Biology Reference
In-Depth Information
In the assay described above, the screen-printed carbon chips
were first pretreated with covalent agent [ N -hydroxy succin-
imide (NHS) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide
hydrochloride (EDC)] to immobilize streptavidin on the transducer
surface.Thesurfacewasinactivatedandblockedwithethanolamine
and bovine serum albumin (BSA) to avoid non-specific adsorption
of antibody-HRP-conjugate onto the electrode surface. The labeled
PCRampliconsproducedfromamplificationof V. cholerae DNAwere
captured onto the transducer surface without hybridization (rapid
method).Thebiotinylatedhybridswereboundtostreptavidinonthe
transducer surface and hybridized with antibody-HRP-conjugate.
Addition of the substrate 3,3'5,5'-tetramethylbenzidine (TMB)-
hydrogen peroxide (H 2 O 2 ) resulted in a signal that was detected
using amperometry (IPA)measurement [8].
Signals were produced only with the specific-labeled PCR
ampliconslabeledwiththecorrespondinghaptens.Thebackground
signal was low and negligible compared to the signal produced by
positive samples. The enzyme-based electrochemical genosensor
assay concept has shown promising results in the detection of
various analytes [16, 17, 24]. The combination of horseradish per-
oxidase (HRP)-coupled hybridizationschemes withelectrochemical
biosensors allow highly sensitive detection of targets because the
signal is amplified [18, 24].
15.3 Advantages of the Enzyme-Based Electrochemical
Genosensors in Detecting Bacteria on
Screen-Printed Carbon Chips
Conventionally, PCR amplicons are detected by agarose gel elec-
trophoresis which takes 45 minutes to one hour and the use of
expensive chemicals, such as SYBR Green dye or harmful agents
such as UV light and ethidium bromide. As an alternative method
for PCR amplicon detection, many enzyme-based electrochemical
genosensor assays have been developed and have shown promising
results [16, 18, 25, 26]. Electrochemical DNA hybridization sen-
sors have been reported for pathogens such as Cryptosporidium ,
Escherichia coli , Giardia , Mycobacterium tuberculosis Salmonella
 
Search WWH ::




Custom Search