Biology Reference
In-Depth Information
Not all bacteria are pathogenic or harmful to humans. Some
microorganisms are harmless or even some are very useful for
human beings. An example is the lactobacilli in human stomach
that helps in converting lactose and other sugars to lactic acid.
However, these bacteria will cause disease if they are detected in
environments that are not their normal habitat. Thus, the presence
of certain bacteria out of their normal habitat is an indicator of a
certaindiseaseorcontamination.Forexample, Enterococcus species
is used as an indicator of fecal pollution in environmental waters,
while the detection of species-specific Enterococcus faecium is used
as an indicator of human fecal pollution [13]. On the other hand,
thepresenceofsomebacteriaalmostcertainlyindicateaninfection;
for example, Mycobacterium tuberculosis causes tuberculosis, and
Streptococcus and Pseudomonas cause pneumonia.
An enzyme-based genosensor for amperometric detection of
PCR amplicons on screen-printed carbon (SPC) chips was recently
described in Ref. 8. The SPCs were pretreated with streptavidin
before each experiment. Covalent agent (200 mM 1-ethyl-3-
[3-dimethylaminopropyl]carbodiimide and 50mM N -hydroxy-
succinimide prepared in 0.05 M phosphate buffer) was added to the
working electrode of the SPC and incubated at room temperature.
The electrodes were washed by dipping them once in deionized
water. Streptavidin (0.05 mg/mL) was then pipetted onto the
working electrode again to form a meniscus and incubated at room
temperature. The electrodes were washed by dipping them once in
deionized water (a schematic diagram is shown in Fig. 15.1A). The
unbound area on the streptavidin-treated SPC reservoir area was
blocked with1 M ethanolaminechloride.
ThePCRampliconswerecapturedontheelectrodesanddetected
using a portable pulse amperometric reader (AndCare, Durham,
NC). A schematic diagram of the detection process is shown in Fig.
15.1B. Briefly, the biotin- and fluorescein-labeled PCR amplicons
were diluted with an equal volume of 0.05 M phosphate buffer,
and the diluted PCR amplicons were applied to the surface of the
working electrode for 5 min. During the incubation step, the biotin-
labeled strands of the PCR amplicons were specifically captured
on the streptavidin precoated working electrode. The excess PCR
amplicons were removed by dipping the electrode 10 times into
 
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