FCS Classroom
Advantages of FCS for Assaying Molecular Interactions
The following sections describe the advantages that make Fluorescence Correlation Spectroscopy (FCS) an ideal technique for measuring and assaying molecular interactions.
For a comparison of the advantages and disadvantages of FCS, ELISA, and PCR, see FCS vs. Conventional Bioassays (ELISA & PCR)
Applications of Molecular Interaction Assaysback to top
Types of Molecular Interactions
The interaction and complexing of biomolecules is fundamental to biological functions. Important examples of molecular interactions include:
- Proteins binding other proteins
- Polynucleic acids (DNA and RNA) binding other DNAs and RN’s
- Proteins binding polynucleic acids
Utilizing Interaction Specificity in Industry
These interactions and binding events are extremely specific to the particular molecules involved and, as a result, the high specificity and selectivity of these interactions can be used to form the basis of biochemical assays. Biochemical assays utilize specific biomarker probes to identify and measure the presence of specific target molecules (e.g., an enzyme probe against the target glucose molecule will bind only glucose, not other sugars).
All of the following industries involve research of molecular interactions:
- Drug discovery is the quest for small molecules that bind and affect the function of medically-important biomolecules
- Medical diagnostics utilizes specific probes (e.g. antibodies or DNA sequences) to detect disease states (e.g. the presence of specific proteins, genes, or pathogens)
- Environmental and bioterrorism screening utilize specific probes (e.g. antibodies or DNA sequences) to detect the presence of dangerous pathogens or biotoxins.
Unique Advantages of FCSback to top
Advantages of the FCS Assay Format
Even in its simplest form, autocorrelation mode, Fluorescence Correlation Spectroscopy (FCS) provides a highly flexible, easy-to-use assay format. These properties of FCS measurements make it an incredibly convenient assay method:
- Very small sample volumes (< 10 µl) and concentrations (picomolar)
- Direct measure of particle number with no need for calibration, a major disadvantage of immunoassays such as ELISA
- No signal amplification required, a major disadvantage of PCR technology
- Single-molecule sensitivity
- Availabile multiwell plate & autosampling formats for increased throughput
Advantages of FCS vs. Averaging Forms of Assays
Most techniques for measuring molecular interaction are intensity-based, which means that they measure the average intensity of a sample, and therefore the average amount of an assay probe without distinguishing bound from unbound material. In contrast, FCS is a nonaveraging technique, offering simultaneous measurement and distinction of bound and unbound fractions. As a result, FCS:
- Can extract complex signals from high background: Any intensity-based measurement is limited when the signal become lost in the background noise. Because FCS is nonaveraging, one can better distinguish between signal and noise based upon the different characteristic time scales over which signal and noise occur.
- Does not require washing: No separation of unbound probe is required as is the case in many other assay systems, such as ELISAs.
- Provides a direct measure of stoichiometry: Stoichiometry, the fundamental ratio of the number of molecules in a complex (i.e., molecules may form a one to one complex or a one to two complex, etc.), is fundamental information that is difficult to obtain by any other technique.
- Dramatically reduces false negatives: This advantage follows from the improved ability to separate signal from noise.
Added Advantages of Cross-Correlation FCS
When operated in cross-correlation mode, FCS offers the possibility of assaying interactions of multiple targets simultaneously. These properties of cross-correlation assays introduce significant advantages to FCS-based assays:
- Reduced false-positives: A positive event must occur at a specific time scale and simultaneously in two channels
- Simple, direct assay for complexes: Complexes can be detected by simultaneous correlation in two detection channels, without need to determine its size.
- Improved separation of signal versus background noise: Noise events in the two channels are uncorrelated, so they do not appear in cross-correlation.
- Ability to create highly multiplexed assays: Cross-correlation allows for distinction based on size and color simultaneously. The QuantumXpert FCS spectrometer is unique among commercial FCS systems in that it offers two excitation wavelengths and three simultaneous detection channels for further increased flexibility.