Transcript Luminescence

detect and identify
Luminescence
Bioanalytik
Light
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Two ways of light emission:
“Hot” Light: Thermal excitation of atoms or
molecules (eg incandescent
lamp)
“Cold” Light: Non-thermal excitation of
molecules (eg Fluorescence,
luminescence)
Luminescence
Bioanalytik
In Chemiluminescence the source of energy
to excite the molecule is a chemical reaction,
frequently oxidation.
In Bioluminescence the energy generating
chemical reaction is catalysed by an enzyme
Examples of Bioluminescence
Arabidopsis
Ctenophore
Deiopea
Photinus
pyralis
Bioanalytik
Examples of Chemiluminescence
1,2-Dioxetanes
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Glow
Luminol (Enhanced Chemiluminescence) Glow
Acridinium Ester
Flash
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Cellular Luminescence /Phagocytosis
in fact CL but applied to living cells
usually injection of stimulus
luminol
or
lucigenin
Flash and Glow Type Kinetics
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For chemistry of Acridinium Ester and
other flash kinetic reactions (e.g.
Aequorin based Calcium assays) reagent
injectors are required
Reagent Injectors
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injection of 10 - 100 µl may
take ~ 100 ms (> time for
flash to reach maximum):
•intrinsic rise-time of
flash is slowed down
•until injection is
completed, mixture is in
a transient state of
rapidly changing
concentrations
Reagent Injectors 2
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good precision only with reproducible volume, time
and pressure profile
rotation and shaking do not mix fast enough
“jet” type injection with 98 - 99 % precision
sequential luminescent reactions like DLR assay
injection of a internal standard following for e.g.
low amounts of ATP or simultaneous measurement
of ATP, ADP and AMP
materials used
TEFLON, TEFZEL
Polypropylen
Advantage: stainless steel can harm live cells
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For chemistry of Enhanced Luminol and
other Glow kinetic reactions reagent
injectors are NOT required
Advantages of Glow chemistry
Instrumentation is much simpler
Samples can be re-measured
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Dis-Advantages of Glow chemistry
Sensitivity is usually less good
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Advantages of Chemiluminescence
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Best sensitivity with large dynamic range
Non radioactive
Low cost
Low background
No protein-quenching problems (fluorescence)
Dynamic Range
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Luminescence
Log RLU/OD
Colourimetric
Log Concentration
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How is the light produced?
Excited State
Chemical Energy
Light
Ground State
Example of a Reporter Gene Reaction Bioanalytik
Light
Excited states: Luciferin-Luciferase-AMP complex
Oxy-Coelenterazine-Luciferase complex
Light Detection
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PhotoMultiplier Tube PMT
photocathode
dynode 2
anode
dynode 1
photoelectric effect
release of 4 - 6 secondary electrons per dynode
up to 14 dynodes
Spectral sensitivity
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30
RbCs
QE [%]
20
10
0
300
500
700
wavelength [nm]
900
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Types of measurement modes
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current measurement: output signal depends on
applied high voltage
amplifier gain
digitizing factor converting current (voltage) into
RLU´s
photon counting:
released photoelectrons = photons(cathode) x QE
stability of signal (less temperature dependent)
so-called RLU-factors for identical RLU reading of
different luminometers
lower dark current (1/2 of current mode)
possibility to discriminate background via
threshold
Amplitude-Time Function
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pulse height
3 categories of pulses:
•Amplitudes between A + B
signals (RLU)
•Smaller than B: noise events
from dynodes
•Greater than A: radioactivity
B
in material, cosmic radiation
time
Dynamic Range
Photon Counting
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> 106 decades/orders of magnitude
Itrue =
Im
1 - Im
Imax
photon counter:
saturation effect due
to its dead-time
approx. Im/Itrue = 0.5
Dynamic Range
Current Counting
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Relative Light Units/s [RLU/s]
10000000
1000000
100000
10000
Photon Counting
1000
100
10
1E-18
1E-17
1E-16
1E-15
1E-14
1E-13
1E-12
ATP [moles]
1E-11
1E-10
1E-09
1E-08
Advantages of Photon Counting
vs Current Measurement
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No need to calibrate
No need to set HV range
Single Dynamic range (current mode typically has 3 settings)
More stable backgrounds
Improved sensitivity
Instrument is stable over years
Detection Limit
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minimum amount of analyte detectable
characteristic of both luminometer and reagent
quality
A * 3BG
Sometimes 2 is used
S=
SA - SBG
S = sensitivity (same unit as A)
A = amount/concentration of analyte in g, mol ...
SA= result of measurement of A in cps
BG = mean value for background in cps
BG = standard deviation of background
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Some competitors claim they use
photon counting
but do not !
If we are measuring the light we
are in a sense counting the
photons
If they have to set the
High Voltage then Current
mode is used
Units
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RLU ... Relative Light Units
a priori: one cannot expect identical
RLU readings from different
luminometers when
measuring the same sample
Crosstalk (X-talk)
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spillover of light between
wells
quasi constant signal Sm
apparent signals in
neighboring wells
Sd diagonal position
Sa adjacent position
CTa = Sa / Sm
CTd = Sd / Sm
Cross talk is minimized by:
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Design of the detector optics
Using opaque microplates
White (or black)
Cross talk is <10-5
Black plates at least 10x lower signal
Compared to white plates