Fluorescence Sensing

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Fluorescence Sensing

• Chapter 19

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Introduction

• Sensing of chemical and biochemical analytes
• Rapid low cost testing clinical, bioprocess,
  environmental applications
• Examples DNA sequencing, DNA fragment analysis,
  fluorescence immunoassays
• Absorbance comparison to reference beam
• Fluorescence - directly measured
  concentrations - 10-10 M

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Optical Clinical Chemistry

• Biological sample - Measure analyte
  concentrations in real time
• Point of care instruments LED light sources,
  fluorescence intensity, polarization, lifetime
• Photolithorgraphic methods arrays of DNA
  sequences or proteins two dimensional surface
• Point-of-care testing immediate answer

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Spectral Observables

• Fluorescence property changes in response to
  analyte intensity, ex. or em., anisotropy, or
  lifetime
• Intensity difficult to use as a sensor
• Fluororphore concentrations
• Photobleaching
• Methods independent of fluorophore concentration
• wavelength ratiometric probes
• Anisotropy
• Lifetime
• Phase modulation

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Optical Properties of Tissues

• Water and tissues
• Tissues lower for longer wavelengths
  hemoglobin, melanin absorption decreases
• Absorption of water increases above 1000 nm
• Region of 600-1000 nm solid state lasers can be
  used in this range

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Lifetime-Based Sensing

• Measurements need to be made in non-cuvette
  systems
• Scattering properties of syringe or medical
  device
• Independent of intensity

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Mechanisms of Sensing

• Collisional quenching analyte directly quenches
  the fluorophore change in lifetime or intensity
• Energy transfer - protein-protein associations,
  immunoassays, pH change in acceptor absorbance
  results in change in donor due to FRET

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• pH probes equilibrium between ionized and
  non-ionized
• Cation binding probes photoinduced electron
  transfer (PET) exciplex formation quenching,
  removed when zinc binds
• Polarization Changes - immunoassays

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Sensing by Collisional Quenching

• Oxygen sensing collisional quencher results in
  decrease in intensity or lifetime
• Long-lifetime probes transition metal complexes
• Dissolved in Silicon oxygen can diffuse into
  but other molecules cannot
• Shift in lifetime and reduced intensity when
  quenched

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Lifetime-Based Sensing of Oxygen

• Oxygen-sensitive MLC filter used to absorb near
  450 nm
• Fiber optic to collect fluorescence
• Vary intensity phase angle unchanged
• Mechanism of oxygen selectivity long lifetime
  probes are sensitive (if under 5 ns little
  quenching by oxygen)
• Silcon support only permeable to 02

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Other Oxygen Sensors

• Any long lifetime probe dessolved in organic
  solvent
• Phosphorescence long decay time
• Camphorquinone in PVC intensity or lifetime
  changes (lifetime more reliable)
• Platinum II octaethylporphyrin large stokes
  shift probe embedded in polystyrene
• Osmium MLC lifetime near 300 microseconds,
  different results in two different silcones
  (supporting media important)

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Chloride Sensors

• Quinine contains quinolinium ring which is
  quenched by chloride
• Probes designed to sense Cl-, different
  sensitivities to Cl-
• Measurement of Cl- transport
• Can also be quenched by free amines decrease in
  quenching constant in cells
• Non-ratiometric probes

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Energy Transfer Sensing

• Changes due to analyte proximity or
  analyte-induced changes in absorption or emission
  spectrum of acceptor
• Flexibility - exciting light wavelength (donor)
• pH and pC02 eosin as donor and phenol red as
  acceptor (pKa near 7, 546 nm in base)
• Eosin intensity decreased as pH increased
• Can use lifetime of donor to measure
• Uptake of analyte into supporting media

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Glucose Sensing by Energy Transfer

• Long term health in Diabetics currently must
  sample blood
• Glucose Detection - Con A (Donor) and Dextran
  (Acceptor) decrease in donor intensity of
  lifetime
• Glucose competes for binding site on ConA
  intensity and decay time increases

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Glucose Binding Assay

• Glucose binding released FITC dextran from
  surface in light path
• Donor fluorescence not-completely recovered at
  high glucose concentration

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Theory of FRET Sensing

• Covalently linked donor and acceptor
• Unlinked donor and acceptor donor can be in two
  forms with different absorbance spectra - two
  R0s
• Simulate expected results with certain
  concentrations

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pH Sensors

• Fluorescein measure pH and pC02 in bicarbonate
  couple
• Fluorescein leaks from cells charged
  derivatives used (BCECF) ratiometric

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• SNAFL and SNARF pH probes shifts in absorbance
  and emission pKa 7.6 to 7.9
• Acid and base are fluorescent lifetimes useful
  but short (can be modified to CNF large shift
  in spectra)

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Photoinduced Electron Transfer (PET)

• Polynuclear aromatic hydrocarbons quenching
  fluorophore by linked side chain
• Transfer of electrons from nitrogen to aromatic
  group if nitrogen has a bound proton then
  transfer is inhibited
• Phosphate sensor hydrogen bonding to amino
  group results in increase fluorescence
• Quenching inefficient at long wavelengths

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Design
pH Sensor
Phosphate Sensor
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Analyte Recognition

• Design of specific probes to bind an analyte
• Specific Cation Probes
• Development of intracellular cation probes

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Theory of Analyte Recognition

• Probe can exist in two state binding
  stoichiometry of 11
• A Dissociation constant (KD)
• The relative concentrations of free and bound
  forms can be expressed
• If the probe fluorescence increases when analyte
  binds but does not contain a spectral shift (TITC
  state formation)

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Wavelength Ratiometric Probes

• Can calculate analytic concentration, where R
  F(?1)/F(?2), ratio of intensities at the two
  excitation wavelengths
• RMIN and RMAX are the ratios for the free and
  complexed probe
• For excitation wavelength shift
• For emission wavelength shift
• Can measure concentration independent of probe
  concentration

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Ca and Mg Indicators

• All based on BAPTA chelator binds Ca near 100
  nM affinity
• Intracellular Ca
• In blood Ca 5 mM
• Does not diffuse across cell membrane
• Many varieties have been designed

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Immunoassays
Couple an associated antibody and antigen to some
other event that yields an observable spectral
change
ELISA coat a surface with an antibody specific
for an antigen of interest, allow antibody to
capture antigen, exposed to 2nd antibody linked
to an enzyme, provide substrate to observe
fluorescence change
Time resolved Immunoassay 2ndary antibody is
conjugated to MLC lanthanide (europium)
enhancer solution which chelates the Eu3 - light
is absorbed by chelators which transfer the
energy to the lanthide
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Energy Transfer in Immunoassays

• Thyroxine 4 (T4) competes for binding site on
  the acceptor
• Follow changes in donor (phycoerythrin) becomes
  unquenched as function of T4

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r rF(fF) rB(fB) Unlabeled cortisol competes
off labeled cortisol
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Genetically Engineering Proteins

• Maltose binding protein cysteine engineered
  into the cleft
• Labeled with NBDSensitive to maltose binding

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• Calcium Sensing
• M13 peptide binds Calmodulin in the presence of
  Ca
• Conf. change results in FRET