Flow Cytometry on a Chip - Point-of-Care Diagnostics on a Chip - page 56

Flow cytometry as an effective and well-established method for counting cells on
a large scale is also used to detect microorganisms in water [ 36 ]. Flow cytometers
allow sensitive and reliable quantification of individual cells; however, as noted
earlier, this technique requires expensive equipment and is skill- and labor intensive.
Microfluidic devices have the potential to increase ease of use by integrating
sample pretreatment and separation strategies. Recently, flow cytometer research
with microfluidic devices has shown detection and quantification of bacteria [ 37 , 38 ],
by using fluorescently labeled anti- E. coli antibodies to selectively detect E. coli .
Flow cytometry has also been used to detect quantitatively Giardia cysts and
Cryptosporidium oocysts [ 39 - 41 ]. These studies evaluated the staining efficiencies
for commercial antibodies and suggest that flow cytometry is a precise method for
the detection of Giardia and Cryptosporidium in water. Antibodies for immunoflu-
orescence staining are available for most of the targeted waterborne pathogens.
The monitoring of drinking water is currently mostly performed by filtering
and culturing techniques and represents a substantial part of workload of micro-
biology laboratories [ 42 ]. Waterborne bacterial pathogens and indicators are often
physiologically altered/stressed and sometimes cannot be cultured efficiently with
standard techniques [ 43 ]. This can lead to a considerable underestimation of the
concentration of these bacteria in water and therefore of their risks to human
health. In contrast, flow cytometry can detect bacteria in all stages. With appropriate
staining (e.g., propidium iodide (PI)), flow cytometers can be used to distinguish
between viable and nonviable cells [ 44 ].

3.3.2 Pathogen Detection in Water with Spatially Modulated

Emission

Our prototype instrument can also be used to reliably identify and count specifically
tagged pathogens (e.g., E. coli , Giardia, and Cryptosporidium ) in water. For
example, Fig. 3.6 a shows the intensity histogram and the speed profile for Giardia
lamblia stained with an anti- Giardia monoclonal antibody conjugated with Cy3.
Incubation studies were performed to determine the required staining time and
amount of reagent. As shown in Fig. 3.6 bfor G. lamblia , a reagent-to-analyte
volumetric ratio of 1:100 and an incubation time of less than 2 min are sufficient
for reliable detection. Note that even for this data point the pathogen signal
( 10 5 MEPE) is more than two orders of magnitude separated from the noise. A

one-step staining method consisted of addition of the fluorescently tagged antibody
to the G. lamblia sample, mixing, and incubation steps. No additional steps, such
as washing to remove unbound antibody, were necessary prior to measurement.
The results from the incubation study indicate that this particular application is
compatible with rapid on-chip sample preparation.