Transcript Biosensors for rapid pathogen detection

Cell-Based Biosensors for Rapid
Pathogen Detection
Ivica Arsov, Ph.D.
Biosensor
Integrated receptor-transducer device
Convert biologically-induced recognition event (e.g.,
enzyme, antibody) into a detectable signal via a
transducer and processor
Usually displays both the presence and concentration of
target analytes
Key Biosensor Components
Transducer
Analyte
Bioreceptor
Enzyme
Microorganism
Antibodies
Chemoreceptors
Tissue
Organelles
Nucleic acids
Electrochemical
(clark electrode, ion
sel. electrode, etc)
Optical
(absorbance,
luminescence,
fluorescence, etc)
Piezoelectric
(quartz crystals,
surface acoustic
wave device, etc)
Calorimetric
(thermometric)
Recording
device
Cell-Based Biosensors
• B cell lines were genetically engineered to
express cytosolic aequorin, a calcium sensitive
bioluminiscent protein from jellyfish, as well as
membrane-bound antibodies specific for
pathogens of interest (Rider et al., 2003)
• Cross-linking of the antibodies with specific
pathogens increases intracellular calcium
concentrations within seconds, causing the
aequorin to emit light.
• This sensor was named CANARY (cellular analysis
and notification of antigen risks and yields)
Cell-Based
Biosensor
Analytes
Surface
Immunoglobulin
Data analysis
Signaling cascade
Ca++
Aequorin
5
Excellent Speed, Sensitivity, and
Specificity
• The CANARY Cell-based sensor detected as
few as 50 CFU of Y. pestis in a total assay time
of 3 minutes.
• The system was also tested in one food
sample-E. coli O157:H7 in lettuce
• 500 CFU/g of E. coli O157:H7 in lettuce was
detected in less than 5 minutes, including the
sample preparation.
• 1000 CFU of B. anthracis spores
Cell-Based Biosensors
• The CANARY system can be easily custom
tailored to respond to specific strains.
• It can distinguish pathogenic E. coli O157:H7
from nonpathogenic E. coli strains.
www.innovativebiosensors.com
Biosensor Based on Immobilized Indicator Cells
• A B-lymphocyte cell line was encapsulated in a
collagen gel matrix (Banerjee et al., 2007).
• This assay measures alkaline phosphatase or
lactate dehydrogenase released by cells
infected with pathogens or exposed to
different toxins.
• The system was tested using different strains
of Listeria, listeriolysin O, and enterotoxins
from Bacillus species.
Banerjee et al., Laboratory Investigation
(2007) 1-11. Reproduced with permission.
Biosensor Based on Immobilized
Indicator Cells
• The assay provides quantitative and
qualitative information
• Very fast (1-6 hours)
• Good sensitivity (MOI>10:1)
• Portability-collagen entrapped cells remain
viable in 48 well plates for 48 hours.
The cryo-SEM images of pathogen- or toxin-induced damage of
Ped-2E9 cells in collagen gel matrix.
FROM:
A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a
biosensor for rapid detection of pathogens and toxins (2007)
Pratik Banerjee, Dominik Lenz, Joseph Paul Robinson, Jenna L Rickus and Arun K Bhunia
Laboratory Investigation (2007) 1-11.
Reproduced with permission
Future…
• Only few biosensors were tested with food
samples so far
• At this point it may not be a cost-effective
technology
• Sample preparation plays a critical role
Future…
• The main advantage of cell-based biosensors
is that they provide information about the
physiological effects of pathogens/toxins.
• Capable of distinguishing between viable
pathogenic strains from nonpathogenic ones
or dead cells.
• Sensitivity and specificity comparable to
current methods.
• Expected to have broad applications in food
testing, animal health, biodefense, disease
diagnosis etc.
Collaborative Study to evaluate a Total Bacteria Count Assay using
Quantitative Real-Time PCR. Thomas L. Romick and Rafael C. Marfil.
Industrial Microbial Testing and Grupo Sinergia
Introduction A collaborative study was undertaken to demonstrate the
precision and accuracy of a total bacteria count (TBC) assay developed to
run with commercially available PCR reagents and a rapid thermocycling
platform. The TBC assay can be completed within one hour. The TBC assay
has many applications for enhanced HACCP applications.
Purpose This study was designed to show how diverse laboratories can use
the TBC assay in their labs for specific applications. Once a standard curve is
constructed for a sample type, it can be used time again for quantifying
unknown samples.
Methods A collaborator kit was supplied to each lab with all the necessary
reagents and consumables that were detailed on a chain of custody
document.
Collaborating laboratories were: Brigham Young University California
Department of Public Health NASA Jet Propulsion Laboratory NASA Ames
Research Center Nestle USA Quality Assurance Center North Carolina State
University San Francisco Water Quality Division
Results Results from the 7 collaborative labs were analyzed using
ANOVA and Signal to Noise ratio statistics (Taguchi Method) and
showed excellent results for the standard curve and some variation in
the unknown data. The β value indicated how close the average result
was to the target and ranged from 0.87 to 1.07 (1.00 target). The signal
to noise ratio (S/R) which includes the variance and β factor as the
indicator ranged from 14.22 to 23.18 (20.00 target).
Significance The overall conclusions are that all the labs performed
well in constructing the standard curve and had some variance with the
unknown samples. However, the results show that all collaborators
successfully used the TBC assay to quantify bacterial load accurately
within one hour. The TBC assay can be used for a rapid real-time
measure of bacteria in many diverse applications and support HACCP
for real-time statistical process control.