Reduction of Background Fluorescence from Impurities in Protein Samples for Raman Spectroscopy

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15310%2F22%3A73615662" target="_blank" >RIV/61989592:15310/22:73615662 - isvavai.cz</a>

Výsledek na webu

<a href="https://downloads.hindawi.com/journals/jspec/2022/1928091.pdf" target="_blank" >https://downloads.hindawi.com/journals/jspec/2022/1928091.pdf</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1155/2022/1928091" target="_blank" >10.1155/2022/1928091</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Reduction of Background Fluorescence from Impurities in Protein Samples for Raman Spectroscopy

Popis výsledku v původním jazyce

Background fluorescence remains the biggest challenge in Raman spectroscopy because of the consequent curvature of the baseline and the degradation of the signal-to-noise ratio of the Raman signal. While the concentrations of the fluorophore impurities are usually too low to be detected by other analytical methods, they are often sufficient to prevent Raman data collection. Among the different existing methods to remove the fluorescence signal, photobleaching remains the most popular due to its simplicity. However, using the spectrometer laser to photobleach is far from optimal. Most commercially available instruments have little or no choice of wavelength, and their output powers are in many cases not suitable for highly fluorescent samples such as those from biological systems (e.g., proteins). In this article, we assess practical aspects of photobleaching such as the apparent reversibility of the process and the effect of convection currents due to what we speculate to be temperature gradients across the bulk of the solution. We also introduce an affordable custom made external photobleaching unit with a choice of excitation wavelength and demonstrate its viability with a highly fluorescent bovine serum albumin protein solution, which had proved most challenging for Raman spectroscopy as it contained ∼10% w/w impurities.

Název v anglickém jazyce

Reduction of Background Fluorescence from Impurities in Protein Samples for Raman Spectroscopy

Popis výsledku anglicky

Background fluorescence remains the biggest challenge in Raman spectroscopy because of the consequent curvature of the baseline and the degradation of the signal-to-noise ratio of the Raman signal. While the concentrations of the fluorophore impurities are usually too low to be detected by other analytical methods, they are often sufficient to prevent Raman data collection. Among the different existing methods to remove the fluorescence signal, photobleaching remains the most popular due to its simplicity. However, using the spectrometer laser to photobleach is far from optimal. Most commercially available instruments have little or no choice of wavelength, and their output powers are in many cases not suitable for highly fluorescent samples such as those from biological systems (e.g., proteins). In this article, we assess practical aspects of photobleaching such as the apparent reversibility of the process and the effect of convection currents due to what we speculate to be temperature gradients across the bulk of the solution. We also introduce an affordable custom made external photobleaching unit with a choice of excitation wavelength and demonstrate its viability with a highly fluorescent bovine serum albumin protein solution, which had proved most challenging for Raman spectroscopy as it contained ∼10% w/w impurities.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10306 - Optics (including laser optics and quantum optics)

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Spectroscopy

ISSN

2314-4920

e-ISSN

2314-4939

Svazek periodika

2022

Číslo periodika v rámci svazku

NOV

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

8

Strana od-do

"1928091-1"-"1928091-8"

Kód UT WoS článku

000893515000001

EID výsledku v databázi Scopus

2-s2.0-85143404943