Table 4 Comparison between developed Gel-FITC collagenase activity assay and current techniques with the same intended use
Technique | Description | Advantages | Disadvantages |
|---|---|---|---|
Developed Gel-FITC collagenase activity assay | Rapid collagenase activity detection technique based on immobilizing fluorescent quenched gelatin at the bottom of the wells of a microplate. When incubated with collagenases, they digest the gelatin, liberating the fluorophore, which can be measured, allowing the quantification of the collagenase activity. Its advantages make it a very good alternative to be used in clinic. | - User-friendly, easy, and fast assay. No need of expertise. | - Specific instrumentation (fluorescent lector) is needed. |
- Quantitative results achievable with appropriate calibration standards. | - It is not yet a well-established method with commercial availability. | ||
- Sensitive detection of enzymatic activity based on changes in fluorescence intensity. | - No yet validated with various types of samples. | ||
- Real-time monitoring of enzyme activity. | - Difficult to provide information about the identity of the analyzed collagenases. | ||
ELISA involves the immobilization of a substrate specific to collagenase on a solid surface, followed by detection using an enzyme-linked antibody. | - Well-established method with commercial availability. | - Time-consuming procedure requiring several hours to complete. | |
- High specificity for collagenase detection. | - Limited dynamic range, challenging for samples with low enzyme activity. | ||
- Compatible with various sample types and formats. | - Reliance on antibodies and substrates may introduce batch-to-batch variability and increase assay costs. | ||
- Quantitative results achievable with appropriate calibration standards. | - Sensitivity may vary depending on antibody affinity and substrate efficiency. | ||
Western blotting involves separating proteins from a sample by gel electrophoresis, followed by transfer to a membrane and detection using antibodies specific to collagenase proteins. | - Established methodology with documented protocols. | - Requires specialized equipment and expertise for gel electrophoresis and membrane transfer. | |
- High specificity for detecting collagenase proteins. | - Time-consuming and labor-intensive procedure. | ||
- Suitable for analyzing protein expression levels and post-translational modifications. | - Sensitivity may vary depending on antibody quality and detection method. | ||
Zymography involves embedding a substrate specific to collagenase in a gel matrix, followed by separation by gel electrophoresis and incubation to allow enzyme activity, visualized as clear bands. | - Specifically designed for detecting enzyme activity. | - Gel-based technique may not provide quantitative data. | |
- Allows visualization of active enzyme bands directly on the gel. | - Sensitivity may vary depending on gel composition and staining method. | ||
- Can detect multiple enzymes simultaneously. | - Limited resolution for distinguishing closely related enzymes. | ||
Fluorescence Resonance Energy Transfer (FRET) Assays [39,40,41,42] | FRET assays utilize changes in fluorescence intensity between fluorophores linked by a peptide sequence susceptible to collagenase cleavage to detect enzymatic activity in real-time. | - Sensitive detection of enzymatic activity based on changes in fluorescence intensity. | - Design and optimization of FRET substrates may be challenging. |
- Real-time monitoring of enzyme activity. | - Background fluorescence and assay conditions may affect signal-to-noise ratio. | ||
- Potential for high-throughput screening applications. | - Requires specialized equipment for fluorescence detection. | ||
Mass Spectrometry [76] | Mass spectrometry is used to detect and quantify peptide fragments generated by enzymatic cleavage of substrates, providing information about the identity and quantity of collagenase activity in a sample. | - High sensitivity and specificity for detecting peptide fragments generated by enzymatic cleavage. | - Expensive instrumentation and expertise required. |
- Can provide information about the identity and quantity of collagenase activity in a sample. | - Sample preparation may be complex and time-consuming. | ||
- Suitable for identifying novel enzyme substrates. | - Interpretation of mass spectrometry data may require bioinformatics expertise. |