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

More on ChemiluminescenceFluorescence Chemiluminescence

• See energy gain through

chemical reaction

• Lower signal (intensity), but

little to NO background =

beter S/N

• Light must be absorbed by

fluorophore to reach

excited state

• Brighter, but MORE

background

(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&g=19&m=264:

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

(Figure 2) Citation 2

Yellow-green light λmax =

560 nm

How do you detect the signal? The GloMax® 20/20 Luminometer is designed to provide

ultra-high 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?

(Figure 3) Citation 2

HTS: Luciferase Concentration

• 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

• Luciferin is not naturally linked to physiology (vs.

ATP)

• Use a Pro-luciferin

– Enzyme of interest must convert this to luciferin link

luminescent signal to enzyme of interest

(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

– ~ 100X more sensitive and significantly faster than some dye assays used to look

at cell metabolism(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)

Luciferin + ATP + O2

oxyluciferin + AMP + pyrophosphate

+ CO2 + light

(Mg2+) (luciferase)

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-luciferase-ATP-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): 373-89.

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

Example of QC in GPCR assay

• 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