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Microplate readers, also known as plate readers or microplate spectrophotometers, are essential laboratory instruments designed to detect biological, chemical, or physical events within microtiter plates. Among the different detection modes available in microplate readers—fluorescence, luminescence, and absorbance—absorbance is one of the most widely used and foundational techniques. It offers researchers a reliable, efficient, and high-throughput method to quantify the concentration of analytes in solution based on the amount of light absorbed by the sample.
What Is Absorbance in Microplate Reading?
Absorbance refers to the amount of light that a substance absorbs at a particular wavelength. This principle is based on the Beer-Lambert Law, which states that the absorbance is directly proportional to the concentration of the absorbing species and the path length through which the light passes. In the context of microplate readers absorbance means that when a light beam passes through a well in a microplate, the amount of light absorbed can be measured to determine the concentration of specific molecules in the well.
Microplate readers equipped for absorbance use a light source (typically a xenon flash lamp or LED) to pass light through the sample. The light that exits the sample is captured by a detector, and the difference between the emitted and transmitted light is calculated as absorbance.
How Microplate Readers Measure Absorbance
A typical absorbance-based microplate reader operates in a range of UV-visible wavelengths (usually 200–1000 nm). It can scan a full spectrum or focus on specific wavelengths optimal for the analyte of interest. The reader shines light vertically or horizontally through each well and records the transmitted light. The output is an absorbance value, which is a unitless number that represents how much light was absorbed.
Modern plate readers come with monochromators or filters to select desired wavelengths, making them versatile for different assays such as ELISA, nucleic acid quantification, and enzyme kinetics.
Applications of Absorbance in Microplate Readers
ELISA (Enzyme-Linked Immunosorbent Assay):
Absorbance is the most common detection mode for ELISA, a widely used immunoassay to detect antigens or antibodies in a sample. The enzymatic reaction in microplate readers absorbance produces a color change measurable by absorbance, usually at 450 nm or similar wavelengths.
Nucleic Acid and Protein Quantification:
DNA, RNA, and proteins absorb UV light at characteristic wavelengths (260 nm for DNA, 280 nm for proteins). Microplate readers facilitate rapid and parallel measurement of multiple samples, essential for molecular biology workflows.
What Is Absorbance in Microplate Reading?
Absorbance refers to the amount of light that a substance absorbs at a particular wavelength. This principle is based on the Beer-Lambert Law, which states that the absorbance is directly proportional to the concentration of the absorbing species and the path length through which the light passes. In the context of microplate readers absorbance means that when a light beam passes through a well in a microplate, the amount of light absorbed can be measured to determine the concentration of specific molecules in the well.
Microplate readers equipped for absorbance use a light source (typically a xenon flash lamp or LED) to pass light through the sample. The light that exits the sample is captured by a detector, and the difference between the emitted and transmitted light is calculated as absorbance.
How Microplate Readers Measure Absorbance
A typical absorbance-based microplate reader operates in a range of UV-visible wavelengths (usually 200–1000 nm). It can scan a full spectrum or focus on specific wavelengths optimal for the analyte of interest. The reader shines light vertically or horizontally through each well and records the transmitted light. The output is an absorbance value, which is a unitless number that represents how much light was absorbed.
Modern plate readers come with monochromators or filters to select desired wavelengths, making them versatile for different assays such as ELISA, nucleic acid quantification, and enzyme kinetics.
Applications of Absorbance in Microplate Readers
ELISA (Enzyme-Linked Immunosorbent Assay):
Absorbance is the most common detection mode for ELISA, a widely used immunoassay to detect antigens or antibodies in a sample. The enzymatic reaction in microplate readers absorbance produces a color change measurable by absorbance, usually at 450 nm or similar wavelengths.
Nucleic Acid and Protein Quantification:
DNA, RNA, and proteins absorb UV light at characteristic wavelengths (260 nm for DNA, 280 nm for proteins). Microplate readers facilitate rapid and parallel measurement of multiple samples, essential for molecular biology workflows.
