CAS No.: 54827-17-7
Chemical Formula: C₁₆H₂₀N₂
Structural Features: Consists of two benzene rings linked by a single bond, with methyl substituents at the 3 and 5 positions of each ring.
Physical Properties: White to pale yellow crystalline powder, poorly soluble in water but soluble in organic solvents (e.g., DMSO, ethanol).

Advantages

1. High Sensitivity:
   When catalyzed by horseradish peroxidase (HRP) in the presence of hydrogen peroxide, TMB produces a blue-colored product (absorbance peak at 450 nm) that turns yellow after acidification (650 nm). Its high color contrast enables detection sensitivity down to the picogram (pg) level.

2. Low Toxicity:
   Compared to traditional substrates like carcinogenic o-phenylenediamine (OPD), TMB exhibits significantly lower toxicity and mutagenicity, aligning with laboratory safety protocols.

3. Excellent Stability:
   The colored reaction product is stable and resistant to spontaneous degradation, allowing delayed measurements (e.g., ELISA plate readings can be delayed for hours).

4. Dual-Wavelength Detection:
   Supports measurements at both 450 nm (pre-acid) and 650 nm (post-acid), minimizing background interference and improving data accuracy.

Applications

1. Immunoassays:

   • ELISA Assays: Widely used for colorimetric detection of viruses (e.g., HIV, hepatitis B) and tumor biomarkers (e.g., PSA, CEA).

   • Lateral Flow Assays: Applied in point-of-care testing (POCT), such as pregnancy tests and drug screening.

2. Molecular Diagnostics:

   • Paired with nucleic acid probes for enzyme-linked signal amplification in genetic testing.

3. Environmental Monitoring:

   • Detects peroxidase-active pollutants (e.g., phenolic compounds) in water or soil.

4. Biosensors:

   • Serves as a signaling molecule in electrochemical or optical sensors for detecting glucose, heavy metal ions, etc.

5. Research:

   • Used in immunohistochemistry (IHC) to label antigens in tissue sections.

Impact on Future Development

1. Advancing Point-of-Care Diagnostics:
   TMB’s stability and efficiency make it ideal for home-testing kits and portable devices, especially in resource-limited regions.

2. Integration with Nanotechnology:
   Combining TMB with nanomaterials (e.g., gold nanoparticles, quantum dots) could enable ultrasensitive detection platforms approaching single-molecule limits.

3. Green Chemistry Innovations:
   Developing water-soluble TMB derivatives or eco-friendly stop solutions (e.g., acid-free) to reduce environmental waste.

4. Multimodal Detection Systems:
   Coupling TMB with fluorescence or chemiluminescence could enable multi-signal outputs for complex samples (e.g., whole blood, saliva).

5. Automation Compatibility:
   Integration with high-throughput systems and microfluidics to enhance clinical diagnostics and drug discovery workflows.

Precautions

• Storage: Protect from light and moisture; store at 2-8°C to prevent degradation.

• Waste Disposal: Post-reaction products must be treated as chemical waste per laboratory safety guidelines.

TMB’s efficiency, safety, and versatility have solidified its role as a cornerstone reagent in life sciences and medical diagnostics. Continued interdisciplinary innovation promises to expand its applications and refine its utility in emerging technologies.