Molecular Probes: Detection and Quantification

Molecular Probes

• Molecular probes include nucleic acids and proteins.
• Usual tracers include isotopes, enzymes, and fluorochromes.
• Examples of applications are in fluorescence.

Description of Molecular Probes

• A probe has a specific affinity for the molecule you want to detect or quantify.
• The probe has a tracer for stable, sensible, and specific detection.
• The tracer does not modify the affinity of the probe for its target.
• The tracer isn't naturally present in the analysed sample.
• Probes are most of the time made to detect and sometimes quantify various molecules that aren't visible to the naked eye, like DNA, RNA, proteins, lipids, ions, or metabolites.

Example of Molecular Probe (DNA)

• A probe contains an oligonucleotide paired with a fluorescent tracer to detect the DNA from samples such as genes responsible for oncogenesis, resistance, or from a transgenic plant.
• It can be used on DNA chips.
• An oligonucleotide has been chemically coupled to Cy3 which is a fluorescent tracer.

Example of Molecular Probe (Protein)

• An antibody (protein) is used here as a molecular probe.
• It's paired to a fluorescent tracer allowing for the detection of an epitope molecule that we want to detect.
• This can be used for Western blotting, ELISA detection, or fluorescence microscopy.

Antibody Structure

• Antibodies are immunoglobulins of types IgG, IgE, and IgM.
• IgG consists of paratope (variable area that fixes the epitope) and Fc region (constant area of the heavy chains)

Common Tracers for Marking Probes

• Isotopes are used for marking probes.
  • Unstable isotopes such as 14C, 3H, 125I, 32P, and 35S are radioactive elements that are less and less used.
  • Stable isotopes such as 13C, 31P, 15N, and 18O, are very commonly used, for example in NMR and mass spectrometry.
• Enzymes are also used as tracers (colorimetry, luminescence) such as alkaline phosphatase, horseradish peroxidase (HRP) and β-galactosidase.
• Fluorochromes are also popular tracers.
  • Small molecules like Cy3 and Cy5.
  • Proteins include Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP).

Atomic Structure (reminder)

• Atoms consist of electrons orbiting a nucleus of neutrons and protons.
• The number of protons is equal to the number of electrons.

Number of Protons Per Element

• All atoms of an element have the same number of protons (atomic number) and same chemical properties.
• They will however have different nuclear properties.
  • Hydrogen (1H) has 1 proton
  • Deuterium (2H or D) has 1 proton + 1 neutron
  • Tritium(3H or T) has 1 proton + 2 neutrons
• The equation for water is H2O vs D₂O vs T₂O

Nucleus Stability

• Nucleus stability depends on the neutron to proton ratio (N/Z).
• If there are too many or few neutrons, the atom is unstable and emits radiation to stabilize itself and the disintegration of the atom doesn't depend on environmental conditions.

Types of Radioactive Radiation α (alpha)

• An unstable nucleus transforms by ejecting 2 neutrons and 2 protons at once, corresponding to a helium nucleus (He²+).
• alpha radiation is often associated with elements in the periodic table with Z > 52.
• The radiation is not often used in biochemistry.
• Americium 241 is in smoke detectors (half-life of 432 years).

Types of Radioactive Radiation β (beta)

• The unstable nucleus ejects a beta particle with a negative charge (negatron) or positive charge (positron), and an antineutrino (νe).
• The beta particle is identified by β- or β+ to identify its charge.
• β- is an electron that is identical to the atom's electrons.
• Most unstable radioactive tracers used in biochemistry emit β- radiation.
• If the number of protons change, there is a transmutation transforming one chemical element into another.

Types of Radioactive Radiation (γ) gamma and X Rays

• These rays are highly energetic (short wavelengths).
• These releases may accompany the release of α or β particles.

Dangers of Radioactivity

• The penetrating power of particles are as follows:
  • Alpha: few centimeters
  • Beta: few meters
  • Gamma: few hundred meters
• The protective screens are:
  • Alpha: Paper
  • Beta: Aluminium
  • Gamma: Concrete or lead

Radio Protection in the Laboratory

• The Canadian Nuclear Safety Commission controls all manipulations using radioactivity.
• Employers must have a utilization permit and have all employees go through basic training.
• At Université Laval, the basic training on using radioactive material, lasting approximately 4 hours is mandatory alongside an examination.

Enzymes: Colorimetric Tracers

• A tracer is an enzyme covalently bound to the probe.
• The enzymatic reaction with a substrate is specific and makes a "colored" product that can be measured with spectrophotometry.
• Color intensity is usually proportional to the amount of the probe fixed on the target.

Main Colorimetry Systems Used

• HRP (horseradish peroxidase)
• ABTS (2,2'-azino-di-(3-ethylbenzthiazoline-sulfonic acid)) staining turns green.
• AEC (3-amino-9-ethylcarbazole) stains PPT red
• TMB (tetramethylbenzidine) stains yellow if pH is acidic
• Alkaline phosphatase
  • PNPP (p-nitrophenyl phosphate) stains yellow
  • BCIP (+NBT) stains PPT mauve
• β-galactosidase
  • X-Gal (BCI-Galactose) stains blue
  • ONPG (o-nitrophenyl galactose) stains yellow

Examples of Results with Colorimetric Probes

• Alkaline phosphatase probe with NBT and BCIP as an enzyme used as a tracer for RNA in situ hybridization.

Examples of Colorimetric Tracers: ELISA (Enzyme-Linked Immunosorbent Assay)

• Consists basically of well coating with an antigen (direct test) or capturing an antigen with an antibody (sandwich method) and blocking non-specific bonding sites.
• Add the specific antibody which fixates to the antigen (primary antibody, direct detection0 or a specific antibody and a secondary antibody (indirect detection).
• Measure the signal.

Enzymes: Luminescent Tracers

• Instead of using a reagent generating a colored product when transformed by the probe enzyme, one uses a substrate producing light when it's transformed into a product.
• It's known as chemiluminescence, such as with luminol oxidation by the HRP.
• Light signal intensity is proportional to the quantity of probes fixed on their target.

Luminescence Detection

• Luminometer uses photomultiplier tubes, photodiodes, and CCD cameras.
• Autoradiography uses emitted light to create an impression on film where light was emitted and this needs a special film applied against the membrane used for blotting.

Example of Luminescence Probe

• Detection and quantification of ATP, using Luciferin + ATP + O2, with luciferase and magnesium to produce oxyluciferin + AMP + pyrophosphate + CO2 + light.

Fluorophores: Molecules Emitting Fluorescence

• Fluorescence is a substance's emission of light from absorption of radiation and emits light in bigger lengths.
• A fluorochrome (or fluorophore) is a molecule emitting fluorescence.
• Light stops getting emitted when illumination ceases.
• Phosphorescence is the continuing emission of light after illumination has stopped.

Fluorescence Cycles

• The fluorescence cycle consists of absorption, excitation, and emission.

Fluorescence

• Emission is always at a higher wavelength than used for excitation.

Utilisation of Filtering Light when Using Fluorescence

• White light is a source of light and we can use filters to specifically excite with wavelengths on a narrow band.
• The excitation can utilise LEDs or lasers in a short wavelengths narrow band or a precise wavelength.

Utilisation of Filtering Ligh when Using Fluorescence

• Wide bands of filters allow for the excitation and measurement of emitted fluorescence in narrow wavelength bands, which can detect a fluorescence signal from a specific molecule originating from a potentially emitting compound's mix.

Fluorescence for Simultaneous Detection of Multiple Tracers

• Fluorescence is possible with good filters and is very useful for fluorescence microscopes.
• An image can be reconstructed from a multiple images acquired by excitation and/or emission from various wavelength pairs.

Fluorescence Detection

• Spectrofluorimeters are necessary to know the wavelengths of absorption and emission peaks.

Common Applications Involving Fluorochromes

• Some molecules that emit fluorescence (fluorochromes) are used as is, without needing pairing with a reconnaissance molecule, therefore they aren't probes.
  • Examples: Detection of acid nucleic on gel (ethidium bromide, SYBR green that fixates to DNA), DNA sequencing (BigDye, which is a fluorescent nucleotide), and Fluorescence microscopy (DAPI bonds with DNA).

Common Applications Involving Fluorescent Probes

• Used in flow cytometry (antibodies bonded to fluorescent tracers used to count circulating cells).
• Hybridization of DNA chips (Oligonucleotides bonded to Cy3 and Cy5). Used in immunofluorescence microscopy (antibodies bonded to fluorescent tracers).
• Used in FRET (Fluorescence energy transfer) where proteins are bonded to fluorescent tracers.

Marking by Chemical Conjugation

• The stable chemical bond forms between a tracer and reconnaissance molecule for generating a probe.
• This can be seen with marking with iodine.

Marking Probes Through Synthesis

• Biosynthesis of complex compounds from precursors of a tracer.
• Proteins are marked with 35S or 14C using marked amino acids during their synthesis.

Marking Probes By Biochemistrty Synthesis

• Connecting a gene to one coding for a fluorescent protein to obtain a merged protein.
• Used to observe a protein's cellular location by using FRET.

Probes for Nucleic Acids (With Radioactivity)

• A single strand of DNA can specifically bond (through base complementarity) to a single strand of DNA (or RNA).
• To be employed as a probe, this fragment needs to be bonded an the tracer can not alter the nucleotide ability to bond and this will allow it to be detected.
• Most of the original nucleic acids probes were paired with radioactive tracers detectable using X-ray films and developed in a darkroom.
• This forms the basis for Southern and Northern hybridizations (Southern and Northern blotting)

Southern Blot Principle

• Genomic DNA is fragmented by the Restriction Endonuclease.
• Then Agarose Gel Electrophoresis occurs.
• Use blotting buffer
• Use nitrocellulose or nylon filter
• Expose to X-ray Film

Probes for Nucleic Acids (Without Radioactivity)

• The disadvantages using radioactivity have led to the development of nonradioactive alternates such as probes bonded to digoxigenin. Digoxigenin is a plant hormone mostly found among living systems (Digitalis purpurea). This hormone can be detected using a specific antibody. That antibody in turn is bonded with an enzyme capable of transforming a non-colored substrate.

Nucleic Acid Probes (Fluoroscence)

• Nowadays acid nucleic probes are often bonded to fluorescent tracers.
• These probes may be very sensible and specific.
• Examples include FAM, TET, and TAMRA.

Summary

• Probes are made of reconnaissance compounds bonded to a tracer.
• Types of molecular probes (based on tracer): Probes marked with a isotope, a enzyme (colorimetry, luminescence, or fluorescence), or a fluorochrome.
• Fluorochromes are not probes.
• The fluorescence principles are excitation and emission.
• Probe marking includes: Chemical Conjugation and biochemical synthesis
• Applications include ELISA, the Southern/Northern blot, and microscopy and cytometry.