B C R Diagnostics BCR Diagnostics Bacterial Chain Reaction for Biomolecular Amplification Jamestown, Rhode Island - bcrbiotech.com Bacteriological Biosensor for Rapid Screening of Platelets Shortly Before Transfusion Boris Rotman, Ph. D. Vice President and CSO

B C R Diagnostics Technical Rationale Biosensor integrates three technologies: 1. LEXSAS TM (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.

B C R Diagnostics 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.

B C R Diagnostics 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 Germination Signals Analyte + germinogenic buffer SPORE IMAGE ANALYSIS Signal Receptor DATA PROCESSING AND DISPLAY Transduced Fluorescent Signals Spores Engineered as Fluorogenic Nanodetectors Bacteria

Response of Engineered Spores to Germinants

Schematic of 80K-bioChip [Cross Section] Bacterium

SEM image of 80K-bioChip [Micro-colanders ® are 20 µm dia]

SEM image of Micro-colander ® with about 200 Spores

80K-bioChip mounted on filtration cartridge

Micro-colander® Scanner

B C R Diagnostics 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 37 o C 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

B C R Diagnostics 80K-BioChip showing ten (10) micro-colanders with significant fluorescence above baseline

Photomicrographs of 80K-bioChip Left: Normal light Right: Fluorescence light

B C R Diagnostics 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

B C R Diagnostics 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: ,  Rotman, B. and Cote, M. A. Application of a real-time biosensor to detect bacteria in platelet concentrates. Biochem. Biophys. Res. Comm., 300, ,  Rotman, B. Using living spores for real-time biosensing. Gen. Eng. News, 21, 65, *Additional patents listed at