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Tetramethylbenzidine (TMB), CAS 54827-17-7, is a chemical with the molecular formula C16H20N2, used as a staining agent for peroxidase and hepatitis B antigen. White to yellow powder. TMB is used as a substrate for catalase in ELISA reactions. This substrate can produce soluble light blue final products, which can be read by a spectrophotometer at 370 or 620-650 nm. The TMB reaction can be terminated with 2 M H2SO4 (yellow) and can be read at 450 nm. Therefore, TMB is a sensitive and specific reagent used for the determination of blood, hemoglobin, and peroxidase. As a non carcinogenic chromogenic reagent, TMB is widely used in enzyme-linked immunosorbent assay (ELISA), clinical biochemical testing, and other fields. Its detection sensitivity is high and stability is strong, which can replace traditional carcinogenic benzidine reagents. It is mainly used for more than ten detection projects such as hepatitis virus detection, pregnancy testing, fecal occult blood analysis, etc., covering medical diagnosis, forensic identification, and environmental monitoring scenarios.
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Chemical Formula |
C16H20N2 |
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Exact Mass |
240 |
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Molecular Weight |
240 |
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m/z |
240 (100.0%), 241 (17.3%), 242 (1.4%) |
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Elemental Analysis |
C, 79.96; H, 8.39; N, 11.66 |
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As a key reagent in the fields of biochemistry and medicine, tetramethylbenzidine (TMB) has completely replaced traditional benzidine compounds and become the core material of modern detection technology due to its high sensitivity, strong stability, and non carcinogenic properties. Its applications cover multiple fields such as clinical diagnosis, forensic criminal investigation, environmental monitoring, biological research, and industrial sensing, forming a complete detection technology system.
1. Infectious disease diagnosis system
TMB, as the core substrate of ELISA technology, achieves a sensitivity of over 98% in hepatitis virus detection. By reacting with HRP labeled antigen antibody complexes, stable blue products are generated that can be quantitatively analyzed at a wavelength of 650nm. The technology has been extended to detect more than 30 pathogens, such as AIDS virus and dengue virus. The cost of a single test is 60% lower than that of PCR, and it has become a standardized diagnostic tool for grass-roots medical institutions.
2. Pregnancy and reproductive health monitoring
In pregnancy testing, the immunochromatographic test strip formed by the binding of TMB and β - HCG antibody can complete the detection within 5 minutes, with a sensitivity of up to 25mIU/mL. The intensity of its blue signal is linearly related to hormone concentration, effectively distinguishing between normal pregnancy and ectopic pregnancy. In addition, among the six tests for sex hormones, the TMB substrate system enabled the lower limit of testosterone detection to exceed 0.1ng/mL, providing key data for the diagnosis of polycystic ovary syndrome.
3. Metabolic disease management
In the field of blood glucose detection, the glucose oxidase method participated by TMB expanded the detection range to 0.05-600mg/dL, covering the whole course of neonatal hypoglycemia to diabetes ketoacidosis. In urine analysis, TMB protease complex can simultaneously detect microalbumin (3-30mg/L) and hemoglobin (0.02-0.5mg/dL), which increases the early diagnosis rate of diabetes nephropathy by 40%.
4. Hematological system analysis
In terms of granulocyte detection, the TMB and myeloperoxidase reaction system achieves an absolute neutrophil counting error of less than 5%, which is three times more accurate than traditional staining methods. In blood parasite screening, TMB enhanced immunofluorescence technology reduces the detection limit of malaria parasites to 5 per μ L, making it an important tool for tropical disease prevention and control.
1. Visualization of occult blood fingerprints
The TMB luminol composite system exhibits excellent performance in crime scene detection:
Sensitivity: Can detect 0.001 μ L of residual blood
Specificity: Distinguishing human blood from animal blood by adjusting pH value
Durability: The reaction product can be stored on a porous surface for more than 3 months
This technology has been successfully applied to the re examination of physical evidence in over 200 long-standing cases, with a development success rate of 92% on special carriers such as silk and leather.
2. Enhanced DNA evidence
In the pre-treatment of STR typing, TMB serves as an oxidation-reduction mediator, which can increase DNA extraction by 3-5 times. The purification technology combined with silica membrane increased the removal rate of PCR inhibitors to 99.7%, significantly improving the genotyping quality of complex samples.
3. Innovation in Toxic Analysis
The saliva alcohol detection test strip adopts TMB ethanol dehydrogenase system to achieve linear detection in the range of 0-0.8 ‰ blood alcohol concentration, with an error rate of<8%. In drug detection, TMB enhanced colloidal gold immunochromatography reduces the detection limit of methamphetamine to 1ng/mL, which is 100 times more sensitive than traditional methods.
1. Biological toxicity assessment of water quality
The luminescent bacteria method involving tetramethylbenzidine can quickly detect the comprehensive toxicity of water bodies
Detection range: 0.1-100% toxic equivalent
Response time: 15 minutes to complete 96 well plate testing
Sensitivity: Can identify heavy metal pollution of 0.01mg/L
This technology has been included in the revised draft of the "Surface Water Environmental Quality Standards" (GB 3838-2002) and has become the preferred method for emergency monitoring of sudden water pollution incidents.
2. Traceability of atmospheric pollutants
In the analysis of PM2.5 components, the TMB horseradish peroxidase system can specifically detect the content of polycyclic aromatic hydrocarbons (PAHs). By optimizing the reaction conditions, the detection limit of benzo [a] pyrene was reduced to 0.05 ng/m ³, meeting the stringent requirements of WHO air quality guidelines.
3. Soil remediation monitoring
TMB enhanced enzyme-linked immunosorbent assay technology can simultaneously quantitatively analyze six organic pollutants in soil:
Polychlorinated biphenyls (PCBs): detection range 0.1-100 μ g/kg
DDT: Recovery rate 95-105%
Phthalate esters: within batch coefficient of variation<5%
This technology shortens the evaluation cycle of soil remediation efficiency from 30 days to 72 hours.
1. Proteomic research
TMB, as the standard chromogenic substrate for Western blot, has the following advantages:
Dynamic range: 0.1-100ng protein detection
Background inhibition: non-specific binding signal reduced by 80%
Compatibility: Perfectly compatible with commonly used carriers such as PVDF and NC membranes
In signal transduction research, the sensitivity of TMB chromogenic phosphorylation protein detection reaches fg level, promoting the study of JAK-STAT and other pathway mechanisms.
2. Cell imaging technology
TMB participates in enzyme-linked immunofluorescence technology to achieve subcellular structure visualization:
Mitochondrial membrane potential detection: resolution up to 50nm
Apoptosis analysis: Caspase-3 activity localization error<1 μ m
Live cell imaging: continuous observation time exceeding 72 hours
This technology has been applied to the study of tumor stem cell heterogeneity, revealing the regulatory mechanism of microenvironment on cell fate determination.
3. Drug screening platform
The high-throughput screening system constructed by TMB has the following characteristics:
96/384 hole plate compatibility
Z-factor value>0.7 (excellent screening model standard)
Daily processing sample size:>10000
In the development of anti-tumor drugs, this platform has increased the efficiency of lead compound discovery by 5 times and reduced research and development costs by 40%.
1. Food quality monitoring
TMB sensors achieve real-time monitoring of food freshness:
Meat spoilage detection: Total volatile basic nitrogen (TVB-N) detection limit of 1mg/100g
Determination of acidity in dairy products: pH measurement error ± 0.02
Fruit maturity assessment: Ethylene release detection range of 0.1-100ppm
This technology has been integrated into intelligent packaging systems, achieving an accuracy rate of 95% for predicting food shelf life.
2. Process control optimization
In the field of biopharmaceuticals, the TMB online monitoring system achieves:
Real time feedback on enzyme activity: response time<1 second
Fermentation endpoint judgment: error<2 hours
Cell density monitoring: range 10 ⁵ -10 ⁹ cells/mL
This system increases antibiotic fermentation yield by 18% and reduces production costs by 22%.
3. In the field of security protection
TMB gas sensors are equipped with:
Lower explosive limit (LEL) detection: sensitivity up to 1%
Toxic gas identification: can distinguish 8 gases including CO, H ₂ S, etc
Anti interference ability: humidity impact<5% RH
In the application of chemical industrial parks, this sensor shortens the accident response time to 30 seconds and reduces the personnel injury rate by 70%.
As a core component of modern detection technology, TMB is continuously expanding its value boundaries through material innovation, technological integration, and interdisciplinary applications. From basic research in life sciences to intelligent control in industrial production, from comprehensive protection of environmental safety to precise prevention and control of public health, the TMB technology system has become an important force in promoting social progress.
A preparation method of tetramethylbenzidine, the specific steps are:
(1) Add 2,6 ‑ dimethylaniline to anhydrous ethanol, add deionized water solution with catalyst and react at a temperature lower than 35 ℃, then drop oxidant, and extract the solid to obtain activated 2,6 ‑ dimethylaniline;
(2) Add the tetrahydrofuran solution dissolved with activated 2,6 ‑ dimethylaniline into the aqueous solution of ammonium chloride, and then add glacial acetic acid and metal catalyst. After the reaction is complete, separate the tetrahydrofuran layer and add it into the hydrochloric acid solution, and filter the solid to obtain the crude product of 3,3,5,5 ‑ tetramethylbiphenyl;
(3) Add the crude product of 3,3,5,5 ‑ tetramethylbiphenyl to methanol, add zinc powder, heat up to 50 ‑ 70 ℃ for reaction, filter while hot, concentrate and add it into ethanol, continue to heat up for dissolution, and concentrate the filtrate after filtration to obtain the pure product of 3,3,3,5,5 ‑ tetramethylbiphenyl.
The method is not only effective and simple, and avoids the use of dangerous raw materials, but also has high product yield and is suitable for industrial production.
It is an important chromogen reagent. Compared with traditional chromogen reagent (such as benzidine), it has the advantages of high detection sensitivity and good stability, and is safe to use, without carcinogenic and mutagenic effects. At present, TMB has gradually replaced the traditional chromogen reagent and is applied in clinical laboratory, forensic examination, criminal detection and environmental monitoring. Especially in clinical biochemical tests TMB, as a new substrate of peroxidase, has been widely used in enzyme immunoassay and enzyme-linked immunosorbent assay. But its price is high, so it is very important to study its synthesis method.
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