Prepared by-

Vedshree Raole

PQA, M.PHARM 1

• Introduction

• Principle

• Instrumentation

• Applications

Contents

What happens to the EM energy subjected on a sample?

After the absorption of ultraviolet-visible light, the excited

molecules species are extremely short-lived and deactivated

occurs due to:

a. Internal collisions(internal conversion)

b. Cleavage of chemical bonds, initiating photochemical reaction

c. Re-emission as light( luminescence)

Re-emission of energy as luminescence occurs from molecules

in which the electron system is shielded from normal

deactivation processes so that complete deactivation by

collisions is discouraged.

Introduction

• Fluorescence

– Some energy is lost through various processes (e.g.

non-radiative transitions) and then light is given off.

• Phosphorescence

– The molecule transitions from an excited triplet state

to an lower energy singlet state and gives off light.

Non-radiative transitions intervene.

10-5 to 10-8 s fluorescence

10-4 to 10 s phosphorescence10-14 to 10-15 s

- Excitation of e- by absorbance of hn.

- Re-emission of hv as e- goes to ground state.

- Use hn2 for qualitative and quantitative analysis

• Luminescence

- A spontaneous emission of radiation

from an electronically excited species (or

from a vibrationally excited species) not in

thermal equilibrium with its environment.

If the excitation energy is obtained from

chemical energy of reaction the process is

termed as Chemiluminescence

A + B C* C + hn

Criteria for a “successful” chemiluminescent

reaction

1. Sufficient excitation energy provided (by the chemical

reaction) for red emission (λ = 600 nm) 47.6 kcal/mol for

blue emission (λ = 450 nm) 63.5 kcal/mol

• The liberation of this much energy usually comes from

bond cleavage or electron transfer.

2. Formation of products capable of forming an excited

state multiple bonds, conjugation, aromatic systems.

3. Presence of an emitter (doesn’t have to be a product)

multiple bonds, conjugation, aromatic systems

4. Rapid kinetics (of the chemical reaction)

rate is more important than high yield (φCL)

φCL = φrxn(rxn) × φf(fluor.) << 10% (typical)

5. Reaction coordinate system must favor formation of an

excited state over ground state of product(s)

• Chemiluminescent reactions can be grouped

into three types:

1. Chemical reactions using synthetic compounds

and usually involving a highly oxidized species

such as a peroxide are commonly termed

chemiluminescent reactions.

2. Light-emitting reactions arising from a living

organism, such as the firefly or jellyfish, are

commonly termed bioluminescent reactions.

3. Light-emitting reactions which take place by

the use of electrical current are designated

electrochemiluminescent reactions.

• Chemiluminescent and bioluminescent reactions usually

involve the cleavage or fragmentation of the O-O

bond an organic peroxide compound.

• Peroxides, especially cyclic peroxides, are prevalent in

light emitting reactions because the relatively weak

peroxide bond is easily cleaved and the resulting

molecular reorganization liberates a large amount of

energy.

• Some electron transfer reactions ( such as with aromatic radical ions from rubrene or p-benzoquinone or from metal complexes such as tris(2,2’-bipyridyl) ruthenium(II) ) result in CL emission without bond cleavage or rearrangement, so those systems can be recycled, as in common with some types of electrogenerated chemiluminescence.

• In order to achieve the highest levels of sensitivity, a

chemiluminescent reaction must be as efficient as

possible in generating photons of light. Each

chemiluminescent compound or group can produce no

more than one photon of light.

Luminol and peroxidase before adding H2O2

Chemiluminiscence after addition H2O2

Luciferin + O2

LuciferaseO C

O O

C R2

R1

SpontaneousCO2 + O C*

R2

R1

Light

• Light emitted by the chemical reaction can e

detected and measured by various devices such as

photomultiplier tube, photographic films,

photocells and multiple channel detectors.

• The attractiveness of Chemiluminescence as an

analytical tool is the simplicity of detection. The

fact that a chemiluminescent process is, by

definition, its own light source means that assay

methods and the instruments used to perform them

need only provide a way to detect light and record

the result.

• Luminometers need consist of only a light-tight

sample housing and some type of photo-detector.

Instrumentation

• Microwell plates :- Light emitted in chemiluminescent

reactions is isotropic - it is emitted equally in all

directions. If a chemiluminescent assay were conducted

in the wells of a trans-parent microwell plate, light

would radiate out not only vertically, in the direction

of the detector, but also laterally in the direction of

other wells. Light is easily transmitted through the inter-

well gaps and throught the plate material itself, a

phenomenon termed light piping. Relatively bright

wells will introduce significant interference in adjacent

wells and beyond.

• For this reason,

CHEMILUMINESCENCE SHOULD NEVER BE MEASURED

IN CLEAR PLATES.

• Opaque microwell plates and strips are commercially

available from several suppliers. They come in two

kinds - white and black.

• Photomultiplier tubes have traditionally been the

workhorse detector in luminometers. Their advantages

include good sensitivity, a broad dynamic range and

applicability over a reasonably broad spectral range.

PMTs are known for their very low dark currents leading

to excellent signal to noise for low intensity samples.

• PMT based systems operate in two basic modes, single

photon counting and current sensing. There are examples

of hybrid systems which are single photon counting to a

light level in the low millions of photons/second and then

switch to current sensing above that level.

Photomultiplier-based Detection

• PMT single photon counting systems are capable of

exquisite sensitivity. Use of this type of detector is

the method of choice for low light detection and

quantitation as in, for example, detecting the

ultraweak luminescence associated with

phagocytosis. The greater sensitivity comes at a

cost however. Sample housings must be very light-

tight. Moderate light levels saturate the detector;

high levels can cause physical damage.

• PMT current sensing systems are also capable of

excellent sensitivity and will often read higher light

levels than single photon counting systems without

damage.

Photodiodes are capable of recording higher light intensities than

photomultiplier tube detectors. This facet makes them an excellent

choice for applications where high light intensities are to be

measured. However, the inherent dark current in solid state detectors

is generally much higher than that of photomultiplier tubes. One

method of mitigating this problem is to cool the solid state detector

via a Peltier or other thermoelectric cooler. Dark currents in solid

state detectors drop dramatically with temperatures in the 0 to -30

degree celsius range. Cooled detectors can then be used to integrate

the light intensity for one to hundreds of seconds without the signal

being overwhelmed by dark current.CCD and other solid state

detectors, although , possess several inherent advantages.

1. Solid state detectors typically offer a "flatter" optical response over the

visible range. Luminescent reactions emitting red and even near

infrared light can be detected with enhanced sensitivity.

2.Camera systems allow imaging of variety of objects. Virtually any kind

of sample or container can be accommodated ranging from

microwell plates and test tubes to bacterial or cell cultures in Petri

dishes, electrophoresis gels and blotting membranes.

Solid State Detection

3. Single PMT systems must have the sample position well

defined before it can be read. A sample tube has to be

brought to a reproducible position to be read

repeatably. Microwell plate PMT readers rely upon the

standard spacing of microwells and will generally move

the plate around to a precalculated position so the

wells can be read one by one. Camera systems have

the advantageof being able to read a sample without

knowing its position in advance, as in the example of a

band on a blot. The camera imaging system gives

positioning information along with sample intensity.

4. CCD camera systems allow imaging of numerous

objects simultaneously. In the present era of 96, 384

and higher number well plates, parallel data collection

is no longer a luxury. Solid state camera imaging

systems have the potential to permit imaging and

quantitation of entire plates in one pass.

APPLICATIONSChemiluminescence immunoassay

DNA hybridization detection

Western blotting

Forensic science

Food analysis

Provides a sensitive, high throughput alternative to

conventional colorimetric methodologies

Principle: -same as ELISA

-uses chemiluminescent substrate,

hydrogen peroxide, enhancers

-stopping reagent is not required

-Incubation period is small

CHEMILUMINESENCE IMMUNOASSAY

Monoclonal antibody coated well

Test specimen (serum)

HRP labelled antibody conjugate

Test antigen: sandwich between solid phase

Ab and enzyme labelled Ab

Incubate for 1 hr at 37° C

Remove unbound enzyme labeled Ab

Chemiluminescence reagent added

Read relative light unit with luminometer

• The widely used enzymes for luminescent

immunoassays are AP(Alkaline

phosphatase) and HRP(Horseradish

peroxidase) and pyruvate kinase(in

bioluminescence)

The suitable substrates for luminescent

immunoassays include

(i) luminol (ii) acridine esters (iii)Isoluminol

(iv)Luciferin (v)AMPPD

• USES

Hormones: insulin, thyroxin, estradiol,

testosterone, progesterone

Vitamin: Vit B12

Tumor markers: bone morphogenic

protein-2, carcino embryonic antigen

(CEA), alpha fetoprotein (AFP)

Human beta chorionic gonadotropin

C-reactive protein

Tumor necrosis factor

DNA hybridization detectionSouthern blotting Involves DNA separation,

transfer and hybridization

Hybridization - Process of forming a double-

stranded DNA molecule between a single-

stranded DNA probe and a single-stranded

target DNA

3.The restriction

fragments present in

the gel are denatured

with alkali and

transferred onto a

nitrocellulose filter or

nylon membrane by

blotting.

• This procedure

preserves the

distribution of the

fragments in the gel,

creating a replica of

the gel on the filter.

4. The filter is incubated

under hybridization

conditions with a specific

HRP labelled DNA probe.

• The probe hybridizes to

the complementary DNA

restriction fragment.

• Detection reagent

containing H2O2 &

luminol is added onto the

membrane

• USES

Identifying DNA in crime case

Paternal Dispute

Classify DNAs of various organism

Steps in southern blotting

1.The DNA to be

analyzed, such as the

total DNA of an

organism, is digested

to completion with a

restriction enzyme.

2.The complex mixture

of fragments is

subjected to gel

electrophoresis to

separate the fragments

according to size.

Western blottingWestern blotting (or protein

immunoblotting) is a technique widely used

to detect specific proteins in samples of

tissue homogenate, cell lysates, cell culture

supernatants or body fluids.

Western blotting

Forensic scienceChemiluminescence is used by criminalists to

detect traces of blood at crime scene

Solution: luminol powder (C8H7O3N3),

hydrogen peroxide, and a hydroxide (eg.

KOH) sprayed where blood might found

Tiny amount of iron from Hb in blood serves

as catalyst for the chemiluminescence

reaction, luminol to glow

A trail of blood made visible with the use of the reagent luminol.

Food analysis

Organophosphorous most popular

pesticide

Most commonly used

organophosphorous: QUINALPHOS (O,O

diethyl-o-quinoxalinly phosphorothioate)

Quinalphos +H2O

2 peroxophosphonate

Peroxophosphonate+ luminol 3aminophthalate

anion*

3-aminophthalate* 3-aminophthalate

+

observed emission

Food analysis

Clinical Utilities

• Pesticide Analysis

Other Applications

Determination of nitrogen monoxide

NO + O3→ NO

2* + O

2

NO2* + → NO

2+ hn ( = 600 – 2800 nm)

Determination of sulfur

4H2

+ 2SO2→ S

2* + 4H

2O

S2* → S

2+ hn ( = 384 and 394 nm)

• Light leaks from assay reagent & reaction

vessels

• Ultra sensitive – stringent controls on

purity of reagents

• High intensity light emission leads to pulse

pileup in photomultiplier tubes leads to

underestimation

Limitations

1. Estimation of glipizide by CL:

• Luminol + K3Fe(CN)

6 3-aminophthalate ion* (3-

AP*)

3-AP* 3-AP hv (λmax

= 425 nm)

• GP + dissolved oxygen + NaOH

superoxide radical + other reaction product

• Superoxide radical + luminol + K3Fe(CN)

6(3-AP*)

• 3-AP* 3-AP + hv (λmax =425 nm)

Contd...Other Applications

• The fluorescence spectra of luminol,luminol–

K3Fe(CN)6, and luminol– K3Fe(CN)6–GP reaction

were scanned in the range of 280– 650 nm, using

an RF5301 fluorescence spectrophotometer. It had

been reported that 3-aminophthalate ion(3-AP),an

oxidized product of luminol, peaking at 425nm

was known as the emitter in the luminol–

K3Fe(CN)6 system. The results obtained were all

found to have the same maximum emission

appearing at 425nm; the luminophor was

confirmed to be 3AP. This indicated that the

luminant in the luminol– K3Fe(CN)6–GP system

was still 3-AP.

• The CL reaction of luminol with superoxide radical

can be catalyzed by potassium ferricyanide in

alkaline solution.

2. Estimation of Atropine

• A sensitive and fast chemiluminescence (CL)

method has been described for the direct

determination of atropine. The method is

based on the quenching effect of atropine on

the CL signals of the luminol– H2O

2reaction

catalyzed by hemin in an alkaline medium.

Hemin could strongly enhance the

chemiluminescence of the luminol–H2O

2

system. The luminophor of the luminol–

H2O

2–hemin CL system was identified as the

excited state 3-aminophthalate anion. The CL

from luminol–H2O

2–hemin system is strongly

inhibited by the presence of atropine.

3. Estimation of Vitamin B12

4. Estimation of Estrogen:

Adsorption of H2O

2on the surface of gold

nanoparticles, we can deduce there must

be competitive adsorption on the surface

of gold nanoparticles between estrogens

and H2O

2, which leads to inhibition of CL.

Estrogens can, on the other hand, react with

hydrogen peroxide or oxygen-related

radicals generated from H2O

2, which might

consume the oxidant and inhibit CL

intensity.

• It has been found that gold nanoparticles

(nano- Au) enhance the chemiluminescence

(CL) of the luminol-hydrogen peroxide system

and that estrogens inhibit these CL signals in

alkaline solution. CL spectra, UV–visible

spectra, X-ray photoelectron spectra (XPS),

and transmission electron microscopy (TEM)

were used to investigate the mechanism of the

CL enhancement. On the basis of the

inhibition, a flow-injection CL method has

been established for determination of three

natural estrogens.

• This method has been used for analysis of

estrogens in commercial tablets and in urine

samples from pregnant women.

5. DNA hybridization by electro-chemiluminescence

Quantum Dots ECL in aqueous solution and SWASV*

were employed for DNA detection. Through the

biotin–avidin-system, avidin–QDs bind tightly to

the biotin-modified cDNA oligonucleotides. By

utilizing the ECL property of QDs, the DNA

hybridization event was converted into a

detectable ECL signal. On the other hand, by

detecting cadmium content in the bound QDs, the

target DNA was indirectly detected through the

SWASV assay. It provided a relatively simple, time-

saving and multi-approach for DNA detection.

*SWASV :square wave anodic stripping voltammetric technique.

6. TOPICAL COMPOSITIONS AND METHODS

OF DETECTION AND TREATMENT

A topical composition includes a nanoemulsion

of a plurality of hydrophobic particles having

a hydrophilic coating therein. The

hydrophobic particles are derived from the

same or different hydrophobic material and

each hydrophobic particle has a melting point

below the melting point of the respective

hydrophobic material. The nanoemulsion

further includes one or more pharmaceutically

active agents and/or one or more

chemiluminescent disease-detecting systems.

• The nanoemulsion can also include one or more

chemiluminescent disease-detecting systems incorporated

therein for detecting a disease or condition in a host. For

example, hydrogen peroxide (H202) is a reactive oxygen

metabolic by-product that can serve as a key regulator for a

number of oxidative stress related states. Hydrogen

peroxide is believed to be over-produced by cells at the

early stages of most diseases such as asthma, infammatory

arthritis, athero sclerosis, diabetic vasculopathy,

osteoporosis, and a number of neurodegenerative diseases.

• overproduction of hydrogen peroxide can occur in the

development of damage caused to skin by exposure to

ultraviolet radiation. Thus, detecting low levels of hydrogen

peroxide in the skin could serve as an early Warning

indicator for skin cancer. The topical composition of the

present invention can therefore be used as a simple, all-

purpose diagnostic tool for detecting diseases.

• chemical reaction that produces

chemiluminescence. In general, the

mechanism is that first the phenyl oxalate

ester and hydrogen peroxide (H202) react to

form a peroxy acid ester and phenol; and

then the peroxy acid ester decomposes to

form more phenol and a highly energetic

intermediate, presumed to be a cyclic

compound containing a four-membered ring

dimer of CO2.As the cyclic dimer decomposes

into two CO2 molecules, it gives up its energy

to a waiting dye molecule, Which then

fluoresces.(fig.1)

• the chemical reaction taking place includes a

solution of a phenyl oxalate ester (commonly

bis(2,4,5-trichlorophenyl-6-

carbopentoxyphenyl)oxalate (CPPO), a fluorescent

dye that determines the color of light, and

hydrogen peroxide (H2O2). The hydrogen

peroxide reacts With the phenyl oxalate ester

producing carbon dioxide, a phenol and, most

importantly, releasing energy. This energy is

absorbed by the dye, exciting electrons in the dye’s

molecules to a higher energy level.

• Once at the higher energy level, the electrons

immediately lose the energy they absorbed and fall

to lower energy levels. As the electrons fall back to

loWer energy levels, the energy that is lost is

transformed into electromagnetic radiation, some

of Which is visible light.(fig. 2)

7. A simple and rapid flow injection (FI) method for the

determination of retinyl acetate is reported based on its

enhancing effect on the luminol-periodate chemiluminescence

(CL) system in an alkaline medium.

8. A simple and rapid flow-injection method is reported for the

determination of retinol, its derivatives and a-tocopherol by its

enhancement effect on a potassium permanganate–formaldehyde

chemiluminescence system in an acidic medium.

9. A novel chemiluminescence (CL) system was evaluated for the

determination of hydrogen peroxide, glucose and ascorbic acid

based on hydrogen peroxide, which has a catalytic-cooxidative

effect on the oxidation of luminol by KIO(4). Hydrogen peroxide

can be directly determined by luminol-KIO(4)-H(2)O(2) CL

system. The present method provides a source for H(2)O(2),

which, in turn, coupled with the luminol-KIO(4)-H(2)O(2) CL

reaction system. The CL was linearly correlated with glucose

concentration. Ascorbic acid was also indirectly determined by the

suppression of luminol-KIO(4)-H(2)O(2) CL system.

10. The proposed CL-FIA system has been applied to the

determination of sulphadiazine (a sulphonamide

mainly used in the treatment of urinary tract infections

for human and veterinary use) using bis[2,4,6-

trichlorophenyl]oxalate (TCPO) as CL precursor, H2O2

as oxidant, imidazole as a catalyst and fluorescamine as

the fluorescent derivatizing agent.

• The use of the PO-CL reaction in micellar medium,

coupled to a FIA manifold has been proposed as an

alternative detection system for quality control of

sulphonamides in pharmaceutical samples. The method

implies the off-line formation of a fluorescent

derivative (fluorophore) with fluorescamine and the

subsequent oxidation of TCPO by H2O2 using

imidazol as catalyst in alkaline medium, in presence of

the fluorophore, whose CL emission is proportional to

the sulphonamide concentration.

• Development of a novel luminol

chemiluminescent method catalyzed by gold

nanoparticles for determination of estrogens

Yongxin Li & Ping Yang & Po Wang & Lun Wang.

• Luminol–K3Fe(CN)6 chemiluminescence system

for the determination of glipizide Xin Chena, Li-

LiXingb, Yu-HaiTangb,n, Guang-BinZhang.

• Quantum dot-based DNA hybridization by

electroc-hemiluminescence an anodic stripping

voltammetry Haiping Huang, Jingjing Li, Yanglan

Tan, Jinjun Zhou and Jun-Jie Zhu*

Refereces

• Analytical Applications of Flow Injection

Chemiluminescence for the Determination of

Pharmaceuticals–A Review Amir Waseema*,

Mohammad Yaqoobb and Abdul Nabib.

• Vitamin A Determination in Milk Samples Based on the

Luminol-Periodate Chemiluminescence System Lubna

Rishi1, Mohammad Yaqoob1, Amir Waseem2,* and

Abdul Nabi1.

• Evaluation of luminol–H2O2–KIO4 chemiluminescence

system and its application to hydrogen peroxide,

glucose and ascorbic acid assays Yanxiu Zhou ,Tsutomu

Nagaoka,Feng Li,Guoyi Zhu

• Flow injection methods for the determination of

retinol and α-tocopherol using lucigenin-enhanced

chemiluminescenceMohammad Asgher,Mohammad

Yaqoob,A. Waseem,Abdul Nabi

• Chemiluminescence determination of sulphadiazine in

drugs by flow injection analysis using the peroxyoxalate

reaction in micellar medium. Giuseppe Lattanzio a, Ana

M. Garc´ıa-Campa˜na b,∗, Jorge J. Soto-Chinchilla

b,Laura G´amiz-Gracia b, Steffano Girotti a.

• Chemiluminescence Determination of Atropine using

Luminol-Hemin-H2O

2 System Seyed Naser Azizi*,

Mohamad Javad Chaichi, Maryam Heidarpour.

• Chemiluminescence-based detection: principles and

analytical applications in flowing streams and in

immunoassays W.R.G. Baeyens a,*, S.G. Schulman b, A.C.

Calokerinos c, Y. Zhao a, A. Ma Garcı´a Campan˜a d, K.

Nakashima e, D. De Keukeleire f

• Chemiluminescent flow-through sensor for 1,10-

phenanthroline based on the combination of molecular

imprinting and Chemiluminescence Jin-Ming Lin* and

Masaaki Yamada.

• Chemiluminescence Analyses of Biological Constituents

using Metal-Complex Catalysts -A Review,Tadashi HARA

and Kazuhiko TSUKAGOSHI.

• Effect of Catlyst on Luminol - Hydrogen Peroxide–Water

Chemiluminescence System V.K. Jain*

• Topical compositions and methods of detection and treatment Jennifer L. Sample, Chevy Chase, MD (US); Julia B. Patrone, Ellicott City, MD (US); Jason J. Benkoski, Ellicott City, MD (US); James C. Crookston, Falls Church, VA (US); Huong Le, Olney, MD (US); Jennifer L. Breidenich, Atlanta, GA (US); Lisa A. Kelly, Ellicott City, MD (US)(PATENT 2012)