Grantee Research Project Results

Final Report: A New Biosensor for Rapid Identification of Bacterial Pathogens

EPA Contract Number: 68D02051
Title: A New Biosensor for Rapid Identification of Bacterial Pathogens
Investigators: Tabacco, Mary Beth
Small Business: Echo Technologies Inc.
EPA Contact: Richards, April
Phase: II
Project Period: June 1, 2002 through June 1, 2004
Project Amount: $224,965
RFA: Small Business Innovation Research (SBIR) - Phase II (2002) Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

Echo Technologies, Inc., completed a Phase II research project in which a new type of optical biosensor was developed and demonstrated. The biosensors are rapid, specific, sensitive, and require little sample preparation or user intervention. The approach minimizes the need for culturing to identify and quantify bacterial pathogens. The biosensors are based on the use of fluorescently labeled virus probes (FLVPs). Highly species-specific bacteriophage particles are labeled with a fluorescent probe and selectively interact with their host bacteria. The sensor is monitored using wavelength-specific fluorescence spectroscopy to indicate the presence of the phage/host complex. Application of the fluorescent bacteriophage technology represents a new approach to rapid identification of bacterial pathogens and an important departure from immunoassay- and DNA-based sensing concepts. The sensors under development exhibited a high degree of specificity and sensitivity for the bacterial pathogens of interest. In a prototype rapid bacteria identification system (RBIS), multiple phages can be used for the simultaneous detection of several pathogens using a single analytical instrument. The numerous probes provide inherent sensor redundancy to ensure against false negatives and false positives.

Summary/Accomplishments (Outputs/Outcomes):

The primary technical objectives were met, and some important additional experiments were conducted to demonstrate the potential utility of the sensors. The key technical accomplishments are summarized as follows:

• Rapid response time: identification of bacterial pathogens can be accomplished in less than 30 minutes. The reaction time between FLVPs and host cells is less than 1 minute.


• Excellent sensitivity: the lower detection limit (nonoptimized) is approximately 8,000 bacteria.


• Good specificity: detection of target bacteria is achieved in a biological background of more than 10⁷ bacteria/mL.


• Simplicity: the phages are commercially available and can be easily propagated and prepared. Pathogens can be identified without the need for culturing.


• Cost: FLVPs are easy to prepare and are stable over a long period of time.


• Multi-analyte sensing: FLVPs demonstrated true potential for simultaneous detection of multiple pathogens.


• Matrix interference: FLVP sensors can detect host cells in distilled water, tap water, and seawater.

Conclusions:

These results lay an excellent foundation for a continued development effort. Nucleic acid stained phages (FLVPs) display a unique affinity for host cells in solution. The attachment of FLVPs to host cells is irreversible, fast, and free from matrix interference with the current sensor protocol. The phages are commercially available, and FLVPs are easy to prepare and stable over a long period of time. The identification of host cells removes the need for laboratory culturing. The approach also offers an ideal complimentary sensor technology that could easily be incorporated into existing optical sensor platforms and prototype instrumentation.

Further development would focus on optimization of the FLVP reaction, design and test of a microfluidic handling system, and prototyping of the RBIS. The RBIS would feature multichannel detection capability with improved sensitivity. With modification of RBIS’ data acquisition and data analysis software to incorporate a chemometric classification algorithm, positive identification of multibacteria cells can be achieved.

Supplemental Keywords:

small business, SBIR, EPA, biosensor, rapid identification, bacterial pathogens, fluorescently labeled virus probe, FLVP, bacteriophage, rapid bacteria identification system, RBIS, monitoring, drinking water,, RFA, INTERNATIONAL COOPERATION, Water, Ecosystem Protection/Environmental Exposure & Risk, POLLUTANTS/TOXICS, Ecosystem/Assessment/Indicators, Ecosystem Protection, Monitoring/Modeling, Ecological Effects - Environmental Exposure & Risk, Drinking Water, Engineering, Chemistry, & Physics, Microorganisms, monitoring, cryptosporidium parvum oocysts, pathogens, microelectromechanical systems (MEMS), biosensing, microbial monitoring, waterborne disease, pathogenic microbes, in situ sensor, cryptosporidium , biosensing system, DNA probe, microorganism, DNA, biosensors, biosensor

SBIR Phase I:

A New Biosensor for Rapid Identification of Bacterial Pathogens  | Final Report