An impedance biosensor was designed, fabricated and tested for detection of viable Escherichia coli O157:H7 in food
samples. This device consists of interdigitated microelectrode array (IDEA) fabricated using thin layer of sputtered gold,
embedded under a polydimethylsiloxane (PDMS) microchannel. The array of electrodes is designed to detect viable EColi
in different food products. The active surface area of the detection array was modified using goat anti-E.coli
polyclonal IgG antibody. Contaminated food samples were tested by infusing the supernatant containing bacteria over
the IDEA's, through the microchannel. Antibody-antigen binding on the electrodes results in impedance change. Four
serial concentrations of E.coli contaminated food samples (3x102 CFUmL-1 to 3x105 CFUmL-1) were tested. The
biosensor successfully detected the E.coli samples, with the lower detection limit being 3x103 CFUmL-1 (up to 3cells/μl).
Comparing the test results with an IDEA impedance biosensor without microchannel (published elsewhere) indicates that
this biosensor have two order of magnitude times higher sensitivity. The proposed biosensor provides qualitative and
quantitative detection, and potentially could be used for detection of other type of bacteria by immobilizing the specific
type of antibody.
Glucose binding protein (GBP) is a monomeric periplasmic protein. It is synthesized in the cytoplasm of
Escherichia coli which functions as a receptor for transport D-glucose. GBP binds glucose with high affinity. The
binding mechanism is based on a hinge motion due to the protein conformational change. This change was utilized as an
optical sensing mechanism by applying Fluorescence Resonance Energy Transfer (FRET). The wild-type GBP lacks
cysteine in its structure, but by introducing a single cysteine at a specific site by site-directed mutagenesis, this ensured
single-label attachment at specific sites with a fluorescent probe. The other sites were amino sites, which were labeled
with second fluorophore. The near IR FRET pair, Alexa Fluor 680 (AF680) and Alexa Fluor 750(AF750), was utilized.
The AF680 targeted the amine sites, which was the donor fluorophore, while the AF750 labeled the single cysteine site,
which was the acceptor fluorophore. The sensing system strategy was based on the fluorescence changes of the probe as
the protein undergoes a structural change upon binding. This biosensor had the ability to detect down to 10 uM
concentrations of glucose. Next the probes were uploaded into red blood cells via hypo osmotic dialysis. The sensor
responded to glucose while encapsulated with the red cells. These results showed the feasibility of an intracellular
glucose biosensor.
Conference Committee Involvement (2)
Smart Biomedical and Physiological Sensor Technology IX
26 April 2012 | Baltimore, Maryland, United States
Smart Biomedical and Physiological Sensor Technology VIII
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.