Biomedical Engineering Reference
In-Depth Information
[Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the gold surface were blocked due to the procedures
of self-assembly of 4-aminothiophenol and antibody immobilization, which were investi-
gated by cyclic voltammetry and impedance spectroscopy [79].
21.2.3
Immunomagnetic Technique
The coupling of an immunological separation (using immunomagnetic beads) with ampero-
metric now injection analysis detection of viable bacteria is presented [80]. Using a solution
containing E. coli 0157, the electrochemical response with two different mediators [potassium
hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system.
Antibody-derivatized Dynabeads were used to selectively separate E. coli 0157 from a matrix.
The kinetics and the capacity parameters regarding the attachment of bacteria to the
immunobeads were studied. The immunomagnetic separation was then used in conjunction
with electrochemical detection to measure the concentration of viable bacteria. A calibration
curve of colony-forming units (du) against electrochemical response was obtained. The detec-
tion limit for this rapid microbiological method was 10 5 cfu/ml, and the complete assay was
performed in 2 h. Some advantages over ELISA methods are the direct detection of viable
cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled),
thus making the assay faster (only one washing step is necessary) and less expensive.
An immunomagnetic capture reverse transcription-PCR (IMC-RT-PCR) assay was eval-
uated to recover and detect enteric virus in sewage and to remove PCR inhibitors [81]. The
procedure was applied along with a simple sample processing consisting of an initial sep-
aration of solids followed by polyethylene glycol precipitation and solvent extraction. This
procedure reduced sample volumes by about 65-fold without eliminating RT-PCR
inhibitors. Paramagnetic beads coupled to pooled human immunoglobulins were used to
simultaneously capture poliovirus 1 (PV-1) and HAV from seeded-sewage concentrates.
The IMC was efficient in removing PCR inhibitors and further reducing sample volumes
by approximately 10-fold, allowing the analysis of 6-7 ml of sewage sample per RT-PCR
reaction. The detection limits of IMC-RT-PCR from seeded concentrates were 0.1-1 PFU
for PV-1 and 1 MPNCU for HAV. The described procedure could be applied successfully
for the detection of enteroviruses, HAV, and rotaviruses in field-sewage samples [81].
21.2.4
Atomic Force Microscopy-Immunosensor Assay
Bionanotechnology can be viewed as the integration of tools and concepts in nanotech-
nology with the attributes of biomolecules. We shall discuss here an atomic force
microscopy-immunosensor assay (AFMIA) that couples AFM with solid-phase affinity
capture of biological entities for the rapid detection and identification of group B cox-
sackievirus particles. Virus identification is based on type-specific immunocapture and the
morphological properties of the captured viruses as obtained by the AFM. Representatives
of the six Group B coxsackieviruses have been specifically captured [82].
21.2.5
Flow Cytometry
This group has developed a rapid, duplexed microsphere-based immunoassay for the char-
acterization of influenza-virus types, which has the potential to overcome many of the
limitations of the current detection methods. The assay uses microspheres of two sizes, each
coupled to an Influenza type A- or type B-specific monoclonal antibody (MAb), to capture
influenza virus in the sample. A cocktail of fluorescently labeled, influenza-specific
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