<p>The presence of <i>Escherichia
coli</i> in water is an environmental indicator that the water is contaminated
with faeces. Approximately, 30% of the world population drink water from
sources contaminated with human faeces. Consequently, this percentage comprises
of people that are highly vulnerable to <i>Escherichia
coli</i> infection. While most strains of <i>Escherichia
coli</i> are harmless or maintain a symbiotic relationship with humans, the
pathogenic strains are responsible for injurious health effects, such as
diarrhoea and kidney failure. The traditional method of detecting <i>Escherichia coli</i> takes about 24 – 48
hours, does not detect viable but non-culturable cells, and requires advanced
equipment and great technical skills. Most other available detection techniques
lack specificity, as observed with enzyme-based techniques, or are not very
sensitive, as observed with most impedance-based techniques with clogged
surfaces.</p>
<p> </p>
<p>As a result of the health effects due to this
microorganism and the basic limitations of available detection techniques,
there is need for a specific, sensitive and rapid detection technique to ensure
a sustained and timely access to <i>E. coli</i>-
free water. Therefore, the aim of this research work is to develop a detection
technique devoid of the basic limitations of available methods. In this study,
the antibody-antigen relationship was taken advantage of to ensure the
specificity of the technique is guaranteed. This was achieved using <i>Escherichia coli</i> polyclonal antibodies
that target the O and K antigens found in most pathogenic strains. These
antibodies were functionalized on carboxyl group modified superparamagnetic
fluorescent microparticles immobilized with streptavidin. The sensitivity of
the technique was ensured by utilizing the low detection limit feature offered
by the use of microfluidic devices. Two microfluidic devices, glass-based and
PDMS-based, were fabricated with easily accessible materials. </p>
<p> </p>
<p>On introducing the sample reagents and test samples
into the microfluidic devices, and passing an alternating current frequency
through the system, the antibodies specifically isolated the target organisms
from the pool of water contaminants and a drop in the device electric potential
proportional to the bacteria concentration was observed. The success of this
procedure depends on the identification of the alternating current frequency
beyond which manipulation of the samples would not be easily carried out. As a
result, the flow field analysis of the microparticles was carried out to study
the particle behavior by varying the alternating current frequency from 15 kHz
– 75 kHz. </p>
<p> </p>
<p>The optimum frequency observed was 35 kHz. Using the
glass-based microfluidic device, the voltage drop observed for the serial
dilutions, 10<sup>1</sup> to 10<sup>6</sup> ranged from 200 mV to 420 mV while
that for the serial dilutions, 10<sup>-7</sup> to 10<sup>-1</sup> ranged from
90 mV to 285 mV. To ascertain if a lower detection limit could be obtained, the
PDMS-based microfluidic device, with a channel with of 300 µm, was used to
analyze the response of the device to 10<sup>-7</sup> to 10<sup>-1</sup><b> </b>serial
dilutions. The result ranged from 10 mV to 30 mV respectively. A comparative
analysis with the conventional detection method showed that it was able to
detect less than 300 <i>Escherichia coli</i>
colony-forming units. This result indicates that an optimized PDMS-based
microfluidic device with higher resolution microchannel could potential detect tens
of bacteria colony-forming units. These results were obtained in about 60 secs
of introducing the sample in the device.</p>
<p> </p>
<p>The rapidity and consistency of the results observed
by the continuous increase in voltage drop with increasing concentrations of <i>Escherichia coli</i> indicate that this
detection technique has great potential in addressing the time, specificity and
sensitivity issues observed with most available detection methods.</p><br>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/9202043 |
| Date | 16 October 2019 |
| Creators | Uzumma Ozeh (7110116) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Coupling_Immunofluorescence_and_Electrokinetics_in_a_Microfluidic_Device_for_the_Detection_and_Quantification_of_Escherichia_coli_in_Water/9202043 |
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