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.
Quantitative compositional analysis of specific primary amines is applied in food science and agriculture to characterize samples for quality control. Primary amines in soil samples provide information about the available sources of organic and bioorganic N in an ecosystem [
For compositional amine analysis, a separation method must typically be applied to resolve specific amines within a sample. Many applications require field-deployable amine compositional analyses, such as clinical devices, detection of harmful biological agents, and
Fluorescence detection of primary amines provides a quick and potentially highly sensitive, quantitative analysis. Particularly, fluorescence detection of amines by CE-LIF with excitation at 488 nm has demonstrated limits of detection (LOD) from
Here, we describe a novel method for amine analysis using MEKC-LIF in conjunction with a labeling protocol employing AF488 NHS-ester. We prepared and analyzed samples of nonylamine, hexadecylamine, and octadecyl amine to test the applicability of this method to aliphatic amines with reduced solubility in aqueous media and no detectable autofluorescence. Samples were separated and detected using a commercial Beckman Coulter P/ACE MDQ system with 488 nm LIF detection. Labeling conditions were optimized, including organic solvent, the concentration of the base diisopropylethylamine (DIEA), and the incubation time. Suitable separation characteristics were found using MEKC with sodium dodecyl sulfate as the surfactant, and the resulting analytical technique was characterized.
All chemicals were of analytical reagent grade and were used as received. AlexaFluor 488 succinimidyl ester (AF488 NHS-ester) was purchased from Invitrogen Corporation (Carlsbad, CA), diluted to 20 mM in N,N-dimethylformamide (DMF, Sigma-Aldrich, St. Louis, MO), and stored at −20°C. Sodium carbonate (NaCO3, Sigma-Aldrich) was used to prepare 50 mM aqueous solutions with 18 MΩ·cm water. The pH was adjusted using 1 M NaOH (Sigma-Aldrich) and measured using a glass electrode and a digital pH meter (Orion 290A, Thermo; Waltham, MA). Sodium dodecyl sulfate (SDS) was acquired from Sigma Aldrich and used to prepare a 100 mM stock in 18 MΩ·cm water. Amines for standard solutions, including nonylamine (C9-NH2), dodecylamine (C12-NH2), hexadecylamine (C16-NH2), and octadecylamine (C18-NH2), were purchased in pure form from Sigma Aldrich and used to prepare 10 mM solutions in ethanol (Sigma-Aldrich). N,N-diisopropylethylamine (DIEA, Sigma-Aldrich) was diluted to 10 mM in ethanol, DMF, and dimethylsulfoxide (DMSO, Sigma-Aldrich). The stock solutions were combined as needed to result in the solutions used.
Labeling reactions were conducted by combining the appropriate volumes of 20 mM AF488, 10 mM DIEA, amine, and solvent. Reactions were incubated in the dark overnight (16–24 hrs) unless otherwise indicated. After incubation, reactions were diluted into the separation buffer at a 5 : 100 ratio unless otherwise indicated.
Capillary electrophoresis (CE) separations were conducted on a Beckman Coulter P/ACE MDQ capillary electrophoresis system equipped with 488 nm laser-induced fluorescence (LIF) detection. The capillary was rinsed using pressure with the separation buffer for two (2) minutes, and then the sample was injected (pressure) for 5 seconds. Separations were conducted at 15 kV for 15 minutes. After separation, the capillary was rinsed using pressure with pure water for five minutes. Capillary conditioning using 1 M NaOH was conducted with a 5-minute rinse as needed. Separation buffers tested included 10 mM carbonate (pH 10) and 10 mM carbonate with 12 mM SDS (pH 10).
Resulting electropherograms were generated and exported in comma-separated values format using 32 Karat software (Beckman Coulter Inc.). These files were imported into PeakFit (Systat) for smoothing (0.1% Loess) and baseline correction prior to peak fitting. The resulting smoothed and baseline corrected electropherograms or data from peak fitting was imported into Origin (OriginLabs) to generate figures. Chemical equations were drawn in ChemBioDraw Ultra. All raw figures were imported into Adobe Illustrator for image cleanup.
Figure
AlexaFluor 488 (AF488) succinimidyl ester and its reaction with primary amines. (a) The chemical structure of the reaction of AF488 succinimidyl ester with primary amines. N-hydroxysuccinimide acts as a leaving group to promote the formation of an amide bond to link AF488 to the primary amine group.
Normalized total fluorescence intensities of amines analyzed by CE-LIF and labeled with AF488 in ethanol (EtOH), dimethylformamide (DMF), and dimethylsulfoxide (DMSO). Labeling was conducted at 25
To explore the impact of DIEA concentration on labeling efficiency, its concentration in ethanol was varied from 0 to 48.75
Effect of varying DIEA concentration on peak intensity (a measure of labeling efficiency). Relative fluorescence intensities of dodecylamine are plotted against varying DIEA concentration. Labeling was conducted at 1
While overnight incubations are logistically simple for an operator and for potential automated implementations, sometimes it is preferable to obtain results from a sample within a shorter window of overall time. Therefore, we examined the impact of incubation time on the labeling reaction extent. Figure
Effect of incubation time on labeling efficiency. Normalized fluorescence intensities of nonylamine and octadecylamine are plotted against incubation time. Labeling was conducted at 1
Based on the above experiments, the optimum conditions for our work using AF488 NHS-ester as a fluorescent probe for amine analysis are 12.5
Table of separation characteristics.
Amine | Amplitude | Area | Elution |
Peak efficiency |
Peak effici. |
LOD (nM) |
---|---|---|---|---|---|---|
Nonylamine | 2.2 × 107 | 3.5 × 107 | 5.5 | 2.7 × 105 | 550000 | N.D.b |
Hexadecylamine | 4.5 × 107 | 1.1 × 108 | 7.0 | 1.7 × 105 | 350000 | 17 |
Octadecylamine | 4.4 × 107 | 1.1 × 108 | 7.0 | 1.1 × 105 | 220000 | 5.7 |
bN.D.: not determined.
Electropherograms of amines labeled with the optimized labeling conditions (1
Amine detection using LIF with AF488 as the reactive probe enables potentially novel analytical techniques as it provides an alternative to other probes’ fluorescent at 488 nm. The optimized reaction conditions are mild and fast (90% efficiency within 6 hrs) while enabling excellent limits of detection (nM or ppb). While AF488 is not fluorogenic and therefore cannot be used as a fluorescent probe without some form of postreaction separation, we have demonstrated that this separation can be achieved using a simple, standard MEKC solution and a commercial CE instrument. The limits of detection achievable using the commercial system and initial CE separation conditions were in the low nM range, or single parts-per-billion, making this technique more than sufficiently sensitive for multiple applications. Further work remains to fully characterize the kinetics of the labeling reaction. Additionally, the current separation method is insufficient for the simultaneous analysis of C16-NH2 and C18-NH2; thus, further work is required to develop application-specific optimized separation methods.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The research described in this paper was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (NASA) and was supported by the NASA Astrobiology Science and Technology Instrument Development program. The JPL author’s copyright for this paper is held by the California Institute of Technology. Government sponsorship is acknowledged.