Biomedical Engineering Reference
In-Depth Information
of uncleaved substrate molecule. The substrate could employ a pair of variants of
green fluorescent protein (GFP) with fluorescence properties consistent with FRET
detection. Alternatively, utilization of a pair consisting of a luciferase and an acceptor
fluorophore protein would provide a substrate with BRET properties. Utilization of
a cell-penetrable luciferase substrate would make it possible to perform live cell
measurements and determination of a time course of the reaction. On the other
hand, conversion of a nonfluorescent reporter substrate could also be assessed using
TR-FRET or AlphaScreen approaches concomitant with lysing the cells.
A binding cell-based assay is usually limited to study of protein-protein inter-
actions (PPIs) (Figure 12.6b); achieving consistent intracellular concentration of a
fluorophore-tagged small molecule may be challenging due to membrane permeabil-
ity. Proteins of interest could be tagged with a GFP variant or a luciferase/fluorophore
pair for FRET or BRET detection, respectively. These assays would be able to mea-
sure protein-protein interaction and its disruption with a compound within live cells.
Two-hybrid systems are also popular for screening PPIs visualizing the interaction
though reporter gene activity.
Reporter gene assays are perhaps the most popular modality among cell-based
assays. A wide variety of gene reporters is currently available. Two major groups of
reporters are illustrated in Figure 12.6c and d. One of them supplies a reporter with a
directly measurable optical property, such as the fluorescence of GFP-tagged protein
(Figure 12.6c). The second group provides a reporter with a catalytic activity that is
measured through the use of fluorogenic or luminogenic substrates (Figure 12.6d).
Similar to biochemical assays, the cell-based assays are also prone to interference
from compound groups consistent with the detection method utilized. In addition,
all cell-based assays rely on the network of cellular proteins and pathways that may
be sensitive to compound inhibition. One major effect of compounds resulting in
assay-specific artifacts of cell-based assays is cytotoxicity of compounds. It can
be especially pronounced when the cells are exposed to a high concentration of
compounds for an extended time. On the other hand, cytotoxicity might be a sought-
after event, as in HTS for an anticancer project. In these assays, the number of live
cells is monitored through a variety of cellular parameters, such as intracellular ATP
concentration or the respiratory activity of mitochondria. Alternatively, parameters
associated with cell death such as activity of caspases or cellular membrane leakage
could be utilized to measure the number of damaged or dead cells.
Cytotoxicity and reporter gene, along with a number of other assays, such as
proliferation and cell motility assays, are part of a large group of “black-box” pheno-
typic assays. In these assays, the same outcome could be achieved through affecting
numerous proteins and pathways. Some of these assays could be handled by tra-
ditional plate reader assays; however, many of them are more robust when special
technologies such as label-free and image-based detection technologies are utilized.
The aforementioned RWG detection approach is applicable not only for biochem-
ical assays but also for cell-based assays. This approach represents a relatively new
but mature field called
label-free biosensor technology
. Another representative of
this technological field is an impedance-based approach that requires the use of
special screening plates and is only applicable to cell-based assays. In cell-based
