02.02.2011- 1 -

Luminescence

Presentation byKathrin Eder and Isabel Denzer

02.02.2011

02.02.2011- 2 -

Overview

1. Definition of luminescence

2. Luminescence process

3. Types of luminescence

4. History of luminescence

5. Bioanalytical application of luminescence

02.02.2011- 3 -

1. Definition of luminescence

“Luminescence means a process at which

electromagnetic radiation of the visual light

area is emitted through different occurrences.

Thereby the temperature of the process is clearly

below the glow temperature of the involved

substances. Therefore luminescence is also

known as “cold light”.”

02.02.2011- 4 -

2. Luminescence process

• An electron in the ground

state S0 is activated to an

excited state S1 by the

impact of energy.

• The excited state of the

electron has a higher

energy and is only stable

for a short time until the

electron goes back to the ground state S0.

• The energy difference between these two states is released in

form of a light emission.

02.02.2011- 5 -

Phosphorescence and fluorescence are related. Both processes belong to the same type of luminescence → photoluminescence.

Fluorescence:

• Fluorescence is a process where the excitation energy is absorbed from a source of light meaning a photon and the released energy is a photon, too.

Phosphorescence:

• Unlike fluorescence, a phosphorescent material does not immediately reemit the radiation it absorbs.

• The electron from the excited state S1 changes its spin and goes to a forbidden energy state T1: Inter system crossing (ISC).

• There is no light emission while the ISC.• The electron from the T1 state falls back to ground state and emits

light. • This process takes longer than fluorescence because the electron

has to change its spin again when it returns from T1 to S0.

02.02.2011- 6 -

3. Types of luminescence

photoluminescence (fluorescence, phosphorescence)

UV/Vis- radiation

Cathodoluminescence (television screen) cathode rays (electron radiation)

sonoluminescenceultrasonics

triboluminescence, crystal luminescence mechanic

electroluminescenceelectric

X-ray luminescence X- rays

radioluminescence, scintillationα-, β-, γ- radiation

thermoluminescencethermical

chemiluminescence, bioluminescence,

oscilloluminescence (oscillatingluminescence)

chemical

LuminescenceType of energy

02.02.2011- 7 -

Chemiluminescence

• Excitation is produced

in the course of a chemical

reaction.

• Thereby the energy is released

as light emission.

Example: oxidation of luminol,

disintegration of 1,2-dioxetanes

02.02.2011- 8 -

Bioluminescence

• Special form of chemiluminescence.• Mostly the chemical reaction leading

to excitation is caused through an enzymatic reaction.

• Limited to flora and fauna.• Example: bioluminescence in

beetles e. g. fireflies, bioluminescence of bacteria and fungi and bioluminescence of deep sea organism

02.02.2011- 9 -

4. History of luminescence

• The history of bioluminescence is notational recorded for 3500 years.

• H. BRAND obtained the first synthetic chemiluminescentcompound by evaporation of human urine under exclusion of air in 1669:

� (NaPO3)n is generated out of (NaNH4HPO4 x 4 H2O)

during the evaporation.

� (NaPO3)n is reduced to colourless elementary phosphor.

� The elementary phosphor emits the famous greenish blue light.

• SCHELLE prepared white phosphor by reduction of bone earth

with magnesia powder in laboratory scale in 1769:

2 Ca3(PO4)2 10 Mg 6 CaO 10 MgO P4

02.02.2011- 10 -

5. Bioanalytical application of luminescence

Bioluminescence systems

The luciferin/luciferase system of Photinus pyralis:

• Most efficient known bio- and chemiluminescent system.• First total synthesis of Photinus-luciferin worked out by

WHITE et al. in 1961. • The bioluminescence rapidly supplies a strong yellow green

emission in vitro and in vivo: attributed to the excited dianion.

• At temperature rise, addition of acid or urea, Zn2+ or Hg2+

ions a weak red luminescence occurs: referable to the excited monoanion.

02.02.2011- 11 -

• The oldest application of

this luminescence system

is the determination of the

ATP concentration.

• This bioluminescent system

is used as a screening

method for microorganism

in the clinical microbiology

alternatively in the monitoring

of hygiene and food.

02.02.2011- 12 -

Bacterial bioluminescence:

• The light producing system consists of two enzymes: a oxidoreductase and a bacterial luciferase.

• FMNH2 and a long-chain aldehyde (C10-C16) are involved in this process.

• The oxidoreductase catalyzes the reduction of FMN:

• FMNH2 reacts with the bacterial luciferase to an activated flavin-enzyme-complex which oxidates the aldehyde to the corresponding fatty acid under light emission by use of atmospheric oxygen.

• The emitted light has a greenish blue colour.• Used as sensible detection system for oxygen.• In 1982 the first successful transfer of luciferase gene out of marine

light bacterium Vibrio harveyi into the genome of the usually not shining bacterium Escherichia coli worked out.

02.02.2011- 13 -

Chemiluminescence systems

Luminol:

• The oldest known and longest examined chemiluminescence reaction.

• Emission of brilliant blue light during theoxidation of an alkaline solution of luminolwith hydrogen peroxide and an one-electron-cooxidans.

• In aqueous solutions the luminolchemiluminescence reaction can also be advantageously catalyzed by enzymes like catalases and peroxidases.

• 3-Aminophthalate-dianion is the emitting species.

NH

NH

O

O

NH2

Luminol

02.02.2011- 14 -

Applications of the luminol system:

• The luminol luminescence is used in acid-base titration of severe dyed solutions.

• A variety of inorganic ions that have a catalytic or an inhibitory influence on the luminol reaction can be proved.

• One of the oldest application fields of the luminolchemiluminescence is the criminology and the medicine for the proof of occult blood traces.

02.02.2011- 15 -

Overview of chemi- and bioluminescence

measurements:

• Detection of toxic trace elements

• Detection of toxic aromatic compounds

• Detection of aminoacids

• Detection of NOx• Detection of SOx

• Immunoassays• Ligands binding assays• Cell metabolism assays• Lipid peroxidation

assays• Enzyme-catalysed

reactions• Detection of small bio-

molecules• Western blot

• DNA-hybridisationassays

• Northern blot• Detection of PCR

products• Southern blot• Search for mutations• Reporter gene

assays including luminography of transfected cells

Chemi- or bioluminescence measurements

chemical-analyticaltechnologies

biochemical technologies molecularbiologicaltechnologies

02.02.2011- 16 -

Fields of application:

• Genetics → reporter gene-assays• Medical basic research• Diagnostics• Clinical Chemistry

- Application in established routines- Immunochemistry

02.02.2011- 17 -

Examples for some application fields:

• Test with luminous bacteria– Short-time test to analyse waste water

– Chemical contaminations intensity of light decreases

– cheap, fast, easy handling

– you can measure disturbing substances and interactions between substances

02.02.2011- 18 -

• GPCR-Signaling– Changes of cAMP levels

(=hallmark (Kennzeichen) of GPCR modulation) are measured with an assay kit

– If cAMP level increases intensity of light decreases

– Advantages: High sensitivity, homogenous, amenable to automation, cost effective, broad linear range of detection, fast, whole cells can be used, no radioactivity

– Disadvantages: Temperature-and light-sensitive, colour quenching

02.02.2011- 19 -

• Flow Injection technique with chemiluminescentdetection– Technique for rapid, automated and quantitative analysis

– Advantages: rapid, cheap, broad spectrum of applications

– Disadvantages: limit of detection, interactions → wrong results

2 x 10-7 mol/dm3Cerium(IV)–sulphuric acid CL

Pharmaceuticalpreparations

Captopril

8.6 x 10-9

mol/dm3Luminol–sodium

hydroxide–hydrogen peroxide

CL

TabletsAscorbic acid

Limit of detectionReactionSample matrixAnalyte

Pharmaceutical applications CL = chemiluminescence

02.02.2011- 20 -

Limit of

detection

ReactionSample

matrix

Analyte

1.6 ng/mL

Luminol–I2 (produced frompotassium iodide in acid) CL

Drinkingwater and

food

Nitrite

0.1 mg/mL

Microwave digestion in HNO3 andhydrogen peroxide. Detection using

alizarin purple–ethanol cetyltrimethylammonium

bromide–potassiumhydroxide

HairCo(II) and Ni(II)

0.31 ng/mL

Gallic acid–hydrogen peroxide–sodium hydroxide CL

River/wastewater

Acetaldehyde

Environmental applications

02.02.2011- 21 -

Limit of

detection

ReactionSample

matrix

Analyte

1 x 10-7

mol/dm3Immobilized L-lactate oxidase to produce hydrogen peroxide for

luminol–horseradishperoxidase–luminol CL detection

Food samples

L-lactate

0.1 mg/mL

Acidified permanganate–luminolCL

VegetablesAscorbic acid

2.7 x 10-7

mol/dm3Inhibition of the luminol–hydrogen

peroxide–Co(II) CLWines3,4-

Dihydroxybenzoic acid

Food and beverage applications

02.02.2011- 22 -

References

• http://www.pinktentacle.com/images/firefly.jpg• http://www.chids.de/dachs/expvortr/642/LumiDateien/einleitung.htm• http://www.uni-

siegen.de/fb8/ac/wickleder/lehre/bachelor/vortraegeac10910/lumineszenz.pdf• http://www.chemie.uni-jena.de/institute/oc/weiss/lumineszenz.htm• http://www.itp.uni-hannover.de/~zawischa/ITP/lumineszenz.html• http://de.academic.ru/pictures/dewiki/76/Luminol.jpg• http://www.chemiedidaktik.uni-wuppertal.de/alte_seite_du/material/lichtsp/info24a.htm• Albrecht, Brandl, Zimmermann, Chemilumineszenz, 1996, Hüthig GmbH Heidelberg.• Brolin, Wettermark, Bioluminescence Analysis, 1992, VCH Verlagsgesellschaft mbH,

Weinheim. • http://www.bmglabtech.com/images/apps/an147-3.jpg• http://www.genengnews.com/gen-articles/assay-miniaturizing-gpcr-signaling-

studies/2074/• http://www.genengnews.com/Media/images/Article/block_4746.jpg• http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/8/bc/vlu/biokatalyse_enzyme/che

milumineszenz.vlu/Page/vsc/de/ch/8/bc/biokatalyse/luci_assay.vscml.html• Stratis-Cullum, D.N.; Griffin, G.D.; Anal Bioanal Chem, 2008, 391:1655–1660.• http://de.academic.ru/pictures/dewiki/76/Luciferin_bacterial_reaction.png• http://enfo.agt.bme.hu/drupal/sites/default/files/Vibrio%20fischeri%20lombiban.png

02.02.2011- 23 -

Thank you for yourattention!

02.02.2011- 24 -

Zusammenfassung

• Lumineszenz ist die optische Strahlung eines physikalischen Systems, die beim Übergang von einem angeregten Zustand zum Grundzustand entsteht.

• Es gibt verschiedene Arten von Lumineszenz.• Für die Bioanalytik sind Bio- und Chemilumineszenz von

Bedeutung:– Biolumineszenzsysteme:

• Luciferin/Luciferase-System von Photinus pyralis

• Biolumineszenz von Bakterien– Chemilumineszenzsystem:

• Luminol

• Anwendungsbereiche:– Chemisch-analytische Technologie– Biochemische Technologie– Molekularbiologische Technologie

02.02.2011- 25 -

Zusammenfassung

• Anwendungsbeispiele:– Abwasseruntersuchung mit Leuchtbakterien– Untersuchung der Signalweiterleitung von GPCRs– Fließinjektionsanalyse mit Chemilumineszenz-Detektor zur

Anwendung in der Pharmazie, Umweltanalytik und Lebensmittelchemie.

• Vorteile:– Lumineszenzmarkierung kann die radioaktive Markierung

ersetzen.– Breites Anwendungsspektrum– Viele Methoden sind schnell, günstig und einfach.– Assays können auch an lebenden Tieren durchgeführt werden.

• Nachteile:– Nachweisbarkeit ist limitiert.– Wechselwirkungen zwischen Substanzen können das Ergebnis

verfälschen.