2002;4:425C432. with this paper), the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein connection, which has the potential for providing a practical alternate for the monitoring of O157:H7 and additional pathogens. O157:H7 1.?Intro In recent times, O157:H7 as one of the major foodborne pathogenic bacteria and as such has attracted considerable attention. According to the U.S. Center for Disease Control and Prevention (CDC), outbreak data and the known ability of the organism to be passed from person to person in nursing homes, day-care centers, and additional personal care facilities, indicate that the presence of as few as 10 O157:H7 could result in disease. It has been reported that there may be about 73,000 infections and 61 deaths occurring due to O157:H7 each year in the United States [1] and therefore it is of utmost importance to develop quick and sensitive methods for O157:H7 detection. By far, the most popular detection methods are tradition and colony counting methods, polymerase chain reaction (PCR) and immunology-based methods and biosensors [2C6]. However, they may be labor rigorous and time consuming or professional operation limited. Biosensors, which incorporate a bioreceptor closely integrated with or connected to a transducer [7,8], have been proved to be a promising method for bacteria detection because of the portability, speed, level of sensitivity and possibility of on-the-spot detection [6,7,9], Surface plasmon resonance (SPR) biosensors are one kind of biosensor that has been widely used for bacterial detection [10C14]. A large number of direct SPR immunosensors have been utilized for the detection of bacterial cells by immobilizing antibodies directly on the chip surface to capture the bacterial cells [15C20]. Mazumdar immobilized antibodies Calpain Inhibitor II, ALLM on platinum surface of each glass prism to capture using the Plasmonic? SPR assay having a detection limit of 1 1.25 105 cfu/mL [20]. Subramanian reported the detection limit of direct surface plasmon resonance assay for O157:H7 detection was 106 cfu/mL [17]. The method based on the surface capture of cells offers some limits to reduce the level of Calpain Inhibitor II, ALLM sensitivity of immunosensors [21C24]. Firstly, the effective penetration depth of the evanescent field which occurs under conditions of total internal reflection is approximately 300 nm. It means that only refractive index changes occurring within the 300 nm range from the surface will cause a change in the generated SPR transmission. Bacteria such as O157:H7 with size of about 1 m probably only interact with the top of the dextran coating that coats the platinum surface and therefore only a small portion of the cell which is in close contact with the sensor surface will produce a measurable transmission, which decreases the level of sensitivity of SPR for O157:H7 detection [21C24]. In addition, due to the large size of bacterial cells, direct cell binding requires the cell-antibody binding affinity must be high to withstand the effect of shear push created from the laminar circulation in the microflow channels [24]. Finally, Biacore tools average the SPR angle over an area of Calpain Inhibitor II, ALLM approximately 0.25 mm2 within the sensor surface. As the sizes of bacterial cells are large, they will not equally cover the area measured due to steric hindrance, which will decrease the transmission response [24]. With this paper, to avoid the problems of SPR detection due to the size of bacteria, a new subtractive inhibition assay using SPR detection of O157:H7 was developed. In the proposed assay, O157:H7 cells and antibodies were incubated for a short of time, and the O157:H7 cells which bound antibodies were eliminated by a stepwise centrifugation process. Then the remaining free unbound antibodies were quantified through binding with anti-antibody immobilized within the sensor chip using BIAcore 3000 biosensor, which were inversely proportional to the O157:H7 cell concentration (Number 1). This method simplifies bacterial cells detection to protein-protein connection, which increases level of sensitivity of SPR for O157:H7 detection. Calpain Inhibitor II, ALLM Open in a separate window Number 1. Principle of a subtractive inhibition assay (SIA) using SPR. 2.?Experimental Section 2.1. Regents The reagents were obtained from the following sources: goat polyclonal antibody for O157:H7 and O157:H7 positive control were from KPL BSG (Gaithersburg, MD, USA); DH5 (ATCC PTA-3137) was from College of Food Technology at Zhejiang University or college; Rabbit anti-goat IgG polyclonal antibody was from Boster (Wuhan, China); Sensor chip CM5, HBS operating buffer (10 mM HEPES, 150 mM NaCl, 3.8 mM EDTA, 0.05% (v/v) Tween), 10 mM acetate buffer (pH 4.5) and 1 M ethanolamine Calpain Inhibitor II, ALLM (pH 8.5) were from GE Healthcare Bio-Sciences AB (Uppsala, Sweden); Phosphate buffered.
