measurements, rather than width measurements. In addition, the effect of scanning parameters such as scan rate and size, applied load, and humidity on particle count and dimensions was examined. Increasing the scan rate reduced the number of resolvable Ag particles, decreased the apparent particle height, and increased the apparent particle radius. Under conditions of low capillary force, a net repulsive force of ∼19 nN resulted in subtle tip-induced changes in the Ag surface morphology. The Ag film surface was damaged at a net repulsive force of ∼23 nN. At slow scan rates, the moisture layer did not significantly affect the quality of the AFM images obtained over a broad relative humidity range. Finally, the Ag surface structure was found to be very homogeneous over a relatively large area.

Psoralen-olefin photoproducts: first observation of a photo-ene reaction.

Methyl-substituted psoralens (4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and 4,5',8-trimethylpsoralen) are found to yield an ene product as well as the expected [2 + 2] cycloaddition product from photochemical reaction with simple olefins. As determined by absorbance, liquid chromatography-mass spectrometry and nuclear magnetic resonance, both products are formed at the pyrone side of the respective psoralen. The product distribution is dependent on olefin concentration as well as the nature of the olefin. In deoxygenated solutions, cyclic olefins form as much as 50% ene product, while unsubstituted straight-chain olefins form as little as 3%. In oxygenated solutions, the product distribution is strongly affected by singlet oxygen.