Chemiluminescence,
Bioluminescence, and the
Luciferin-Luciferase ATP
Detection Assay
Ashley Long
February 22, 2011
Bioanalytical Chemistry – Spring 2011
Overview
• Background on Chemiluminescence and
Bioluminescence
• General overview of uses of Bioluminescence
in High-Throughput Screening (HTS)
• Introduction to the Luciferin-Luciferase ATP
detection assay and its importance in HTS
The firefly
Image from:
http://scienceline.org/2010/11/lighting-the-way/
What is chemiluminescence?
• Occurs when an EXOTHERMIC CHEMICAL reaction
releases energy to generate electromagnetic
radiation which gives off light 1,2
• Types1:
– Reactions with synthetic compounds (i.e. H2O2)
– Bioluminescent reactions – from a living organism
– Electrochemiluminescent reactions – use electric current
minescence. However, because more photons can be collected from macroscopic samples, reducing background
associated with the assay method becomes more important. This is the case for assays performed in multiwell
plates, where chemiluminescent assays often outperform
analogous fluorescent assays.2,3 The low background inherent in chemiluminescence allows for a better signalto-noise ratio and thus better assay sensitivity. Such
Fluorescence
macroscopic measurements are the foundation for most
HTS methods, which rely heavily on the use of multi• Light
must
be absorbed
by across
well plates
to rapidly
measure
a single parameter
a large number of samples. Furthermore, fluorescent asfluorophore
tothereach
excited
state
says can also
be affected by
presence
of interfering
fluorophores within the biological samples or in the liBrighter,
but MORE
background
brary •compounds,
whereas
chemiluminescence
is less
hindered.
Firefly (Photinus pyralis) luciferase is a monomeric
enzyme of 61 kDa that requires no posttranslational modifications for activity.4 It acts by first combining beetle
luciferin with ATP, to form luciferyl-AMP as an enzymebound intermediate. This intermediate reacts with O2 to
create another bound intermediate, oxyluciferin, in a
high-energy state. The subsequent energy transition to the
ground state yields yellow-green light with a spectral
Chemiluminescence
maximum of 560 nm (Fig. 2).
Renilla luciferase from the sea pansy Renilla reni• See
energy
gain through
formis
is a 36-kDa
monomeric
enzyme that catalyzes the
oxidation of coelenterazine to yield coelenteramide and
chemical
reaction
blue light
with a spectral
maximum of 480 nm.5 It has
been used primarily as a co-reporter in conjunction with
• Lower
signal
(intensity),
firefly
luciferase.
For HTS,
it is used in but
a duallittle
luciferase
format to provide an internal control for identifying aber-
More on Chemiluminescence
to NO background = beter S/N
FIG. 1. Comparison of fluorescence (left) to luminescence (right). S0, ground state; S1, excited state after vibrational relaxation; S2, excited state.
(Figure 1) Citation 2
What is bioluminescence?
• A type of chemiluminescence that
occurs in living organisms
• Enzyme catalyzed reactions
– Enzymes: luciferases
• Firefly luciferase
• Renilla luciferase (sea pansy)
• Aequorin (jellyfish- Aequorea victoria)
– Substrates (photon-emitting): luciferins
Citation 2; Image taken
fromhttp://www.biosynth.com/index.asp?topic_id=119
Firefly Bioluminescence
• Light Intensity ~ Chemical Concentration
– [Chemical] of interest can be ATP, luciferin, or
luciferase (hold all others constant)
– Very large linear range
• Most common luciferase used in the
development of High-Throughput Screening
(HTS) Assays
Citation 2
Firefly Luciferase Catalyzed Rxn
Yellow-green
light λmax = 560
nm
(Figure 2) Citation 2
How do you detect the signal?
The GloMax® 20/20 Luminometer is designed to provide ultrahigh performance for bioluminescent and chemiluminescent
assays. In addition to high performance, the GloMax® 20/20
blends user-friendly operation and a small footprint with
flexible purchasing options. The result of this design is an
instrument with superior performance that is easy to use,
affordable, and can be customized to your lab’s needs.3
The GloMax®-96 Microplate Luminometer is a state-of-the-art
microplate luminometer that meets the requirement for high
sensitivity and broad dynamic range that is necessary for
chemiluminescent and bioluminescent applications. With
optional Single or Dual Auto Injectors, the GloMax®-96 is a
versatile luminescence system capable of performing both flash
and glow-type luminescent assays.3
Citation 3
How can this reaction be used in HTS?
130
Fan and Wood
(Figure 3) Citation 2
™
130
HTS: Luciferase Concentration
Fan and Wood
• Goal: investigate intracellular events by monitoring
gene transcription
• May include internal control (dual-luciferase assay)
• Simple and efficient (HTS)
• Commonly used for GPCR and nuclear receptor assays
(Figure 3) Citation 2
HTS: Luciferin Concentration
FIG. 3. Bioluminescent assays for HTS. luc, firefly luciferase gene; Ultra-Glo ™ rLuciferase, stable recombinant firefly luciferase (Promega, Madison, WI); MAO, monoamine oxidase.
• Luciferin is not naturally linked to physiology (vs. ATP)
• Use a Pro-luciferin
nilla luciferase) can help mitigate these false hits and imAn alternative strategy for generating dual luciferase
prove data quality. However, introducing both reporters assays was developed based on a standard two-plasmid
into the cell on the same plasmid backbone can result in approach (Fig. 4). One plasmid features both a firefly lu– Enzyme
interest
must
convert
luciferin
linkelement of interaberrant expression
due to of
cross
interference
between
ciferasethis
geneto
regulated
by the 
response
promoters and response elements (authors’ unpublished est (e.g., CRE or NFAT) and a hygromycin-selectable
luminescent signal to enzymemarker.
of interest
data).
The second plasmid expresses the target GPCR
FIG. 4. A diagram of two plasmids involved in
the dual-luciferase assay of GPCRs. RE, response
(Figure 3) Citation 2
HTS: ATP Concentration
•
Enzyme must be consistent!  Often use “stabilized” luciferase enzymes for HTS
•
Used in:
– Cytotoxicity screens  ATP concentrations ~ cell viability
– Kinase activity screens  all kinases consume ATP in phosphorylation rxn
– Real-time detection of ATPase activity4
–
FIG. 3. Bioluminescent assays for HTS. luc, firefly luciferase gene; Ultra-Glo ™ rLuciferase, stable recombinant firefly luciferase
(Promega,
Madison,
WI); MAO,
oxidase. faster than some dye assays used to look at cell
~ 100X
more
sensitive
andmonoamine
significantly
metabolism
nilla luciferase) can help mitigate these false hits and improve data quality. However, introducing both reporters
into the cell on the same plasmid backbone can result in
An alternative strategy for generating dual luciferase
assays was developed based on a standard two-plasmid
approach (Fig. 4). One plasmid features both a firefly lu-
(Figure 3) Citation 2
Examples of HTS assays
• Luciferase Enzymatic Activity
monitoring assays2
– Sensitive & broad detection range
• Protein- Protein interaction assays5,6
– BRET – Bioluminescence resonance
energy transfer
– PCA – Protein fragment
complementation assay
• Real-time bioluminescence to
analyze inhibitors of polymerases
(DNA & RNA)7
(Figure 2) Citation 6
Examples of HTS assays
• BL/CL recombinant whole-cell
biosensors
– Genetically engineered cells
– Create a luminescent signal ~ to
a specific analyte (analyte should
be regulating gene expression)
– Used for monitoring:
• Stress, oxidants, metals,
xenobiotics, receptor activating
molecules, etc.
(Figure 5), Citation 6
ATP Assay Kit - Demonstration
• ATP Determination Kit
– Molecular Probes (Invitrogen )
– Bioluminescence assay
– Quantitative determination of ATP concentrations
– Components:
• Recombinant firefly luciferase (enzyme)
• D-luciferin (substrate)
(Mg2+) (luciferase)
Luciferin + ATP + O2

oxyluciferin + AMP + pyrophosphate + CO2 + light
Citation 8
ATP Assay – Standards
ATP Determination Kit
• Advantages:
– Very sensitive
– Detect down to ~ 0.1 picomoles of ATP (must create a standard curve
within the desired range)
– Readily available & affordable
• Uses:
– Versatile (can be used to look at ATP production in different
enzymatic reactions) – NADPH, ATPase
– Detect contamination in a range of samples (milk, blood, sludge, etc.)
– Many others!
Citation 8
We can detect ATP. So what?
• ATP is required for cellular
metabolism9
• “… each human being recycles
the equivalent of his/her own
mass of ATP every day.”9
• Extracellular ATP
concentrations are critical in
biological receptor response10
Image from: http://www.bris.ac.uk/Depts/Chemistry/MOTM/atp/atp1.htm
Areas of interest for ATP quantitation
• The Mitochondria –
the “powerhouse” of
the cell
– Complex system
– ATP is produced and
sent to the cell
• Implications in
cardiomyopathies
Mitochondrial
ADP/ATP Carrier
(Figure 1) Citation 9
Rapid Hygiene Tester (Biothema)
• Detect ATP (quickly) down to
very low detection
– i.e. ATP in one animal cell (~ 1 pg)
• Also developed to test AMP as
well (bi-product of ATP
breakdown)
• Important in the food
production industry, labs,
hospitals, etc.
Citation 11
Conclusions
• Chemiluminescence and Bioluminescence are
common, extremely versatile, and useful analytical
tools
• HTS methods are becoming increasingly more
dependent upon this “background-free” technique
• The ATP Detection Assay could have huge implications
in pharmacology as it evolves for different types of
detection
Works Cited
1.
"Chemiluminescence." Lumigen, INC - A Beckman Coulter Company. Web. 07 Feb. 2011.
<http://www.lumigen.com/detection_technologies/chemiluminescence/>.
2.
Fan, Frank, and Keith V. Wood. "Technology Review: Bioluminescent Assays for High-Throughput Screening." ASSAY and Drug
Development Technologies 5.1 (2006): 127-36.Fan, Frank and Wood V. Keith. ASSAY and Drug Development Technolgoies. V5, N1. 2007.
3.
"Luminometer Comparison Chart of Microplate/Multiwell and Single Tube Luminometers from Promega." Promega Luminometers,
Fluorometers, and Multimode Readers. Web. 07 Feb. 2011. http://www.luminometer.com/instruments/luminometers-dual-luciferaseATP-ELISA.php?gclid=CIel3d-r9qYCFYbb4Aodw3MjFg.
4.
Karamohamed, Samer, and Guido Guidotti. "Bioluminometric Method for Real-Time Detection of ATPase Activity." BioTechniques 31
(2001): 420-25.
5.
Hoshino, Hideto. "Current Advanced Bioluminescence Technology in Drug Discover." Expert Opinion in Drug Discovery 4.4 (2009): 37389.
6.
Roda, Aldo, Patrizia Pasini, Mara Mirasoli, Elisa Michelini, and Massimo Guardigli. "Biotechnological Applications of Bioluminescence
Adn Chemiluminescence." TRENDS in Biotechnology 22.6 (2004): 295-303.
7.
Gregory, Kalvin J., Ye Sun, Nelson G. Chen, and Valeri Golovlev. "Real-time Bioluminescent Assay for Inhibitors of RNA and DNA
Polymerases and Other ATP-dependent Enzymes." Analytical Biochemistry 408 (2011): 226-34.
8.
"ATP Determination Kit (A22066)." Product Information: Molecular Probe: Invitrogen detection technologies. Revised 29-Nov-2005.
9.
Dahout-Gonzalez, C., H. Nury, V. Trezequet, J. M. Lauquin, E. Pebay-Peyroula, and G. Brandolin. "Molecular, Functional, and
Pathological Aspects of the Mitochondrial ADP/ATP Carrier." PHYSIOLOGY 21 (2006): 242-49.
10.
Seminario-Vidal, Lucia, Eduardo R. Lazarowski, and Seiko F. Okada. "Assessment of Extracellular ATP." Bioluminescence, Methods in
Molecular Biology 574 (2009): 25-36.
11.
"Can You Say It's Clean with Confidence?" BioThema - Luminescence Analysis, We Sell Our Kits and Reagents Worldwide. Web. 14 Feb. 2011.
<http://www.biothema.com/news_newsdetail.do;jsessionid=A047393FA5453A636E59B387B9B173B6?newsentryid=12〈=en>.
Works Cited – Other Helpful
Websites
• http://uvminerals.org/fms/luminescence
– Great website which clarifies VERY well the
differences in luminescence
• http://www.photobiology.info/Branchini.htm
– Great website to visit if you want a better
understanding of the chemistry
mid backbone can result in
ross interference between
ents (authors’ unpublished
approach (Fig. 4). One plasmid features both a firefly luciferase gene regulated by the response element of interest (e.g., CRE or NFAT) and a hygromycin-selectable
marker. The second plasmid expresses the target GPCR
Example of QC in GPCR assay
mids involved in
Rs. RE, response
bilized and optiPro-Glu-Ser-Thr
n virus 40 pronce gene; PCMV,
Neor, Renilla luce gene fusion.
with rapidly de-
• Dual-Luciferase assay
– Helps to account for interferences (i.e. from cytotoxic compounds) which could
effect results (false -/+’s)
– Plasmid 1 – firefly luciferase gene, marker, and response element of interest
– Plasmid 2 – GPCR of interest and Renilla luciferase marker fusion protein
(Figure 4) Citation 2
Example of Luciferin Detection (comparing
fluorescence and bioluminescence)
(Figure 7) Citation 2
Example of an Actual ATP Standard Curve
using an ATP Detection Kit
1600
Standard Curve at 560 nm
1400
Luminescence
1200
y = 1E+09x + 43.478
R² = 0.99675
1000
800
600
400
200
0
-2.00E-07 0.00E+00 2.00E-07
4.00E-07
6.00E-07
8.00E-07
Concentra on of ATP (M)
1.00E-06
1.20E-06