Fluorescein is one of most used fluorescent labels for characterising biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins, then one must account for the fact that the fluorescence of fluorescein labelled protein can be affected by the presence of intrinsic amino acids residues, such as, tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved and this makes it difficult to evaluate quantitatively the photophysics of fluorescein labelled proteins. To address this we have explored the fluorescence of fluorescein in buffered solutions, in different acid and basic conditions, and at varying concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Non-fluorescent complexes with low association constants (5.0 -24.1 M -1 ) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphereof-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3×10 9 M -1 s -1 ) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer mechanism was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7×10 9 s -1 ) and the electronic coupling values (5.7-25.1 cm -1 ) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter complex were then obtained and analysed. This data will be applied to generate a more detailed, quantitative understanding of the photophysics of fluorescein when conjugated to proteins containing the amino acid tryptophan. KEYWORDS:Fluorescein, Tryptophan, fluorescence quenching, electron transfer, bioconjugation.Page 2 of 19 IntroductionWidely applied in fluorescence imaging microscopy, the fluorophore labelled protein can be used to rapidly and easily visualise many different biochemical pathways, which involve protein interactions, protein expression, trafficking, intracellular signalling events, and cellular location. 1,2 Many of the fluorophores used are designed to conjugate with specific amino acid residues or functional groups present in the target biomolecule. In many cases, the fluorophore is simply used as a contrast agent i.e. to show the location of the target biomolecule in a particular environment. However, for quantitative measurements of protein-surface intera...
Using fluorescence excitation-emission matrix spectroscopy and chemometric methods we demonstrate an effective and rapid method for quantitative monitoring of a mammalian cell culture based manufacturing process.
Publication InformationLi, B.; Calvet, A.; Casamayou-Boucau, Y.; Morris, C.; Ryder, A.G. (2015) 'Low-content quantification in powders using Raman spectroscopy: a facile chemometric approach to sub 0.1% limits of detection'. Analytical Chemistry, 87 (6):3419-3428. PublisherAmerican Chemical Society Low-content quantification in powders using Raman spectroscopy: a facile chemometric approach to sub 0.1% limits of detection.Boyan Li, Amandine Calvet, Yannick Casamayou-Boucau, Cheryl Morris, and Alan G. Ryder* Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland, Galway, Galway, Ireland.* To whom correspondence should be addressed.Tel: +3539149 2943 Email: alan.ryder@nuigalway.ie ABSTRACT A robust and accurate analytical methodology for low-content (<0.1%) quantification in the solid-state using Raman spectroscopy, sub-sampling, and chemometrics was demonstrated using a piracetam-proline model. The method involved a 5-step process: collection of relatively large number of spectra (8410) from each sample by Raman mapping, meticulous data pretreatment to remove spectral artefacts, use of a 0-100% concentration range partial least squares (PLS) regression model to estimate concentration at each pixel, use of a more-accurate, reduced concentration range PLS model to accurately calculate analyte concentration at each pixel, and finally statistical analysis of all 8000+ concentration predictions to produce an accurate overall sample concentration. The relative prediction accuracy was ~2.4% for a 0.05~1.0% concentration range and the limit of detection was comparable to high performance liquid chromatography (0.03% versus 0.041%). For data pretreatment, we developed a unique cosmic ray removal method and used an automated baseline correction method, neither of which required subjective user intervention and thus were fully automatable. The method is applicable to systems, which cannot be easily analyzed chromatographically such as hydrate, polymorph, or solvate contamination.
The rapid, quantitative analysis of the complex cell culture media used in biopharmaceutical manufacturing is of critical importance. Requirements for cell culture media composition profiling, or changes in specific analyte concentrations (e.g. amino acids in the media or product protein in the bioprocess broth) often necessitate the use of complicated analytical methods and extensive sample handling. Rapid spectroscopic methods like multi-dimensional fluorescence (MDF) spectroscopy have been successfully applied for the routine determination of compositional changes in cell culture media and bioprocess broths. Quantifying macromolecules in cell culture media is a specific challenge as there is a need to implement measurements rapidly on the prepared media. However, the use of standard fluorescence spectroscopy is complicated by the emission overlap from many media components. Here, we demonstrate how combining anisotropy measurements with standard total synchronous fluorescence spectroscopy (TSFS) provides a rapid, accurate quantitation method for cell culture media. Anisotropy provides emission resolution between large and small fluorophores while TSFS provides a robust measurement space. Model cell culture media was prepared using yeastolate (2.5 mg mL(-1)) spiked with bovine serum albumin (0 to 5 mg mL(-1)). Using this method, protein emission is clearly discriminated from background yeastolate emission, allowing for accurate bovine serum albumin (BSA) quantification over a 0.1 to 4.0 mg mL(-1) range with a limit of detection (LOD) of 13.8 μg mL(-1).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.