The basic concept behind high-throughput screening (HTS) is a numbers game: If one has the technical ability to test thousands upon thousands of
potential drug compounds against a specific target,
it is likely that some compounds will interact with
the target in a positive manner. Playing this numbers game quickly and efficiently is vital as our aging
population faces increased incidences of heart disease, cancer, and respiratory disease. Drug discovery
researchers have responded by increasing their
efforts and placing more demand on HTS reagents,
instrumentation, and software providers: more
information, more sample processing, and more
options. Detectors and related detection technologies, including microplate readers, have evolved at
the same frenzied pace as the field of HTS to provide faster turnaround, higher accuracy and precision, and more efficiency for screening volumes of
drug candidates.
Figure 1
- Fluorescence polarization can provide information on
changes in molecular mobility found, for example, in receptor–ligand interactions, protein–DNA interactions, or membrane fluidity.
As the number of drug compounds has increased,
assay miniaturization has developed to counter rising reagent, drug
candidate, and drug target costs.
Miniaturization to 384-well and 1536-well microplate formats has reduced the cost per test,
although this miniaturization is not without its limitations. Common and
robust assays such as
enzyme-linked immunosorbent assays (ELISAs) are
difficult to miniaturize beyond a certain volume due
to a separation, or washing, step. This has led to a
new generation of “mix and read” or “homogeneous” assay platforms, where
separation is not
required. Some of the most common homogeneous
screening assay technologies are fluorescence polarization (FP), based
on molecular rotation with a
single fluorescent label (Figure 1); fluorescence resonance energy
transfer (FRET), measuring the distance-dependent interaction of two
fluorescent
labels; and time-resolved FRET (TR-FRET), using
a principle similar to FRET with a delay after the
excitation light pulse to reduce background fluorescence. These
fluorescence-based techniques achieve good
sensitivity while using a very simple
assay protocol, and are easy to automate and miniaturize. They are
implemented using HTS microplate readers
with rapid analysis times, advanced
detection systems, and compatibility
with various microplate formats.
Detection technologies available on
these readers are typically fluorescence polarization, fluorescence
intensity (to enable FRET detection),
time-resolved fluorescence (to enable
TR-FRET detection), luminescence
(commonly used for luciferase gene
expression assays), and absorbance.
Prices typically range from about
$60,000 to well over $100,000.
Within life science research laboratories, popular multimode, or multidetection, microplate readers have developed because of
their versatility. These differ from HTS readers in
that there is no need to support high-density plates
(1536-well plates), speed is less important, and
homogeneous assay detection is not critical.
Flexibility is the focus, with various features including kinetic reading, well scanning, reagent injection, monochromators, temperature control, and shaking.
These features are not usually considered necessary
in HTS instrumentation but can be critical for life
science research applications. Prices typically range
from $15,000 to about $60,000.
Figure 2
- Synergy 2 multidetection microplate reader shown with the Bio-StackTM Twister 2.
Although requirements from HTS and life science
research differ, there has been convergence over
time. On one hand, the limited success of massive
screening campaigns thus far has prompted pharmaceutical laboratories and biotechnology companies
to adopt a more thoughtful approach to screening;
the all-speed, all-performance approach is giving
way to more modular and targeted strategies, where
flexibility and pricing become more important factors. On the other hand, new research applications
for detection technologies such as FP and FRET
have pushed researchers to look for instrumentation
with high levels of performance in these modes of
operation. The SynergyTM 2 (BioTek Instruments, Inc., Winooski, VT) (Figure 2) is one of the first of
a new generation of multidetection microplate readers that reflect this convergence. It has been
designed with screening applications in mind but
has retained the need for greater flexibility found in
life science research laboratories. The system is fast,
compatible with 1536-well plates, and has very high
performance in polarization and time-resolved
modes. At the same time, it is equipped with an
extremely precise temperature control system, a
built-in shaker, a monochromator-based spectrophotometer,
and an optional reagent injection
system for applications more traditionally found in
research laboratories. Each of its detection technologies is available
as a module so that researchers
can tailor the instrument to their specific application and budget,
while retaining the ability to
upgrade whenever necessary. This new type of
reader provides the combined benefit of bringing to
research laboratories a level of performance and
technologies usually found on high-end HTS instrumentation, while at the
same time delivering efficient cost control to screening laboratories.
The HTS revolution has had a broad impact on
instrumentation, software, and assay technologies.
Multidetection microplate readers have benefited
greatly from these evolutions, and now offer life science research laboratories technologies and performance previously found only on high-cost HTS
instrumentation. In return, HTS screening laboratories can now access high-performance instrumentation at a cost significantly lower than traditional
HTS readers.
Mr. Amouretti is Product Manager, BioTek Instruments, Inc.,
Highland Park, P.O. Box 998, Winooski, VT 05404-0998,
U.S.A.; tel.: 888-451-5171; fax: 802-655-7941; e-mail:
[email protected].