FCS Classroom
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 | |
|---|---|---|---|
| Quantitative | Sometimes | Yes | Yes |
| Normalization Required | Yes | Yes | No |
| Standard Curve Required | Sometimes | Yes | No |
| Measurement Time | 30 min (Real-time PCR) - 6 hours | 3 - 6 hours | 10 - 20 minutes |
| Time to Assess Assay Quality | End of assay (0.5 - 6 hours) | End of assay (3 - 6 hours) | < 1 minute |
| Detection Mode | Average of all molecules | Average of all molecules | Non-averaged distinction between molecules in different states |
| Distinction Between Multiple Targets | Color-based | Color-based | Color- and size-based |
| Sample Handling: Pre-Assay | DNA extraction | None | None |
| Sample Handling: During Assay | DNA amplification, gel separation (if not using real-time PCR) | Tedious Washing, enzymatic amplification of signal | None |
| Probe Purity Requirements | Medium | Medium | High |
| Target Purity Requirements | High | Low | Low |
| Probe Specificity | High (DNA-based) | High (Antibody-based) | High (DNA- or Antibody-based) |
| Probe Availability | High (DNA-based) | High (Antibody-based) | High (Can use PCR and ELISA probes) |
| Probe Design | Easy | Difficult, but many kits available | Difficult, but can use PCR and ELISA probes |
| Analysis Complexity | Complex (if quantitative) | Simple relation to standard curve | Complex curve fitting unless using a specific kit |
| Potential Pitfalls | Possible errors in amplification due to poor sample/enzyme quality | Results are directly related to effectiveness of washing | Low quality probes can cause ambiguity |
| Instrumentation Cost | > $30K | > $20K | > $100K |