Detection systems Flashcards by Sarah King

uses what as labels

Detection systems RadioImmunoassay

Uses radioactive isotopes as labels

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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

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RADIOIMMUNOASSAY: RIA

Requires special what

Requires licensure for handling radioactive substances
& special disposal

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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

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β Emission – Liquid Scintillation

Parts

Scintillation fluid, Photocathode
Optical window
Scintillator
Light
Radition

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β Emission – Liquid Scintillation

Two PMTs what

Decreases
noise due to thermionic
emission

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Amount of radioactive material A compared to the original amount A0 or any quantity which is proportional to A

Radioactive decay

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Spectroscopy

Beer lambert law

A= EbC

Were A= Absorbance
E= molar extinction
b= path length (1cm)
C= concentration

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2-log(%T)

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Spectroscopy parts

Light source–> Entrance slit–> Monochromomator–> Exit slit–> cuvette–> Detector–> Readout device

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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)

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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.

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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

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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

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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)

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Monochrometer

‣ Isolates the characteristic wavelength of emitted light
‣ Accomplished via: prisms / diffraction gratings or filters

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Detector

(photomultiplier tube or photocell)
‣ converts light emitted by excited atoms into electrical
energyInstrumentation of flame photometer

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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)

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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

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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