Pavel Khramtsov , Anastasia Novokshonova , Zarina Galaeva , Maria Morozova , Tatiana Bezukladnikova , Mikhail Rayev
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引用次数: 0
Abstract
3,3′,5,5′-Tetramethylbenzidine (TMB) remains one of the most widely utilized chromogenic substrates for horseradish peroxidase (HRP) in colorimetric immunoassays, including enzyme-linked immunosorbent assays (ELISA). Despite its introduction into ELISA workflows over four decades ago, limited research has been conducted to systematically optimize TMB substrate formulations. Recent advancements in the field have proposed innovative approaches to enhance HRP catalysis, such as the use of deep eutectic solvents and ionic liquids, alongside investigations into the chemical properties of TMB and its analogs to identify more efficient alternatives. However, the development of stable and high-performance TMB solutions for clinical diagnostics requires a comprehensive understanding of how formulation parameters influence signal intensity and stability. In this study, we address these gaps by conducting a systematic evaluation of key factors affecting TMB substrate performance, including buffer pH, composition and molarity, specific ion effects, incorporation of organic solvents, and the use of polymer stabilizers. Additionally, novel strategies for signal amplification, identified through an extensive review of literature and patents, were experimentally tested. Based on these findings, we developed an optimized TMB formulation comprising 0.2 mol/L sodium citrate buffer (pH 4.5), 5 % DMSO, 0.37 mmol/L CaCl2, 0.4 mmol/L 2-hydroxy-β-cyclodextrin, 0.8 mmol/L TMB, and 1.3 mmol/L H2O2 as final concentrations. The performance of this optimized formulation was evaluated in comparison to previously reported formulations from the literature.
期刊介绍:
The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field.
The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology.
The journal has been particularly active in:
-Analytical techniques for biological molecules-
Aptamer selection and utilization-
Biosensors-
Chromatography-
Cloning, sequencing and mutagenesis-
Electrochemical methods-
Electrophoresis-
Enzyme characterization methods-
Immunological approaches-
Mass spectrometry of proteins and nucleic acids-
Metabolomics-
Nano level techniques-
Optical spectroscopy in all its forms.
The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.