Fluorescence Spectroscopy
Method Introduction
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The fluorescence signal arising from the aromatic amino acids, primarily present in a protein sequence is called “intrinsic”. Intrinsic fluorescence is particularly sensitive to subtle conformational changes: partial unfolding and/or aggregation can lead to changes in the local environment of the tryptophan (Trp) and tyrosine (Tyr) residues (e.g., solvent exposure), leading to changes in fluorescence intensity and maximum emission.
Intrinsic fluorescence is measured upon excitation at 280 nm, and the protein’s fluorescence signal arises, in this case, from both tryptophan and tyrosine residues. Tryptophan fluorescence changes upon burial into the protein core are characterized by an increase in the fluorescence intensity and a blue shift in the maximum wavelength. More protein-specific are, instead, the changes in fluorescence that tyrosine undergoes in the same process because of possible quenching caused by the presence of other amino acids in close proximity in the tertiary structure. Therefore, depending on the protein and the scope of the work, it could be informative to use an excitation at 295 nm to selectively monitor the tryptophan residues, as well as the excitation at 280 nm (excitation of both Trp and Tyr), allowing a more straightforward interpretation of the observed changes in fluorescence intensity.
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Some fluorescent dyes bind to particular structural features and, if these features are present on the protein or peptide aggregates but missing from its monomeric form, and the binding results in a change in fluorescence intensity and/or emission maximum, the dye binding can be used to monitor changes in aggregation state.
Various fluorescent dyes, such as Nile Red, ANS and Bis-ANS, can be used for this purpose and each applicable to detect a peculiar structural feature (e.g., hydrophobic patches, intermolecular beta sheet structures, etc.) appearing during the aggregation pathway
Applications
Fluorescence spectroscopy is a frequently used and versatile technique for the analysis of the integrity of the higher-order structures of proteins or, when aromatic amino acids are present, peptides.
Quality and Biosafety Level
We provide all our analytical services with the highest quality standards. Experienced scientists carry out each project, and a scientific reviewer comprehensively checks every report or data presentation. We offer this technology with the following quality and biosafety levels:
R&D level
We offer this method under R&D. Our GRP system assures the highest-quality research standards.
Up to biosafety level 2
This method can be applied to nucleic acids, viruses, cells, viral vectors, including lentiviruses and more.
Fluorescence Spectroscopy Frequently Asked Questions (FAQs
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Fluorescence spectroscopy is a very sensitive analytical method used to investigate the higher-order structure of proteins by detecting intrinsic or extrinsic fluorescence signals. It is highly sensitive to conformational changes, unfolding, and aggregation, making it a valuable tool in biopharmaceutical development.
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Intrinsic fluorescence arises from aromatic amino acid residues—primarily tryptophan and tyrosine—within a protein. When excited at specific wavelengths (e.g., 280 nm or 295 nm), these residues emit fluorescence signals that shift with changes in their microenvironment, such as solvent exposure during protein unfolding.
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Extrinsic fluorescence involves adding fluorescent dyes like ANS, Bis-ANS, or Nile Red, which bind to, e.g., hydrophobic patches exposed during protein unfolding or aggregation. These dyes fluoresce strongly when bound, enabling detection of early-stage aggregation and structural instability.
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Fluorescence spectroscopy helps identify formulation conditions that promote protein stability. By detecting subtle structural changes under stress conditions—such as changes in pH, temperature, or excipients—it supports the development of robust biopharmaceutical formulations.
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Coriolis applies fluorescence spectroscopy to peptides (when possible), proteins, viral vectors (including lentiviruses), nucleic acids, and other biologics. The method is validated for R&D-level studies under biosafety levels up to BSL-2.
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Yes. Because it detects fine conformational differences, fluorescence spectroscopy is often used in comparability and biosimilarity studies to assess higher-order structure between reference and biosimilar products.
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Excitation at 280 nm captures fluorescence from both tyrosine and tryptophan residues, while 295 nm selectively excites tryptophan. The latter is often preferred due to its heightened sensitivity to microenvironmental changes, providing more precise data on protein structure
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All studies are performed under Coriolis’ GRP (Good Research Practice) system and reviewed by experienced scientists to ensure reliable, high-quality results.
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Yes. It is often used alongside orthogonal techniques such as circular dichroism (CD), nanoDSF, FTIR spectroscopy, and differential scanning microcalorimetry (µDSC) for a comprehensive analysis of protein structure and stability.
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Coriolis offers deep expertise in higher-order structure characterization, flexible method adaptation, and an integrated suite of orthogonal techniques to support clients across early- and late-phase biopharmaceutical development.
Analytical Method Development, Qualification and Validation
For common sample types, we can often apply standardized methods with little setup effort. However, when needed, our experienced analytical experts create or optimize custom methods tailored to your active pharmaceutical ingredient, product type and development phase.
Talk to Our Experts or Request a Quote
Our expert team is ready to answer your questions and guide you to the services best suited to your program’s modality, stage and challenge. If your needs are well-defined, we’ll begin the quotation process.
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