BCR Diagnostics

BCR DiagnosticsBacterial 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

BCR Diagnostics

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.

BCR 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.

BCR 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 Engineeredas

Fluorogenic Nanodetectors

Bacteria

Response of Engineered Spores to Germinants

0

5,000

10,000

15,000

20,000

0 50 100 150 200 250

Time (seconds)

Schematic of 80K-bioChip[Cross Section]

Micro-colander (5 pL volume)

Filter membrane (0.2 µm pore)

Spores

Silicon layer

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

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Biosensor Operation

Platelet sample is processed to separate bacteria, and theresulting 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

BCR Diagnostics

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

Photomicrographs of 80K-bioChip

Left: Normal light Right: Fluorescence light

BCR 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

BCR 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: 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