Tetramethylbenzidine CAS 54827-17-7

In the field of clinical in vitro diagnostics, TMB is the core chromogenic substrate of enzyme-linked immunosorbent assay (ELISA) and the core functional component of the vast majority of immunoassay kits. In the scene of serological detection of infectious diseases, the ELISA kits for screening infectious diseases such as hepatitis B B, hepatitis C antibody, and AIDS antibody based on the TMB chromogenic system, with extremely low background color and high detection sensitivity, have become the mainstream technical scheme for large-scale blood screening in domestic blood stations.

The application data of domestic blood stations shows that the minimum detection limit of hepatitis B B surface antigen can reach 0.1IU/mL with the blood screening kit using TMB as the chromogenic substrate.

Compared with the traditional OPD chromogenic system, the false negative missed diagnosis rate is reduced by 90%, completely blocking the transmission risk of transfusion related infectious diseases from the source. In routine outpatient infectious disease screening in tertiary hospitals, the TMB colorimetric system ELISA kit controls the inter batch difference of detection results within 5%, doubles the detection speed compared to traditional methods, and can complete high-throughput testing of thousands of samples per day.

Perfectly meeting the needs of early screening of infectious diseases in large-scale populations. In the clinical detection of hormones and tumor markers, the high sensitivity advantage of TMB colorimetric system is further highlighted. In the detection of thyroid function hormone indicators such as TSH, T3, T4, etc., the dynamic linear range of TMB color development is three times wider than traditional color development reagents, which can accurately cover the full concentration range from healthy individuals to hyperthyroidism and hypothyroidism patients, avoiding false alarms caused by hook effects in high concentration samples.

In the clinical detection of tumor markers, the TMB based ELISA kit can accurately detect tumor markers such as CEA and AFP with a concentration of only a few ng/mL in serum, providing reliable quantitative results for early screening and postoperative recurrence monitoring of cancer.

Statistical data from the pathology department of a tertiary hospital in China shows that the detection rate of early gastrointestinal tumors using the TMB colorimetric tumor marker combination detection scheme is 25% higher than traditional methods. In the auxiliary diagnosis scenario of rapid detection of COVID-19 antigen, TMB, as the core component of enzymatic color development other than colloidal gold method.

It is applied in the high-throughput ELISA detection system for COVID-19 antigen. The detection sensitivity is one order of magnitude higher than that of traditional colloidal gold method, which can achieve positive detection in the stage of low viral load, greatly reducing the missed diagnosis rate of early infected persons. In the supporting system of fully automated chemiluminescence immunoassay, TMB, as an enhanced substrate for horseradish peroxidase (HRP) direct chemiluminescence, can significantly improve the intensity and stability of the luminescence signal, increase the signal-to-noise ratio of chemiluminescence detection by 4 times, and control the CV value of the detection result within 3%, fully meeting the high-precision detection requirements of high-end fully automated immunoassay equipment.

In the field of biological research, tetramethylbenzidine is the preferred chromogenic substrate for HRP labeled secondary antibodies in Western blot experiments and is one of the most commonly used chromogenic reagents in molecular biology laboratories. In traditional Western Blot experiments, the use of TMB chemiluminescence enhanced colorimetric system can detect target proteins as low as pg level.

Compared with traditional DAB colorimetric method, the detection sensitivity is increased by two orders of magnitude, and it can easily identify the expression changes of low abundance signaling pathway proteins and transcription factors in cells.  Comparative experimental data from a domestic university's biology laboratory shows that using optimized TMB high-sensitivity luminescent solution, the development time of Western Blot has been shortened from the traditional 30 minutes to less than 10 seconds, with clean background bands and no impurities. Two similar protein bands with a molecular weight difference of only 1kDa can be clearly distinguished, greatly improving the reliability of protein immunoblotting experiments.

In the experimental scenarios of immunohistochemistry and immunohistochemistry, the improved TMB staining system can generate stable blue insoluble products, accurately locate the expression positions of target proteins in tissue slices and cell slides. Compared with traditional DAB brownish yellow staining, the blue TMB staining products form a soft color distinction with the blue purple nuclei stained with hematoxylin, without the common edge diffusion and background non-specific staining problems of DAB staining. In the experiments of neurotransmitter protein localization in neural tissue and PD-L1 protein expression interpretation in tumor tissue.

The recognition of TMB staining results is significantly better than traditional staining methods, helping researchers to more accurately observe the morphology of protein subcellular localization.

In the basic research scenario of enzyme activity analysis, TMB is a specific substrate for determining trace peroxidase activity. Based on TMB oxidation colorimetric spectrophotometry, HRP enzyme activity with a concentration as low as 1ng/mL in the sample can be accurately detected. It is a classic experimental method for studying plant peroxidase and microbial antioxidant enzyme activity. In the forefront research of single-molecule enzymology, immobilized TMB molecules can generate localized color precipitates under the catalysis of a single HRP enzyme molecule.

The spatial distribution and catalytic kinetics of a single enzyme molecule can be directly observed through an optical microscope, providing an intuitive observation method for the basic research of single-molecule enzymology. In the detection scenario of nucleic acid hybridization, HRP is labeled on the nucleic acid probe and the target nucleic acid sequence in the sample is quantitatively detected by TMB color intensity. Without the need for complex fluorescence detection equipment, low-cost nucleic acid qualitative and quantitative detection can be completed. It has been widely used in gene typing and screening experiments of genetically modified components in grassroots scientific research laboratories.

In the field of environmental monitoring, tetramethylbenzidine is widely used as a highly sensitive oxidation colorimetric reagent for rapid detection of trace amounts of hydrogen peroxide and heavy metal ions in water. In the scenario of drinking water safety testing, the TMB based peroxidase simulation colorimetric detection system can quickly detect residual hydrogen peroxide disinfectant in drinking water as low as 0.01mg/L, far below the limit requirements of the national drinking water hygiene standards.

The detection process does not require large instruments, and semi quantitative judgment can be completed by observing the blue depth of the solution with the naked eye, which is very suitable for emergency water quality monitoring in the field. In the detection scenario of heavy metal ions such as lead and mercury in water bodies, functionalized nano gold simulated peroxidase can specifically respond to heavy metal ions. Through the change of TMB color intensity, high selective visualization detection of heavy metal ions can be achieved, with a detection limit of up to 1nM and a detection cost of only one percent of traditional atomic absorption spectroscopy.

It has shown extremely high application value in rural drinking water safety screening and rapid monitoring of industrial wastewater discharge sites.

In the field of food safety testing, TMB is the core color developing component of various rapid detection test strips. In the scenario of enzyme inhibition detection of pesticide residues in food, organophosphorus and carbamate pesticides inhibit the activity of acetylcholinesterase. Through the coupling of peroxidase catalyzed TMB color change, on-site screening of pesticide residues in vegetables can be quickly completed within 10 minutes. 

The detection sensitivity meets the limit requirements of national food safety standards and is the most commonly used rapid detection method for pesticide residues in domestic agricultural markets and grassroots regulatory departments. In the colloidal gold immunochromatographic detection of small molecule pollutants such as clenbuterol and melamine illegally added in food, the enzyme catalyzed enhanced colorimetric technology based on TMB can increase the detection sensitivity of traditional colloidal gold test strips by more than 10 times, achieving accurate detection of low concentration illegal additives and greatly reducing the probability of false negatives and missed detections.

In the rapid detection scenario of microbial contamination in food, the use of hydrogen peroxide generated during microbial metabolism, combined with TMB color reaction, can quickly complete semi quantitative screening of the total number of bacteria in food within 2 hours.Compared with the traditional 48 hour plate culture method, the detection time is greatly shortened, which is very suitable for rapid quality control of food production lines in food manufacturing enterprises. In the rapid detection of aflatoxin B1 in grain and oil foods, the TMB based ELISA detection system has a minimum detection limit of 0.1 μ g/kg, fully meeting the strict limit requirements of aflatoxin in national food. The detection cost is much lower than that of mass spectrometry detection methods with large instruments, making it suitable for large-scale grain and oil food safety surveys.