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FCS vs. Conventional Bioassays (ELISA & PCR)

The advantages of Fluorescence Correlation Spectroscopy (FCS) explained in Advantages of FCS for Assaying Molecular Interactions showed that FCS compares favorably with conventional bioassays and elaborated conditions where it exceeds the performance of conventional assays.

Table 1 below lists some relative advantages and disadvantages of conventional polynucleic acid based assays (typified by PCR), immuno-based assays (typified by ELISA), and FCS.

No assay is the perfect choice for all circumstances, however, these tables clearly illustrate that FCS offers unique capabilities not available from competing detection/measurement techniques.

Want to learn more about FCS? See What is FCS?

Table 1: Advantages & Disadvantages of PCR, ELISA, & FCS

PCR ELISA FCS
QuantitativeSometimesYesYes
Normalization RequiredYesYesNo
Standard Curve RequiredSometimesYesNo
Measurement Time30 min (Real-time PCR) - 6 hours3 - 6 hours10 - 20 minutes
Time to Assess Assay QualityEnd of assay (0.5 - 6 hours)End of assay (3 - 6 hours)< 1 minute
Detection ModeAverage of all moleculesAverage of all moleculesNon-averaged distinction between molecules in different states
Distinction Between Multiple TargetsColor-basedColor-basedColor- and size-based
Sample Handling: Pre-AssayDNA extractionNoneNone
Sample Handling: During AssayDNA amplification, gel separation (if not using real-time PCR)Tedious Washing, enzymatic amplification of signalNone
Probe Purity RequirementsMediumMediumHigh
Target Purity RequirementsHighLowLow
Probe SpecificityHigh (DNA-based)High (Antibody-based)High (DNA- or Antibody-based)
Probe AvailabilityHigh (DNA-based)High (Antibody-based)High (Can use PCR and ELISA probes)
Probe DesignEasyDifficult, but many kits availableDifficult, but can use PCR and ELISA probes
Analysis ComplexityComplex (if quantitative)Simple relation to standard curveComplex curve fitting unless using a specific kit
Potential PitfallsPossible errors in amplification due to poor sample/enzyme qualityResults are directly related to effectiveness of washing Low quality probes can cause ambiguity
Instrumentation Cost> $30K> $20K> $100K