Biomedical Engineering Reference
In-Depth Information
immobilization process and labeling with fluorescent dyes (35); and (iii) in most cases, an
immunoassay cannot differentiate between live and dead cells. For example, in food and
water samples, where nonviable cells may not pose a problem, a standard immunoassay
may not provide enough information; and (iv) an immunosensor is designed to detect a
specific target because the antibody for that target is immobilized within the sensor. With
this in mind, it is obvious that the investigator must know what he or she is looking for in
the sample. This of course requires some previous knowledge about the sample, and more
importantly, other potentially harmful pathogens may go undetected. The multiarray sen-
sors reviewed herein were specifically designed to solve or partially solve one or more of
the limitations of existing methods discussed above.
It is pertinent to note that most research laboratories use “simulants” when testing
prototype systems for pathogen detection. For example,
Bacillus globigii
is a nonpatho-
genic, sporulating bacterium, and is a commonly used simulant for the detection of
Bacillus anthracis
(anthrax). Owing to its widespread use,
B. globigii
is being cited in this
review as a model to compare the sensitivities of different systems.
19.4.1
Optical Multiarray Sensors for Pathogen Detection
Portability is an important consideration in pathogen detection; consequently, fluores-
cence-based sensors are the most common type of optical systems reported for this pur-
pose. There is a large variety of commercially available, low-power-consuming diode
lasers, photodiode detectors as well as many different bright fluorophores covering a
wide spectral range. Fluorescent immunoassays are typically performed in the sandwich
format where a capture antibody is immobilized on a surface. After the target antigen is
captured, it is tagged with a second “tracer” antibody that is labeled with a fluorescent
molecule or conjugated to an enzyme that will catalyze a reaction producing a fluores-
cent product.
The two most obvious advantages of a multiarray sensor are as follows: (i) individual
analytes and controls can be simultaneously assayed multiple times; and (ii) capture
surfaces (e.g., antibodies) for different targets can be integrated onto the same sensor
surface permitting multiple sample screening for more than one analyte. Fluorescent
immunosensors designed in this manner will then require an antibody “cocktail” made
up of the required fluorescent-labeled antibodies for detection. This concept has been
applied to a 6
6 array patterned onto a glass slide (36). The immunosensor was
designed to simultaneously detect three different viral and bacterial analytes (37). Anti-
B. globigii
, SEB, and MS2 bacteriophage antibodies were immobilized onto discrete areas
of a glass slide. Detection was accomplished using fluorescent-labeled antibodies and a
charge-coupled array for detection. The system correctly detected and identified the ana-
lytes in 126 blind samples in approximately 14 min with detection limits similar to ELISA
methods. This system was further developed for the detection of nine analytes and
produced a limit of detection of 10
5
cfu/ml for
B. globigii
(38). The feasibility of using the
fluorescent sandwich immunoassay format on glass slides for actual food samples was
examined (39). The system was able to identify the intestinal disease-causing microor-
ganisms,
Campylobacter
and
Shigella
, in a variety of food samples with detection limits as
low as 4.9
10
4
cfu/ml. The planar array immunoassay format was coupled to microar-
ray printing technology to deposit antibodies for different analytes onto the glass slides
(40). The microarray technology has the advantage of automated production and detec-
tion as well as small sample volume requirement. The system used a prototype flow
module containing five channels to introduce samples and fluorescent-labeled antibody
cocktails onto the microarray slide. Each channel could contain up to 88 capture antibody
spots. Using a sandwich immunoassay format with fluorescent-labeled detection
