Drop Coating Deposition Raman Spectroscopy of Fluorescein Isothiocyanate Labeled Protein

Abstract

Using bovine serum albumin (BSA) as the model protein normal Raman spectra of Fluorescein isothiocyanate (FITC) -conjugated protein was systematically studied for the first time using both solution and the drop coating deposition Raman (DCDR) sampling techniques. The FITC-BSA Raman spectra are dominated by the FITC Raman features that are strongly pH dependent. Current DCDR detection sensitivity obtained with a 10:1 FITC-BSA conjugate is 45 fmol in terms of total protein consumption and ~15 attomol at laser probed volume. Unlike the FITC-BSA solution Raman spectra where the FITC Raman features are photostable, concurrent FITC fluorescence and Raman photobleaching is observed in the DCDR spectra of FITC-BSA. While the FITC Raman photobleaching follows a single exponential decay function with a time constant independent of the FITC labeling ratio, the fluorescence background photobleaching is much more complicated and it depends strongly on the FITC labeling ratio and sample conditions. Mechanistically, the FITC Raman photobleaching is believed to be due to photochemical reaction of the FITC molecules in the electronically excited state. The FITC fluorescence photobleaching involves both concentration quenching and photochemical quenching, and the latter may involve a photochemical intermediate that is fluorescence inactive but Raman active.

Structure of bovine β-lactoglobulin–lactoferrin coacervates

“…Fluorescently labeled proteins, FITC-BLG and RITC-LF, were prepared according to Zhang et al 24 Stocks of 5 g L À1 FITC or RITC ethanol solution were added dropwise under mild stirring into 200 mM BLG or LF in pH 8.0, 0.1 M phosphate buffer with a molar ratio (dye-protein) of 2.0. Aer 1 h reaction in the dark, labeling was terminated and free dye was removed by dialysis (10 kDa MW cutoff) against 10 mM Tris-HCl buffer (pH 7.4, 0.6 M NaCl).…”

Raman Enhancement and Photo-Bleaching of Organic Dyes in the Presence of Chemical Vapor Deposition-Grown Graphene

“…Raman peaks from both graphene (ca. 1592 cm −1 ) and FITC (1185 cm −1 , 1334 cm −1 , 1425 cm −1 , 1495 cm −1 , and 1640 cm −1 ) were clearly observed on Gr/FITC sample (Gr: graphene), while the FITC sample in the absence of graphene barely showed any peak except the Si-Si overtone (969 cm −1 ) from the Si substrate [ 9 , 20 , 21 ]. Similar results were also observed on other fluorescence dyes, such as pyrromethene, coumarin 540A, fluorescein 548, disodium fluorescein, and kiton red 620.…”

Fluorescein‐Labeled Starch Maleate Nanoparticles as Sensitive Fluorescent Sensing Probes for Metal Ions

“…This is because FITC is present in dianionic form and more soluble in basic condition [9,28,31]. However, FITC is mainly in monoanionic form [32] and has lower solubility in acidic solution.…”

“…Financial support by the Ministry of Higher Education (MOHE) Fundamental Research Grant Scheme (FRGS), Grant no. 01 (17)746/2010 (32), and MyBrain (MyMaster) Program for graduate scholarship was gratefully acknowledged.…”