BCR Diagnostics Bacteriological Biosensor for Rapid Screening of Platelets Shortly Before Transfusion Bacterial Chain Reaction for Biomolecular Amplification Boris Rotman, Ph. D. Vice President and CSO Jamestown, Rhode Island - bcrbiotech.com
Technical Rationale Biosensor integrates three technologies: 1. LEXSASTM (Label-free Exponential Signal-Amplification System) A recently developed system using engineered spores as fluorogenic nanodetectors. 2.Microfluidics: The detection system is a unique device containing an array of 80,000 microscopic, independent biosensors, each of which is about 5-picoliter volume. This small reaction volume enables detection of individual bacterial cells. The device is termed “80K-bioChip.” 3. Computerized Parallel Imaging The 80K-bioChip provides high-throughput data acquisition and processing.
LEXSAS™Characteristics • Ability to detect and count low bacterial levels. • Quantitative results in less than 20 minutes. • Cost-effective testing. • Linear detection response over a wide dynamic range of one to 10,000 bacteria per sample. • Low instrumentation cost. • Applicability to automated high-throughput operations.
Why using Engineered Spores for Cell-based Biosensing? • Spores are metabolically dormant microorganisms resistant to extreme chemical and physical environmental conditions. • Despite their ruggedness, however, spores remain alert to specific environmental signals that trigger the spores to rapidly break dormancy and initiate germination. • The spores in the LEXSAS™are engineered to be fluorogenic, i.e., the spores are not fluorescent per se, but produce fluorescent light in the presence of nearby bacteria.
Principle of the Biosensor Analyte + germinogenic buffer Bacteria Germination Signals Signal Receptor SPORE Spores Engineered as Fluorogenic Nanodetectors Transduced Fluorescent Signals IMAGE ANALYSIS DATA PROCESSING AND DISPLAY
Bacterium Schematic of 80K-bioChip[Cross Section]
Biosensor Operation Platelet sample is processed to separate bacteria, and the resulting suspension is mixed with engineered spores Mixture is combined with germinogenic substrate and then filtered through the 80K-bioChip 80K-bioChip is incubated at 37oC Fluorescent images of 80K-bioChip are captured at time intervals Digital images are processed to yield number of bacteria in the sample
Schematic of 80K-bioChip[Top View] Micro-colanders with one bacterium show visible fluorescence
80K-BioChip showing ten (10) micro-colanders with significant fluorescence above baseline
Photomicrographs of 80K-bioChip Left: Normal light Right: Fluorescence light
BCR Diagnostics, Inc. • Privately held since 1994 • Technology protected by five U. S. and multiple worldwide Patents (additional U.S. and ROW pending) • Awarded Phase I and Phase II Grants from the Rhode Island Center for Cellular Medicine • Awarded Phase I and Phase II SBIR Grants from the National Institutes of Health
Publications • Rotman, B., Cote, M. A., and Ferencko, L. Phenotypic Engineering of Spores. U. S. Patent pending. • Rotman, B. Analytical system based upon spore germination. U. S. Patent No.6,596,496, March 29, 2005.* • Ferencko, L., Cote, M. A., and Rotman, B. Esterase activity as a novel parameter of spore germination. Biochem. Biophys. Res. Comm., 319: 854-858, 2004. • Rotman, B. and Cote, M. A. Application of a real-time biosensor to detect bacteria in platelet concentrates. Biochem. Biophys. Res. Comm., 300, 197-200, 2003. • Rotman, B. Using living spores for real-time biosensing. Gen. Eng. News, 21, 65, 2001. *Additional patents listed at www.bcrbiotech.com