Biomedical Engineering Reference
In-Depth Information
(A) Sandwich
(B) Competitive
(C) Displacement
(D) Direct
Binding
Target
Alexafluor or Cy5-Dye
Fig. 11.1.
Assay configurations. Four different types of assays are possible using
fluorescence biosensors, with variations such as the binding of antibodies to the
surface and labeling of antigen in competitive assays in a variation of the format
shown in (
b
) or the use of molecular beacons to provide a signal upon direct binding
of antigen in a variation of the format shown in (
d
). The figure was prepared by
Kim Sapsford, George Mason University
that the very small decreases caused by low concentrations of target are dif-
ficult to measure. One way to avoid this problem is to use a displacement
assay. In this configuration, the active sites of an immobilized monoclonal an-
tibody are saturated with a fluorescent analog of the target molecule. Pulses
of sample are introduced over the surface in a continually flowing stream.
When the stream contains the target, the fluorescent analog is displaced from
the immobilized capture molecule and measured downstream. Thus a signal
is generated that increases in proportion to the amount of target present.
Of the most commonly described recognition molecules, oligonucleotides
provide the greatest sensitivity. The principle reason is that the target genes
can be amplified prior to the detection reaction using a polymerase and se-
lective primers. This amplification step not only produces more molecules for
the detection reaction, but also increases the ratio of target to background
molecules in complex samples. Fluorescence biosensors have been developed
that simply measure the process of amplification using dyes that bind to in-
creasing concentrations of double stranded DNA, assuming that only target
oligonucleotides are replicated. For more selective analysis, the products of the
amplification have been measured using capillary electrophoresis [11] or hy-
bridization to selective probes in solution or on a surface (reviews in [1,2,10]).
The biorecognition molecules most frequently used by the optical biosensor
community have been antibodies. Antibodies have excellent selectivity and
are relatively stable - unless they are exposed to too much heat or harsh
chemicals. However, to find the most appropriate antibody for each appli-
cation can be a time-consuming process. Genetic engineering techniques are
being used to create a bigger range of specific-binding molecules. Advances
in this area include the development of single-chain antibodies, in which the
