I N
PA R T N E R S H I P
C & E N
W I T H B M G
P R E S E N T S
L A B T E C H ,
Microplate readers:
solutions and best practices
for chemical assays
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Microplate readers: solutions and
best practices for chemical assays
Are you looking for new ways to improve and optimize your chemical experiments?
Microplate readers offer an efficient and cost-effective measurement platform for your
analytical and testing applications!
The microplate reader has become a key instrument for
many biological, chemical, or physical laboratories today
and covers many assays across a wide range of disciplines.
Microplate readers detect and quantify the light produced
or transmitted by a sample in microtiter plates and
thereby enable to detect and measure chemical, biological
or physical reactions. Since samples are read in small
microcavities (wells) on a single plate instead of individual
sample tubes, this setup reduces the required reaction
volume and read time per sample substantially.
Microplate readers mainly differ by the type of detection
modes they cover, the most popular being absorbance,
fluorescence, and luminescence. Next to dedicated
single-mode readers which allow the detection of only
one of the above-mentioned modes, multi-mode readers
combine several detection technologies in one device
and thereby offer higher flexibility. Some of the more
sophisticated readers cover advanced detection modes like
Time-Resolved Fluorescence (TRF), Förster Resonance
Energy Transfer (FRET), fluorescence polarization or
AlphaScreen®. This broad spectrum enables the use of
these instruments in a wide variety of disciplines including
life sciences, drug discovery, bioassay validation, quality
control, drug safety, toxicity testing, clinical diagnostics and
biopharmaceutics.
Originally developed with a focus on life science approaches
such as vaccine development, microplate readers have
increasingly found their way into chemical applications in
recent decades. Due to the great advantages, they offer
in terms of time, reagent and ultimately cost savings,
they have become an indispensable part of the chemical
industry.
Number of wells
With the introduction of the microplate, a breakthrough was
achieved in terms of handling, measurement times and
sample volume requirements. A microplate is a flat plate
with multiple small cavities named “wells” which allow the
testing of multiple samples in one piece. Microplates are
available in varying formats and typically have 6, 12, 24,
48, 96, 384, 1536 or 3456 sample wells arranged in a 2:3
matrix. Despite the different well capacities and numbers,
the footprint dimensions of the plates adhere to specific
standards, varying only minimally if at all.
1.
Microplate color
Besides their density (number of wells per plate),
microplates are mainly available in 4 colors: clear, black,
white, and grey. The choice of a plate’s color is related to
the nature of the produced light signal and to its detection.
Clear microplates are a prerequisite for absorbance
Microplate format
Classic photometers are operated with a cuvette - a small
tube-like container, made of a clear, transparent material
like glass, plastic, or fused quartz. Cuvettes have at least
two clear opposite sides for light transmission with an
inner width of exactly 1 cm. Through this window, the light
beam passes through the sample horizontally during a
classic absorbance measurement. The pathlength of
1 cm is of importance for the back calculation of sample
concentrations. Based on the Beer–Lambert law,
A = c*d*ε (A = Absorbance, c= concentration, d = pathlength
and ε = extinction coefficient), the concentration of a
substance is linear to the obtained absorbance intensity.
If the extinction coefficient and pathlength are known, the
sample concentration can be calculated directly from the
absorbance. However, since such cuvettes have typically a
large filling volume of at least 0.5 mL and the samples have
to be transferred and measured individually, this method is
considered very time-consuming and costly.
2
As the number of wells increases, the volume per well
decreases. By reducing the sample volume used, fewer
reagents are needed for the respective reactions. While
96-well plates can hold a sample volume of up to 350 µL
per well, 3456-well plates may be used with a maximum of
5 µL per well. This decreases the price per test and enables
a larger number of samples to be carried out at the same
cost when miniaturized. As a rule, the lowest volume
recommended for a microplate well in order to have an
efficient and realistic measurement is generally >1/3 of the
maximum possible volume. Hence, for a standard 96-well
microplate, you should not go below 100 µL. Independent of
the density, microplates may vary in the maximum capacity
per well, depending on the overall height of the plate or size
of the wells (e.g., 96-well half area plates have well sizes
and filling volumes comparable to a 384-well plate).
What must however not be neglected is that with the use
of miniaturized plate formats and lower sample volumes,
there is also a need for highly sensitive microplate readers
that can detect substances even in the smallest volumes
while maintaining the same concentration.
Figure 1: Microplates are available in different formats and colors.
MICROPLATE READERS: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS
Beyond color
Other microplate characteristics to consider are
well coating and binding property. As most chemical
applications are based on the presence of detectable
target substances in suspension, low- to medium-binding
plates are regularly applied in this field. High-binding
plates reduce availability of molecules in solution but are
a good choice if the assay requires fixation of an analyte.
In addition, the culture of adherent cells requires specific
coatings to improve cell attachment and growth on plastic.
Depending on your application, microplates should be
checked for their chemical solvent compatibility and
resistance since these can vary dramatically between
different plate types.
2.
UV/vis spectrometer
The recording of complete spectra instead of individual
wavelengths is accompanied by many advantages,
especially in chemistry. For instance, addition or
subtraction of functional groups during reactions easily
results in absorbance wavelength shifts. By recording
absorbance spectra instead of measuring at a single
3
wavelength, such effects do not stay unnoticed - even if
they were unpredictable. Two options are available for the
acquisition of absorbance spectra: monochromators and
spectrometers. An absorbance monochromator breaks
white light into its individual wavelengths, mechanically
selects a wavelength of a certain bandwidth and directs
it to the sample. Thus, only one wavelength or range of
wavelengths can be recorded at a given time. If a spectrum
has to be acquired, the sample must be scanned step
by step at each wavelength with multiple individual
measurements.
Spectrometer
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measurements as these are based on the detection
of transmitted light. Not everybody is aware of the
distinction between regular and UV-transparent plates, as
measurements in the UV range are relatively uncommon.
However, although perfectly suited for measurements in
the visible range, the classically used polystyrene plates are
only poorly or not at all transparent in the UV range below
320 nm. Therefore, dedicated UV-transparent plates
made of cyclic olefin copolymer (COC) must be used for
measurements in the wavelength range of 200 – 400 nm.
Black objects absorb light of all wavelengths. Similarly,
black microplates partially quench the signal derived
from samples. This property can be exploited to reduce
background signal, auto-fluorescence and well-to-well
crosstalk when measuring fluorescence intensity , FRET
and fluorescence polarization. Assays based on these
detection modes usually yield high signals and the use
of black plates results in a bottom-line improvement of
signal-to-blank ratios.
In contrast, white plates are recommended for luminescent
and TRF assays that usually have a low photon yield.
These plates reflect light and thereby increase the signal
that is directed to the detectors. As a disadvantage, the
background is equally amplified. This is not an issue
for luminescent assays as the background is typically
negligible here. In TRF, fluorophores with long-lived
fluorescence emission are used. This offers the possibility
to delay the measurement period to a time point at which
unspecific background fluorescence is decayed. The
delayed detection excludes the unspecific and short-lived
background signal from the measurement time frame.
And what about grey plates? They are a dedicated solution
for AlphaScreen measurements, where the reduction of
crosstalk, and the amplification of low signal yields are
equally relevant.
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500
550
600
wavelength [nm]
650
Microplate
well
Xe Flashlamp
Figure 2: The UV/vis spectrometer captures absorbance spectra from
220 - 1000 nm in less than 1 second/well, significantly faster than any
monochromator.
Similar to monochromators, spectrometers incorporate a
highly efficient optical grating which equally breaks up light
into individual wavelengths. In contrast, the spectrometer
incorporates a solid-state assay detector which allows the
simultaneous detection of multiple individual wavelengths
on different areas of the detector (figure 2). Thereby, the
spectrometer allows to measure a full UV/visible spectrum
(e.g., 220 – 1000 nm) with a resolution down to 1 nm in less
than a second per well. Pilot spectral scans and selection
of specific wavelengths before the actual measurement are
no longer necessary.
With most monochromator-based devices, this time is
only sufficient for the detection of one wavelength, as a
mechanical shift of the optic components is required. The
potential time savings from using spectrometers are, of
course, significant and become even more apparent when
thinking about high-throughput experiments involving
thousands of samples. Moreover, the spectrometer typically
provides better sensitivity and resolution.
The time-dependent observation of reaction processes is
a common investigation in chemistry. Due to the ultra-fast
speed of spectral capture with a spectrometer, time-course
experiments can be monitored for wavelength shifts with
0.2 second resolution.
MICROPLATE READERS: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS
Despite these advantages, plate readers are commonly
equipped with monochromators for absorbance detection
as these can be employed as well for fluorescence
excitation.
BMG LABTECH is the only plate reader manufacturer to
incorporate a UV/vis spectrometer for spectral absorbance
detection into single- and multi-mode instruments.
3.
“Walk-away” solutions: automatable
processes to simplify detection
In recent years, the manufacturers of microplate readers
have introduced many functions to the market that not only
enable automatic measurement over time, but also expand
the experimental process, such as kinetic detection, the
addition of reagents, as well as temperature control.
Microplate readers can therefore no longer be regarded
as mere measuring devices – they rather represent an
automatable assistant that offers a versatile “walk away”
solution.
Automatic kinetic detection
In kinetic mode, microplate readers offer multiple
measurements at pre-defined intervals for a set time
window. Thereby both, very fast (e.g., a few seconds),
as well as prolonged (e.g., 24, 48, or 72 hours) timedependent changes during an experiment can be
automatically monitored. Slow kinetics are typically
detected in “plate mode”: all samples on a plate are
sequentially measured at each time point. For fast kinetics,
“well mode” is typically used. Here, the full kinetic for one
well is measured before moving on to the next one (see
figure 3).
Blank
NC
0.025 mol
0.050 mol
0.100 mol
0.175 mol
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Range 10 s - 70 s
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OD (410 nm)
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Injection
peak
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10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
Time (s)
Figure 3: Signal curves of esterase-catalyzed reactions using different
concentrations of the substrate pNPA.
Temperature control
All chemical, molecular, or enzymatic reactions show a
temperature dependency – at least to some extent. It is of
particular importance to meet the assay specific optima.
It is of even greater importance to keep the temperature
conditions constant over the course of the experiment
as well as over several runs to enable high quality and
reproducibility of the results.
4
Temperature control is a common feature of microplate
readers. For instance, all BMG LABTECH readers are
equipped with incubation from room temperature up
to 45°C. Furthermore, an extended incubation option
up to 65°C is available on some readers. To prevent
condensation build-up on the microplate lid or sealer
in long kinetic measurements, microplate readers offer
different approaches. BMG LABTECH readers for example
use two heating plates where the upper heating plate is
operated 0.5°C above the lower plate.
Furthermore, some readers allow the implementation of
temperature variations during measurement. This enables
temperature ramps and the simultaneous detection of
temperature-dependent reactions.
Automatic reagent injection
Many of today’s most popular assays require the ability
to monitor a signal directly after or even during the
addition of a reagent. In the past, this could only be done
by manually adding a substance and immediately reading
the plate. This procedure is very tedious, especially with
several injection steps. In addition, some assays produce
signals that decay very quickly after reagent addition,
becoming unreadable within a few seconds upon injection.
Without the ability to read and inject simultaneously, the
user can lose valuable signal information.
Injectors on plate readers enable reagent delivery to
any plate format from 6 - 384 wells. Thereby the time
between reagent addition and first read can be reduced to
a minimum. Some devices even allow to read and inject
simultaneously. In addition, injector flexibility has to be
evaluated depending on one´s needs. Besides multiple
additions from one injector into the same well, some
readers enable to perform two injections from different
injectors at the same time. Moreover, injection timing,
injection speed, delivery volume, and the ability to inject
different volumes in each well of a plate are further
options that have to be considered. Finally, simultaneous
injection and reading ensure that users experience no loss
of data and save valuable laboratory time.
On plate readers, injectors can be added onto/aside or are
built in the instrument, depending on the manufacturer.
Full integration of injection modules comes with several
advantages like light protection for sensitive reagents,
reagent incubation and small dead volumes, saving costs
when working with expensive reagents.
Shaking features
For samples that require high uniformity, or tend to
sediment, different shaking options ranging from linear
to double-orbital are typically available. The flexibility to
define shaking intensity, duration, and assign shaking to
specific time windows within a kinetic
(e.g., immediately following reagent injection into a well)
further expand automation capabilities.
MICROPLATE READERS: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS
4.
Wavelength selection in
fluorescence detection
LVF Monochromator principle
In fluorescence intensity measurements, wavelength
selection plays an important role. The excitation of the
fluorophore needs to take place with light of a wavelength
close to the excitation maximum of the fluorophore.
Selecting the specific wavelengths needed for excitation is
essential to avoid unspecific signals from other fluorescing
assay components. On the other hand, the emission
light must be filtered as well, so that the resulting
fluorescence signals are not polluted by unspecific light
of all wavelengths. This way, only the fluorescence coming
from the fluorophore of interest is guided to the detector
for quantification.
Further enhancement of the sensitivity of a microplate
reader can be achieved by an additional selection event
between excitation and emission: the use of a dichroic
mirror. This dichroic mirror typically blocks excitation
light derived from the light source and transmits emission
light to the detector, thereby reducing background and
increasing specificity.
Monochromators vs. filters
The selection of specific wavelengths can be accomplished
with monochromators.
A monochromator is an optical device, that selects a
wavelength of light or a range thereof. Conventional
monochromators rely on diffraction, with light passing
through a slit, a concave grating, then another slit. All
these steps reduce the light passing through the device,
resulting in a final transmission rate of about 10 % of the
original excitation light to the sample. The outstanding
advantage of this technology, however, is the great
flexibility it provides, covering broad wavelength ranges
and accordingly almost any imaginable assay from the very
beginning.
Filters represent the static alternatives to
monochromators. They are available in various fixed
wavelengths and bandwidths. In contrast to conventional
monochromators, they typically allow a transmission rate
of more than 90 %.
A hybrid alternative that combines the advantages of
both technologies are so called linear variable filter
(LVF) monochromators. These consist of 2 linear variable
filter slides which define the rising and the falling
edge of a selected wavelength range (figure 4). Since
the slides can be moved relative to each other, high
flexibility in the selection of the wavelengths and the
bandwidths is provided. LVF monochromators are the first
monochromators with filter-like performance. As a unique
feature, a Linear Variable Dichroic Mirror (LVDM) slide is
used to separate the excitation from the emission light.
5
Short pass and long pass filter combination SP-LP
SP
LP
Block
Block
Pass
UV
(c)
IR
Figure 4: (A) Schematic of the LVF components including slits and long
and short pass linear variable filter (B) Transmission spectrum of LVF
combination of short and long pass filter slides.
In addition to the outstanding transmission rate, which
exceeds that of conventional monochromators by far, the
LVF monochromator also shows outstanding blocking
of unwanted stray light for best performance and lowest
background noise.
The setup furthermore allows the selection of large
bandwidths (e.g., up to 100 nm) which is extremely
beneficial in luminescence applications. The LVF
Monochromators are so far only available on the
CLARIOstar Plus plate reader, making it the most sensitive
monochromator-based microplate reader on the market.
5.
Simplified detection: EDR for largest
possible dynamic range
In fluorescence detection, fluorophores absorb light and
consequently emit light of lower energy with a longer
wavelength. Highly varying concentrations between the
largest and smallest sample result in highly varying signal
intensities within the same experiment. Also, during timecourse experiments the maximum signal builds up over a
lapse of time during the kinetic. It can easily happen that
the first values are not yet detectable due to insufficient
sensitivity of the device and the last values are already so
intense that they also fall out of the detectable frame. Such
experiments require readers with large dynamic windows.
The dynamic window of a reader defines the ratio of the
largest and lowest sample, that a reader is still capable of
measuring in a single detection run. Classically, different
signal ranges are covered by adjusting the gain setting.
A higher gain amplifies the intensity of lower signals. At
MICROPLATE READERS: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS
the same time, it can also amplify the signal of highly
concentrated samples to the point where it possibly moves
outside the detectable range, saturating the detectors.
6.
Conversely, by reducing the gain, the signal intensity of
very intense samples can be easily detected. However, the
signals of low-concentrated samples may no longer be
properly separated and no longer be distinguished from
negative samples – resulting in a low sensitivity.
In the pharmaceutical industry, high-throughput screening
is an important method for drug discovery. The assessment
of solubility in this process is mandatory to determine the
validity of the pharmacological results and the selection
of promising compounds. Solubility has a major impact
on drug availability, formulation, dosing, and absorption.
Hence, it is very important to analyze it early in the drug
discovery process to avoid time-consuming and costly
ADME screens of low solubility compounds.
10+8
Gain 1
Gain 2
Gain 3
Enhanced Dynamic Range
Laser-based nephelometry:
Dedicated solution for turbidity
measurements
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RFU
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Traditionally, equilibrium solubility assays have been
determined in limited throughput, by shaking and
incubating the compound with a solvent for at least 24
hours, prior to filtration and concentration determination by
HPLC. This approach does no longer fulfil the requirements
of modern drug discovery. Today, automated kinetic
solubility screens run on nephelometers deliver higher
throughput in shorter times.
Fluorescein concentration in nM
Figure 5: EDR allows the detection of very low and very high
concentrated samples in a single measurement. Without EDR, the
concentration range could only be covered in several runs with different
gain settings.
Enhanced dynamic range
Especially in empiric science the full range of the
concentrations or associated signal intensities cannot
always be predicted. Here, the signals may span over
more than three decades. Most microplate readers offer
the option to manually adjust the gain or some sort of
automatic gain identification. To further simplify the
laborious and time-consuming procedure of manual gain
adjustment BMG LABTECH developed the ‘Enhanced
Dynamic Range’ (EDR) technology.
Fixed gain results in a fixed dynamic range. Thereby only
a limited range of signal intensities are reliably detected.
EDR ensures consistent detection of samples over a large
range of concentrations and signal intensities with no
manual intervention. With the application of EDR, the need
for repeated detection runs with different gain settings
is eliminated (figure 5). This does not only save time and
money but furthermore enables to compare data that have
been acquired at different time points with the same kit.
Nephelometers and turbidimeters
Nephelometry (from the Greek nephelo: cloud) describes
an analytical chemistry technique used to measure the
amount of turbidity or cloudiness in a solution caused
by the presence of suspended insoluble particles. When
directed through a turbid solution containing suspended
solid particles, light is transmitted, absorbed (blocked) and
scattered (reflected off the particles).
The amount of scattered light depends on the size, shape,
and concentration of the insoluble particles in solution,
as well as on the incident wavelength of light. This
scattered light can be detected by nephelometry which
typically measures the amount of light that is scattered
by a substance at a 90° angle to the incident light beam
derived from the light source (figure 5). Turbidimeters with
detectors located at an angle to the incident beam are
called nephelometers and are considered the standard
instrument for the measurement of low turbidity values.
Nephelometry
Z-axis control
Plate well
Options like EDR, significantly simplify the experimental
setup, as the measurement can be started directly without
any further adjustment of the gain setting. This offers
significant advantages, especially for approaches with
unpredictable signal intensity, such as assay development
or large-scale screening campaigns.
Analyte in
solution
Solubility change
Ulbricht
Sphere
Photodetector
Beam Block
Figure 6: Schematic of nephelometric measurements in the
NEPHELOstar Plus
6
MICROPLATE READERS: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS
A
Scattered light
Light source
TURBIDIMETRY
Detector
B
Scattered light
Light source
NEPHELOMETRY
Filtration or phase separation of the solution from the
undissolved residues are not required. Samples just have
to be transferred into the wells of a microplate. Since this
measurement method does not modify the substances and
does not require the introduction of additional reagents
for the analysis, the very same samples can subsequently
be used for further assays. Finally, nephelometry can be
employed to determine both the concentration at which a
compound becomes soluble and the point at which a solute
begins to precipitate.
Typically, the detected signal is linear for up to 3 orders of
magnitude of particle concentration and a limit of detection
of about 20 mmol/L can be reached for kinetic solubility
assays 3. For instance, with BMG LABTECH’s NEPHELOstar
Plus
silica particles (from 0.5 - 10 µm) can be detected down
to 800 nM and the dynamic range covers 5 decades.
Detector
Figure 7: Difference between turbidimetric and nephelometric detection.
The world’s first laser-based microplate nephelometer, the
NEPHELOstar Plus, was developed by BMG LABTECH. As the
measurement principle differs significantly from all other
methods mentioned, nephelometers are dedicated singlemode readers.
The turbidity of a liquid can also be detected by measuring
the transmitted light passing through a sample in line with
the light source instead of the scattered light. The decrease
in light transmission is measured compared to a reference,
and the absorbed light is quantified as Optical Density
(OD) units 1. Accordingly, turbidity can be also measured
for instance using an absorbance microplate reader (e.g.,
bacterial growth at 600 nm). Turbidimetry is suited to read
and quantitate high particulate concentrations in liquids.
Small changes may however remain unnoticed, since a
small difference between two intense signals can be hardly
determined by turbidimetry. Nephelometry in contrast,
detects scattered light and is better suited for samples with
low particulate concentrations.
Commonly, nephelometric assays are performed in 96- or
384-well microplates. The optical quality of a microplate is
an extremely important aspect. Imperfections like dust, dirt,
fingerprints, or scratches on the well bottom can scatter
light, generating false positive signals, reducing the assay
window, or leading to a significantly reduced sensitivity.
Nephelometry applications
In pharmaceutical laboratories, nephelometry is mainly
used to assess the solubility of drugs or compounds 2.
Microplate-based nephelometers usually provide a higher
throughput, simplified and low-volume approach to the
collection of turbidity data. These features make them a
very valuable tool for high-throughput compound solubility
screenings. Here it shows substantial advantages over
other methods.
7
MICROPLATE READERS: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS
Chemical applications on microplate readers
The features highlighted above show the advantages
microplate reader-based detection can bring to your
experimental setup. The higher sample numbers,
throughput, automation capabilities and flexibility offered
by microplate readers allow you to analyze samples faster
and cheaper, possibly on one single device. But how can
these advantages be applied in your day-to-day chemical
experiments?
this reaction and has a high molar extinction coefficient in
the visible range. The molar extinction coefficient of TNB is
reported to be 13,600 M-1 cm-1 at 412 nm and pH 8.0 and
can be quantified using a spectrometer.
O
DTNB2-
O
O
O
O
Compound synthesis and analytical
chemistry
The analysis of compounds is of course an important step
in chemical synthesis. Regardless, whether you plan to run
a one-step or complex multi-step synthesis, verification of
the chemical structure of your reagents and the procedure
of the chemical reactions is crucial.
The yields of chemical reactions may vary considerably
with parameters such as solvent, catalyst, temperature,
and reaction time. A challenge for the chemist is to
optimize these parameters to obtain a satisfactory yield at
acceptable time and cost. To aid optimization, microplate
readers can be used to increase the number of reactions
that can conveniently be performed in parallel by an
individual chemist.
Endpoint reactive group detection: thiol chemistry
One possible application is the detection of reactive groups.
An example for this is the Ellman’s assay for in-solution
quantification of sulfhydryl groups. As thiol chemistry is a
rapidly expanding field in basic and applied bioscience, the
quantitative measurement of -SH groups is a routine task
in many applied disciplines where a quick and easy method
is much preferred. While electrochemical and fluorimetric
assays are very sensitive and accurate, they involve lengthy
procedures (complete proteolysis, electrolysis, HPLC
separation). Although spectrophotometric thiol assays
such as Ellman’s are less sensitive in comparison, they
offer a rapid and simple solution for the quantification of
sulfhydryls.
5,5’-dithio-bis-(2-nitrobenzoic acid; DTNB) reacts with a
free sulfhydryl group to yield a mixed disulfide and 2-nitro5-thiobenzoic acid (TNB; see figure 8). The target of DTNB
in this reaction is the conjugate base (R—S-) of a free
sulfhydryl group. TNB is the “colored” species produced in
8
O
N
S
N
O
R
S
O
S
O
O
O
O
O
O
S
S
N
R S
N
O
O
TNB2-
mixed disulfide
Figure 8: Reduction of Ellman’s reagent.
Sulfhydryl groups may be estimated in a sample by
comparison to a standard curve composed of known
concentrations of a sulfhydryl-containing compound
such as cysteine. Alternatively, sulfhydryl groups may be
quantitated by reference to the extinction coefficient of
TNB. The standard curve itself can be a useful indicator
of the strength of the assay. By taking the full spectrum of
absorbance values for each solution, the isosbestic point
can be determined either tabularly or graphically to confirm
that the molar ratio between the Ellman’s reagent and the
test sample are equivalent across each test solution.
The isosbestic point for Ellman’s assay is approximately 356
nm (figure 9). A smooth peak at 412 nm also indicates that
your solution falls within the working range of the assay.
2.5
Absorbance (Blank Corrected)
Depending on the nature of your research and experiments,
plate readers can be used in a multitude of applications.
These comprise the whole range of chemical research
from the chemical analysis to the evaluation of synthesized
compounds. Below, examples from
BMG LABTECH’s application note library highlight the
possibilities and applications offered by the microplate
reader-based measurement format.
2.0
Isobestic Point
Std. A: 1.6 nM
Std. B: 0.8 nM
Std. C: 0.4 nM
Std. D: 0.2 nM
Std. E: 0.1 nM
Std. F: 0
1.5
1.0
0.5
0.0
300
350
400
450
Wavelength [nm]
Figure 9: Absorbance spectra of cysteine standards.
MICROPLATE READER: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS - CHEMICAL APPLICATIONS ON MICROPLATE READERS
500
Similarly, the absorbance spectra of the unknown solutions
can be compared to those of the standard curve to ensure
that the concentrations of the solutions fall within the range
of the standard curve (figure 10). The absorbance values for
unknown solutions A and B were plotted along the standard
curve, corresponding to a sulfhydryl concentration of 1.234
mM and 0.810 mM, respectively (figure 11).
2.5
Unknown A
Unknown B
Absorbance (OD)
2.0
1.5
1.0
In situations where reactants and products differ in their
molar extinction coefficients, the reaction progress over
time can be directly followed by UV/visible spectroscopy.
Rather than determining the concentrations of species
by physical separation and individual quantitation, kinetic
acquisition of spectra in which these species vary in their
relative proportions can be analyzed mathematically and
accompanied by derivation of kinetic rate constants by least
squares fitting.
This principle can be highlighted by monitoring the
metalation of porphyrin with Zn2+ (figure 12) as a function
of solvent. The porphyrin starting material and product
are both highly colored and the reaction proceeds with a
perceptible but subtle color change.
The absorption spectra of the two species overlap
significantly making a global least squares analysis of
spectra desirable for quantitative rate comparisons.
0.5
0.0
300
350
400
Wavelength [nm]
450
500
Figure 10: Absorbance spectra of sulfhydryl-containing unknown
solutions.
Figure 12: Metalation of tetraphenylporphyrin (TPP) with zinc.
Unknown A
1.2
Unknown B
Y = 1.086 * X +0.014
R2 = 0.999
0.6
0.0
0.0
Unknowns
Standards
Standard Curve
0.6
1.2
1.8
Concentration (mM)
Figure 11: Quantification of sulfhydryl groups in three unknown test
solutions.
Ellman’s assay is a useful tool to easily determine the
sulfhydryl concentration in your reagent solutions with
a microplate reader. The assay can also be adapted to
accommodate larger volumes of test samples for readings
in a cuvette by using Beer’s Law and the extinction
coefficient of TNB.
The rate of the metalation reaction of tetraphenylporphyrin (TPP) with Zn2+ (figure 13 and 14) shows solvent
dependence, due to differing degrees of solvation of
the porphyrin and zinc salt and the possibility for bases
to assist in porphyrin deprotonation. Twelve common
laboratory solvents spanning a range of polarity and
chemical properties were chosen for comparison.
Starting material and product were both found to dissolve
adequately in (N,N-dimethyl-formamide; DMF). The
reaction progress could be qualitatively assessed by
visual spectral inspection (figure 13 and 14) using BMG
LABTECH’s MARS evaluation software, which revealed
the most rapid reaction in the halogenated solvents
dichloromethane and chloroform and little change in
N-methyl-2-pyrrolidone (NMP).
0.9
0 min
10 min
30 min
60 min
120 min
240 min
480 min
1440 min
0.8
0.7
0.6
OD
Absorbance (OD)
1.8
0.5
0.4
0.3
Kinetic detection: studies on the metalation of porphyrin
The quantification of reactive groups can be employed as
a qualitative measure for your chemical reactions. Kinetic
studies of chemical reactions can help to reveal reaction
speed and dynamics to optimize your synthesis approaches
even further.
9
0.2
0.1
0
450
500
550
600
wavelength [nm]
650
Figure 13: Changes in visible spectrum accompanying zinc metalation
of TPP in chloroform. Arrows indicate the evolution of the absorption
bands with time.
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0.9
0 min
10 min
30 min
60 min
120 min
240 min
480 min
1440 min
0.8
0.7
OD
0.6
0.5
0.4
0.3
0.2
0.1
0
450
500
550
wavelength [nm]
600
650
absorbance minima and maxima, performing complete
absorbance spectral scans can be used for their analysis
(figure 15). This of course does not replace analysis by more
detailed means like mass spectrometry but can be used
as an easy first measure to assess sample identity. Here,
including reference samples such as educts, products, or
side products allows conclusions on synthesis success and
sample purity if distinctions between their spectra can be
made.
Figure 14: Changes in visible spectrum accompanying zinc metalation
of TPP in NMP.
Analyte 1
Analyte 2
3
Analyte 3
Analyte 4
Analyte 5
2
Analyte 6
Analyte 7
OD
Isopropanol and acetonitrile reactions gave lower
maximum OD values than the other samples which can be
attributed to poor solubility of the TPP starting material in
these solvents. For DMSO, a black precipitate was apparent
in the sampled reaction aliquot, possible evidence of a side
reaction.
2,5
Analyte 8
1,5
1
0,5
220
240
260
280
300
320
340
360
380
400
420
440
Wavelength in nm
These results highlight the advantages of this simple and
straightforward method for assay development. Due to the
microplate-based platform, several reaction conditions can
be performed and compared simultaneously and in kinetic
measurements on a microplate reader, opening up efficient
and cost-effective solutions for your assay optimization.
Relative
initial
rate
Solvent
DMF
8
NMP
1
Toluene
10
Dichloromethane
300
Chloroform
100
Tetrahydrofuran
30
Pyridine
2
Ethylacetate
30
Acetone
50
Dimethylsulfoxide
~1
Solvent
Relative
initial
rate
Table 1: Rates of reaction of tetraphenylporphyrin with Zn(OAc)2. 2H2O in
different solvents, relative to the rate in N-methyl-2-pyrrolidone (NMP).
Authentication and quality testing
Once synthesis is up and running and you have the first
compounds in hand, their properties need to be analyzed.
Scans of absorbance or fluorescence spectra are useful
tools for this task. Since many compounds display distinct
10
Figure 15: UV/visible fingerprint profiles of different analytes.
Identification of fluorescent spectral properties
Fluorescent compounds such as fluorescent probes or dyes
require the identification of excitation and emission maxima
to ensure the best possible measurement settings in
downstream applications. Partial or complete scans of the
excitation and emission spectra of fluorescent compounds
on microplate readers offer a fast and accurate way to
assess these parameters and optimize measurement
settings (figure 16).
100
80
Intensity in %
To quantify the relative reaction rates in the different
solvents, spectra were subjected to singular value
decomposition and globally fitted to a kinetic model in
which the only colored species were TPP and ZnTPP.
Comparison of reaction rates (table 1) showed that the
metalation reaction was fastest in the two halogenated
solvents dichloromethane and chloroform, while rates in
the highly polar solvents NMP, DMF and DMSO were slow in
comparison. Furthermore, the basic and aromatic solvent
pyridine was less effective than non-basic toluene and two
orders of magnitude slower than dichloromethane.
60
40
20
0
420
440
460
480
500
520
540
Wavelength in nm
560
580
600
Figure 16: Spectral scans of PicoGreen reagent bound to DNA. The
excitation scan (blue line) was recorded between 400 and 540 nm with
a fixed emission wavelength at 550 nm. The emission was scanned
between 490 and 600 nm (red line) while a fixed excitation wavelength of
460 nm was used.
Analysis of compound solubility
One of the final steps before testing compounds for their
performance in downstream assays is to determine their
solubility and prepare stock dilutions. Microplate readerbased laser nephelometry offers a unique solution to
qualitatively measure the solubility of compounds under
different conditions or dilutants in low- to high-throughput.
MICROPLATE READER: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS - CHEMICAL APPLICATIONS ON MICROPLATE READERS
Within the drug discovery industry there is a growing trend
towards highlighting potential ADME issues and reducing
attrition as early as possible in the drug discovery phase.
This implies applying high-throughput approaches to
determine ADME and the physical chemical properties of
large numbers of compounds at an early stage.
As solubility is one of the most important properties of a
compound, identifying solubility issues at an early stage in
the drug discovery process is invaluable. Not only are low
solubility compounds more difficult to develop, obtaining
reproducible data for ADME screens such as Caco-2 and
lipophilicity is also more time-consuming and costly.
Laser nephelometry is the measurement of forward
scattered light when a laser beam is directed through a
solution and has been shown to be a reliable technique for
the measurement of kinetic solubility. The more particulate
there is in the solution, the greater the amount of forward
scattered light (measured as counts). In the recent years,
this technique has been advanced to be even used in fully
automated setups for rapid kinetic solubility screens in up
to 384-well plate formats.
Figure 17 highlights the principle of such kinetic solubility
screens with the compound hydrocortisone as an example.
Depicted are the mean nephelometric counts (n=4) plotted
against the concentration of different hydrocortisone
dilutions.
Figure 18: Batch to batch variability for hydrocortisone as a control
compound. Results over 6 different days (n=14) are shown.
Compound testing
Once compounds have been synthesized, their efficacy,
selectivity and other important parameters have to be
tested. Microplate readers offer a very flexible platform
for a near limitless amount of possible test setups and
assays, significantly simplifying the identification of
the right solution for your compound testing. Spanning
the whole range of non-radioactive detection methods
from fluorescence intensity, absorbance, luminescence,
fluorescence polarization, AlphaScreen®, time-resolved
fluorescence to time-resolved FRET, most of the widely
used testing and screening methods can be performed on a
microplate reader.
As microplate-based assays can be used with a plethora of
different reagents and samples, choosing the right method
depends on the compounds you want to test, their targets
and the scientific questions you want to answer. Regardless
of whether you want to investigate interactions with other
compounds in living cells, purified enzymes or receptors,
compound uptake, conversion or degradation, a wide range
of possible applications exists which can already be applied
as they are or can be modified to fulfil your individual
experimental needs.
Figure 17: Kinetic solubilities for hydrocortisone.
The dramatic increase in counts above 260 µg/mL
corresponds to the compound precipitating out of solution.
Subsequently, two linear lines are fitted to the data and the
point at which they cross is taken as the kinetic solubility.
Batch to batch variability of the system was tested for
hydrocortisone run over 6 different days (n=14; figure 18).
This yielded a mean solubility of 289 ± 14 μg/mL, which was
used to set a deviation threshold for further batch testing.
11
These applications can be run as single measurements or
in large screening approaches. Here, the highly adaptable
data capture on microplate readers can be used to perform
kinetic studies of multiple compounds or conditions in
parallel (multiplexing).
Enzyme inhibition screening
A common approach is the compound screening
for enzyme inhibition highlighted by this example
of Pseudomonas elastase (pseudolysin, LasB). This
metalloprotease virulence factor is secreted by the
opportunistic pathogen Pseudomonas aeruginosa. As
one of the main virulence factors of this bacterium, it
contributes to chronic and intractable infection in various
disease states from the cystic fibrosis lung to chronic
ulcers of the skin. The central role of LasB makes it a key
drug target in this process, and so a library of inhibitor
candidates was developed for screening against this
enzyme.
MICROPLATE READER: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS - CHEMICAL APPLICATIONS ON MICROPLATE READERS
ratio (665nm / 620 nm * 10000)
For this purpose, a substrate conversion assay has been
used which measures the amount of LasB substrate
conversion by a fluorescence-based approach. Adding
rising inhibitor concentrations to this approach led to a
decrease in enzyme activity and subsequently substrate
conversion (figure 19).
25000
Control
AFU (Arbitrary Fluorescent Units)
1 mM
20000
3000
1.25 nM
0.625 nM
2000
0.313 nM
0.156 nM
1000
0 nM
0
0
1000 2000 3000 5000 7000 9000
time [s]
2.5 mM
5 mM
Figure 20: Tracer characterization. Association was monitored by TRFRET after combining a lanthanide-labelled kinase and a fluorescent
kinase tracer. Dissociation was recorded after addition of excess
staurosporine to the tracer sample.
10 mM
15000
Blank
10000
5000
0
0
5
10
15
20
25
30
Time (minutes)
Figure 19: Progress curves for hydrolysis of substrate by LasB in the
presence of a range of concentrations of a typical LasB inhibitor.
Kinetic evaluation of such inhibition curves can be used
to calculate kinetic parameters like compound affinity,
efficacy, as well as association and dissociation rates. For
this purpose, many microplate reader system come with
dedicated software solutions which provide easy-to-handle
and comprehendible support for data analysis.
Analysis of binding kinetics
Kinases form another prominent target group in drug
development. A feature associated to the efficacy and safety
of kinase drug candidates is how fast they bind to (kon), and
dissociate from (koff) their targets, and accordingly for how
long they can occupy them (residence time). Ideally these
kinetic parameters are determined in preclinical stages,
creating a need for high-throughput methods to profile
hundreds and thousands of compounds.
One way to determine association and dissociation rates
is the kinetic probe competition assay (kPCA). Association
and dissociation rates of unlabeled test compounds that
compete for the binding to a target kinase (linked to a
lanthanide-based fluorophore) can be detected by timeresolved energy transfer (TR-FRET). In this experimental
setup, a tracer molecule (bound to a dye) with known
binding kinetics is used.
Here, the addition of a test compound leads to a reduction
of the TR-FRET signal if the compound competes and
displaces the fluorescent tracer molecule from the target
kinase (figure 20). In this context, binding of the competitor
molecule alters the signal curve of interaction between
tracer and target in a dose-dependent fashion. Kinetics
and affinity parameters of tracer and competitors can
be derived by fitting the resulting signal to appropriate
mathematical models.
12
5 nM
2.5 nM
Enzymatic protein degradation
Even complex biological processes such as protein
degradation can be investigated with microplate-based
methods. Recent years saw the development of the
proteolysis targeting chimeras (PROTACs) technology.
These small molecules use the cell’s protein degradation
machinery to induce the degradation of a specific target
protein.
PROTACs achieve this by directly engaging a target protein
with an E3 ubiquitin ligase through the action of two
binding ligands that are joined by a chemical linker: one
specific for the ligase, and the other, for the target protein.
The formation of an induced ternary complex between
the E3 ligase, PROTAC, and the target protein is the first
mechanistic step required for targeted degradation (figure
21).
Figure 21: Simplified schematic of ternary complex formation and
ubiquitination assay principles. Kinetic, real-time assessment of
PROTAC mechanism was achieved by co-expression of a fluorescently
labelled HaloTag-VHL (top) or HaloTag-Ubiquitin (bottom) fusion
construct with the endogenously tagged HiBiT-BRD4 protein.
NanoBRET™-based assays can be used to assess the
kinetics of ternary complex formation and target protein
ubiquitination in live cells upon PROTAC treatment.
Exemplary data are provided for BRD4 and the BET family
PROTAC ARV-771, which recruits BET family proteins to the
VHL E3 ligase complex.
MICROPLATE READER: SOLUTIONS AND BEST PRACTICES FOR CHEMICAL ASSAYS - CHEMICAL APPLICATIONS ON MICROPLATE READERS
BRD4 was tagged with the 11 amino acid peptide HiBiT
in cell lines stably expressing LgBiT. The high affinity
association of HiBiT and LgBiT results in the reconstitution
of the NanoBiT® luciferase that is fused to the endogenous
BRD4 protein. PROTAC-induced ternary complex formation
or ubiquitination can then be monitored by expressing
a fluorescently labeled HaloTag® fusion to either the E3
ligase recruiter or ubiquitin.
Endogenously tagged HiBiT-BRD4 cells co-expressing
either HaloTag®-VHL or HaloTag®-ubiquitin showed
rapid and dose-dependent ubiquitination (figure 22) upon
treatment with a concentration series of ARV-771 PROTAC.
BRD4 ubiquitination began to reach a plateau at 4 hours.
NanoBRET™ signal above the baseline was detected down
to 4nM ARV-771, demonstrating high sensitivity and assay
robustness.
BRD4 / UBB
uM ARV-771
BRET Ratio (mBU)
30
25
20
1
0.333
0.111
0.037
0.012
0.004
0
15
10
5
0h
1h
2h
3h
4h
Time in hours
5h
6h
Figure 22: NanoBRET™ ubiquitination assay. ARV-771 induces rapid
and dose-dependent ubiquitination of BRD4. Data are expressed in
milliBRET units (mBU) and error bars represent standard deviation of
n=4 replicates.
References
1 Mary C. Haven; Gregory A. Tetrault; Jerald R. Schenken (1994).
Laboratory Instrumentation. John Wiley and Sons. ISBN 0471285722.
2 Joubert A, Calmes B, Berruyer R, Pihet M, Bouchara JP, Simoneau P,
Guillemette T. Laser nephelometry applied in an automated microplate
system to study filamentous fungus growth. Biotechniques. 2010 May;
48(5):399-404. doi: 10.2144/000113399. PMID: 20569213.
3 B. Hoelke, S. Gieringer, M. Arlt, C. Saal, „Comparison of Nephelometric,
UV-Spectroscopic, and HPLC Methods for High-Throughput
Determination of Aqueous Drug Solubility in Microtiter Plates“,
Anal. Chem. 2009, 81, 3165-3172.
5 Things to consider when choosing
a microplate reader for chemical
research
There is a plethora of microplate reader-based applications
which can be used in your day-to-day chemical research.
However, depending on your experimental setup certain
instrument features are more important than others. This
short overview containing five crucial points to consider
should give you a general idea and help you in finding the
perfect reader for your applications.
1. Plate format: Depending on your application and needs,
experiments can be run in plate formats from 6 to 1536
wells. The 96-well plate is today’s standard for all
plate readers. If you need different or additional
formats, make sure to check the plate format
compatibility of the different instruments.
2. Detection modes: The readouts of your experiments
define the detection modes you need. If you want to
exclusively record absorbance spectra of your
compounds, then choose a single mode plate reader
equipped with a spectrometer. Do you want to use
different detection modes and applications? Choose a
multi-mode reader for maximum flexibility.
3. Wavelength selection: Microplate readers are typically
filter- or monochromator-based systems. While filters
give you the overall best performance, they are limited
to fixed wavelengths. Monochromators on the other
hand enable flexible wavelength selection and scanning
capabilities at the expense of sensitivity.
4. Reagent dispensers: Injecting different sample
solutions into the wells of a microplate allows to
automate a manual task and exactly control the timing
of your experiments for large numbers of samples.
Readers capable of simultaneous injection and
measurement ensure that you never miss a time point
in your reaction, even in fast kinetics.
5. Shaking and incubation: Sample homogeneity and
stable environmental conditions are paramount for
obtaining high-quality data. An optimal sample
incubation temperature is crucial as chemical
reactions are temperature dependent. Different
shaking options guarantee thorough mixing of your
samples. Temperature and shaking options differ
between readers - make sure to choose a suitable
device for your requirements.
For more information feel free to contact us at
applications@bmglabtech.com
BMG LABTECH GmbH
Allmendgrün 8
77799 Ortenberg
Germany
13
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