Detection systems Flashcards
uses what as labels
Detection systems RadioImmunoassay
Uses radioactive isotopes as labels
RADIOIMMUNOASSAY: RIA
Are what
Adapted to many procedures
- Very sensitive ~ 5 pg/ml
- Adapted to many procedures
‣ Has been adapted to solid phase immunoassay for easy
separation of bound and free components
RADIOIMMUNOASSAY: RIA
Requires special what
- Requires licensure for handling radioactive substances
& special disposal
B emission liquid scintillation
-Highly colored compounds (blood)
may need dilution due to interference
(quenching)
* γ counter – similar, but use a crystal
instead of fluidβ
β Emission – Liquid Scintillation
Parts
Scintillation fluid, Photocathode
Optical window
Scintillator
Light
Radition
β Emission – Liquid Scintillation
Two PMTs what
Decreases
noise due to thermionic
emission
Amount of radioactive material A compared to the original amount A0 or any quantity which is proportional to A
Radioactive decay
Spectroscopy
Beer lambert law
A= EbC
Were A= Absorbance
E= molar extinction
b= path length (1cm)
C= concentration
A=
2-log(%T)
Spectroscopy parts
Light source–> Entrance slit–> Monochromomator–> Exit slit–> cuvette–> Detector–> Readout device
Flame Emission
Prinicple
1.If energy (thermal, electrical, electromagnetic) is supplied to
anatom, its e-s can move to an orbit of higher energy
2.Excited atom releases a photon (of light) when its e-s fall back into their original orbits (ground state)
Flame emmission
The wavelength of emission gives the what
The WAVELENGTH of emission gives the IDENTITY of the element
while the INTENSITY of the emitted light is PROPORTIONAL TO THE
# OF ATOMS IN THE ELEMENT.
Instrumentation of flame photometer
- Radiation source (the excited atoms)
Each element or ion emits a גּ or color characteristic for
that element (SQB1)
Na+ = yellow K+ = red-violet Li+ = red Mg2+ = blue
Instrumentation of flame photometer
sample cell or cuvette
- Sample cell or cuvette (the flame!)
The greater the thermal energy of a flame, the greater the number of atoms
that will be excited. BUT increased background noise is often an unwanted
by product
Atomizer- Burner
Advantages
and disadvantages
✦Mixes liquid sample into the flame
✦Sample introduced at a rapid, stable rate
Advantages: delivery of a completely representative aliquot of
sample into the flame
Disadvantages: wide variation of droplet size, aspiration
variance due to sample viscosity, turbulence (results in much
background noise)
Monochrometer
‣ Isolates the characteristic wavelength of emitted light
‣ Accomplished via: prisms / diffraction gratings or filters
Detector
(photomultiplier tube or photocell)
‣ converts light emitted by excited atoms into electrical
energyInstrumentation of flame photometer
Internal standards
are used to
Simultaneous analysis for what
- Internal standards are used to eliminate instrument variance
due to temperature, atomization, oxygen / fuel flow, and
voltage fluctuations - Simultaneous analysis for both (the internal reference
standard & the element of interest)
Internal standards
Because both what
If running what then
- because both internal standard & element of interest are
affected in an identical manner - Internal standard - if running sodium, use lithium or cesium
as internal standard
Atomic absorption spectrophotometry
Differences from flame emission photometry
- Specimen is atomized in flame, not excited
- Spectrally pure light source is also directed at flame
- Atoms in flame that match wavelength of light
(spectral alignment) will absorb that light (atomic
absorption)
Atomic Absorption Spectrophotometry
Benefits
‣ 100 times more sensitive than FEP
‣ Highly specific for element being measured
‣ Used for Al, Ca, Cu, Pb, Li, Mg, and Zn
Atomic Abs spectrophotometry parts
Light source–> atomizer–> emmision–> Detector
Sample–> atomic cell–> atomizer
Atomic absorption spectrophotometry
step one
Hollow cathode (exciter) lamp produces a beam of high spectral
purity (monochromatic)
Atomic Absorption Spectrophotometry
step two
Chopper transforms the specific incident light into a pulsing beam
(AC, alternating current
Atomic Absorption Spectrophotometry
step three
Sample introduced to flame, vaporized (& reduced) to their ground
state
Atomic Absorption Spectrophotometry
step four
Reduced sample atoms absorb the light when bombarded by AC
from chopper
Atomic Absorption Spectrophotometry
step five
Reduced samples emit light that is not in a pulsing beam (DC
Atomic Absorption Spectrophotometry
step 6
DC2 deflected by the Monochromator (grating) to the Detector
(photomultiplier tube
Atomic Absorption Spectrophotometry
step 7
Modulated Meter is synchronized to the chopper so that it
recognizes AC as well as DC2
Atomic Absorption Spectrophotometry
Chemical interferences
(contaminating chemicals might ionize &
interfere or merely interfere with reading)
Atomic Absorption Spectrophotometry
Ionization interferences
(elements other than elements of interest
might ionize similarly)
Atomic Absorption Spectrophotometry
Matrix interferences
(other components of the sample matrix such as
buffer salts or other additives like detergent or solvents may ionize
& interfere or merely interfere with reading)
Atomic Absorption Spectrophotometry
Emission interferences
(other elements might absorb & emit at same
wavelength)
Turbidimetry
Measurement of loss in intensity (dynamic change) of a
light beam through a solution that contains suspended particulate matter
Nephelometry
The measurement of the intensity of reflected light
Light scatter
Nephelometry & turbidimetry are related
techniques
that are dependent on measurement of
light scatter
Quantitative relationships exist between what
Light scatter
size &
number of suspended particles degree of scatter.
Oscillating dipole
When a light beam strikes a particle, its electric
field moves the particle’s electrons in one
direction and the nucleus in the opposite direction
(Oscillating dipole)
Light scatter
the maximum amount of what is
The maximum amount of movement is
proportional to the electric field strength of the
light beam.
Turbidimetry
➡ Turbidimetry can be performed on any standard
spectrophotometer
✦ Wavelength selection criteria.
* Turbidimetric measurement is based on the size of
the particle.
* Particles not more than 40 nm in diameter can be
measured with light of a 400 nm wavelength,
* larger particles (300 nm) as might occur with latex
agglutination are probably going to be measured
with a light of a wavelength in the 500-600 nm
range.
Turbidimetry
Wavelength selection criteria
- Turbidimetric measurement is based on the size of
the particle. - Particles not more than 40 nm in diameter can be
measured with light of a 400 nm wavelength, - larger particles (300 nm) as might occur with latex
agglutination are probably going to be measured
with a light of a wavelength in the 500-600 nm
range.
Nephelometry
Nephelometry is one of the most commonly used
measurement principle for the immunochemical
determination of protein in serum, urine and other body
fluids
Nephelometry
Measurement of
Scattering
Instrumentation
- Measurement of light scatter
- Scattering
– Size of particles
– Number of particles - Instrumentation
– Similar to spectrophotometers, fluorometer
Types of light scatter
Small particles
Light scattered symmetrically but minimally at 90 degrees ( Rayleigh)
d< 0.1 lamda
Large particles
Light scattered preferentially forward ( Rayleigh- Debye)
d< lamda
Very large particles
- Light mostly scattered forward ( Mie) d> lamda
D= particle size
lamda= wavelength of light
Immunonephelometry
- Antigen
- Antibody
- Antigen-Antibody complexes
Nephelometer
angle of detection
light sources
Uses
- Angle of detection
– 5°-90° (depends on light source) - Light source
– Monochromatic (filtered, laser, diffraction gradient)
– Polarized (collminating lens) - Use
– Serum and CSF proteins, antibodies, drugs
