Enzyme-linked immunomagnetic electrochemical detection of Salmonella typhimurium.

There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures. We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry. In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody. With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format. Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection. The electrochemical response was directly proportional to the number of captured bacteria. Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca. 80 min.

Ultraviolet difference spectral studies in the gamma radiolysis of DNA and model compounds. I. Aqueous solutions of DNA bases.

Ultraviolet difference spectra of gamma-irradiated, air-saturated aqueous solutions of DNA bases vs. unirradiated solutions of the same bases are shown to be a very sensitive supplemental tool with which to investigate the yields, postirradiation kinetics, and general nature of DNA base radiation products. Irradiated pyrimidines yield difference spectra which are approximately negative mirror-images of the base absorption spectra in the near-UV, indicating loss of ring conjugation. Difference spectra of irradiated purines yield a more complex pattern containing a positive long-wavelength peak, interpreted as radiation-induced extension of conjugation of the pi electron system beyond that of the unirradiated purine. On the basis of the spectroscopic evidence from these studies, 8-hydroxyguanine appears to be the dominant UV-absorbing radiation product in air-saturated guanine solutions with a G-value of 0.3 molec (100 eV)-1. Difference spectral studies provide isosbestic points which can be used in testing proposed radiation products and their yields. Such spectral studies are a rapid, non-invasive, supplemental tool which can be employed in conjunction with other analytical techniques in radiation-chemical studies, and which is one of the few tools able to detect short-lived chemical intermediates observed in oxygenated solutions of irradiated purines.

Use of a light-addressable potentiometric sensor for the detection of Escherichia coli O157:H7.

We describe the development of an immunoligand assay (ILA) in conjunction with a light-addressable potentiometric sensor (LAPS) for the rapid detection of Escherichia coli O157:H7 cells in buffered saline. The ILA protocol consists of "sandwiching" bacterial analyte between biotinylated and fluoresceinated antibodies, indirect enzyme labeling of the bacteria with urease-labeled anti-fluorescein antibody, and active capture of the immune complex at a biotinylated bovine serum albumin-blocked nitrocellulose filter membrane with streptavidin. Using live E. coli O157:H7, the efficiency of the ILA was compared using various ratios of the biotinylated and fluoresceinated antibodies. Simultaneous addition of equimolar biotinylated and fluoresceinated antibodies effected optimal urease labeling and subsequent active capture of the bacteria in the ILA. Equimolar concentrations of the antibodies were varied to achieve optimal LAPS detection response for the live bacteria. Using ILA with LAPS, a minimum detectable level of ca. 7.1 x 10(2) cells/ml of heat-killed or ca. 2.5 x 10(4) cells/ml of live E. coli O157:H7 bacteria was achieved in Tris-buffered saline in an assay time of ca. 45 or ca. 30 min, respectively.

Immunoelectrochemical assays for bacteria: use of epifluorescence microscopy and rapid-scan electrochemical techniques in development of an assay for Salmonella.

Immunoelectrochemical sensors in which the sensor surface functions as both analyte capture phase and electrochemical detector have recently been developed for bacteria analysis. The speed and sensitivity of these devices make them very attractive for applications such as the detection of pathogenic microorganisms in food and water. However, the development and optimization of assays utilizing these sensors can be complicated by undesired interactions between the capture and detection functions. Modification of the sensor to achieve improvements in one function can have deleterious effects on the other function, and such effects can be difficult to diagnose and correct. In the course of investigations on immunoelectrochemical detection of Salmonella, we developed a rapid, nondestructive epifluorescence microscopy method to determine bacteria capture efficiency. This method enabled us to study capture and detection functions independently and efficiently identify performance-limiting factors. Rapid-scan electrochemical methods were used to optimize detection sensitivity and to provide diagnostic information on detection performance.