Amine Analysis Using AlexaFluor 488 Succinimidyl Ester and Capillary Electrophoresis with Laser-Induced Fluorescence

Abstract

Fluorescent probes enable detection of otherwise nonfluorescent species via highly sensitive laser-induced fluorescence. Organic amines are predominantly nonfluorescent and are of analytical interest in agricultural and food science, biomedical applications, and biowarfare detection. Alexa Fluor 488 N-hydroxysuccinimidyl ester (AF488 NHS-ester) is an amine-specific fluorescent probe. Here, we demonstrate low limit of detection of long-chain (C9 to C18) primary amines and optimize AF488 derivatization of long-chain primary amines. The reaction was found to be equally efficient in all solvents studied (dimethylsulfoxide, ethanol, and N,N-dimethylformamide). While an organic base (N,N-diisopropylethylamine) is required to achieve efficient reaction between AF488 NHS-ester and organic amines with longer hydrophobic chains, high concentrations (>5 mM) result in increased levels of ethylamine and propylamine in the blank. Optimal incubation times were found to be >12 hrs at room temperature. We present an initial capillary electrophoresis separation for analysis using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecylsulfate (SDS) and 5 mM carbonate, pH 10. Limits of detection using the optimized labeling conditions and these separation conditions were 5–17 nM. The method presented here represents a novel addition to the arsenal of fluorescent probes available for highly sensitive analysis of small organic molecules.

Structure-Based Multifunctionalization of Flexuous Elongated Viral Nanoparticles

“…9,47−49 For addressability of these residues, a fluorescent dye was used: Alexa Fluor 555 NHS ester. 50 The conjugation was also followed by UV illumination of a SDS-PAGE gel, as described previously (Figure 5). Conjugation was tested in both virions and VLPs, obtaining conjugation in both.…”

The hydrophobic C‐terminal sequence of transthyretin affects its catalytic kinetics towards amidated neuropeptide Y

“…Among synthetic fluorophores, Alexa Fluor 488 has been successfully used in several labeling techniques [50]. In the labeling process, the Alexa dye reacts with primary amines on protein molecules [51,52], and its conjugate product was claimed to have more fluorescence and be more photostable than the conjugates of conventional dyes [51]. Similarly to other dyes in the Alexa series, Alexa 488 is synthesized in succinimidyl ester form, which allows the reaction to take place efficiently at pH 7.5-8.5 [53].…”

Derivatisation for separation and detection in capillary electrophoresis (2015–2017)

“…The handling of minute samples is difficult, and thus this mode is applied when ample volumes of samples and reagents are available. Pre‐capillary mode was the most commonly applied procedure accounting for 2/3 or 29 of the 46 covered articles .…”

“…This is often the case for fluorescence detection where native fluorescence is rather uncommon and only the labelled species provide a strong signal. There were 24 articles on LIF , 16 on UV , 3 on electrochemical detection , and 4 on MS . In MS detection, derivatisation was used to improve the separation (see Section ) and to enable MS detection in favourable positive ion ESI mode .…”