Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser-induced fluorescence detection.

Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and β-cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.

Amino acids are organic compounds that exhibit a major role in many fields (nutrition, health, cosmetics, biochemistry, etc). Several cosmetic products have proved to bring flexibility, softness and elasticity to human skin due to their content in amino acids. Their level in stratum corneum which is the outermost layer of the epidermis and the protecting barrier of the skin is a potential marker of this organ health. Amino acid systemic level may be used to diagnose inherited metabolic diseases. Thus, quantitative and qualitative analysis of amino acids is critical [1][2][3]. Detection of amino acids from different matrices at trace levels is difficult due to their physicochemical properties, such as absence of chromophores, amphotericity, non-volatile nature, high polarity, and complexation with metal ions [4].
FITC is widely used as a derivatization reagent for primary amine group. Its derivatives are easily formed and generate strong fluorescence signals with excitation and emission wavelengths (488/520 nm) compatible with an argon ion laser. Derivatization can be obtained after incubation, in the dark, at room temperature for 16 h [33,34] or at 50°C for 5 h [35,36].
Liwei Cao et al. [4] also proposed a microwave (MW)-assisted derivatization approach to accelerate labeling reaction kinetics and hence to reduce derivatization time to 150 s. In inorganic and organic chemistry, microwave technology has been used since the late 1970s and the mid-1980s, respectively. The main advantages of microwave assisted organic synthesis are solvent-free use, shorter reaction times and expanded reaction range which make this technique very interesting [37].
Many studies have been done on the derivatization and separation of standard amino acids, but few papers have been devoted to the separation of non-standard or non-protein amino acids by CE and, to our knowledge, no CE-LIF method have been developed so far to separate and detect these amino acids from microalgae. For this, the analysis of amino acids in Dunaliella salina (DS) microalgae was conducted. Indeed, chemical studies on microalgae are complicated by difficulties mainly connected with the influence of nutrients and contaminants in their environment as well as by climatic factors on the biosynthesis of natural products [38]. DS microalga is known to be the best commercial source of natural β-carotene.
It can accumulate significant amounts of valuable chemicals such as carotenoids, lipids, vitamins and proteins. Additionally, it has a high potential value for biotechnological processes such as treatment of wastewater [39]. In regard to amino acid extraction from microalgae a new method based on low voltage (few V/cm, few mA) was developed. Low voltage (or low current) can induce iontophoresis phenomena implicating the motion of ions across the cell membrane under the influence of the electric field. Electroporation and reverse iontophoresis have been successfully used for extraction of transdermal multi-biomarkers, like urea, prostate-specific antigen (PSA), and osteopontin [40]. Reverse iontophoresis across the skin was also shown to be a useful alternative for non-invasive drug monitoring [41].
Moreover, it was shown that electrically based disruption techniques such as pulsed electric 4 field (PEF) (20 kV/cm, 1-4 ms) and high-voltage electrical discharge (HVED) (40 kV/cm, 1-4 ms) allow selective extraction of water soluble ionic components and microelements, small molecular weight organic compounds and water soluble proteins in a short time [42,43].
In fact, electroporation is an effective process to breach cell membrane barrier by applying a voltage, and has been used for the extraction of intracellular protein and small molecules from bacterial and eukaryotes cells. In an electroporation process, cells are exposed to an external electric field which induces a potential difference across the membrane. The induced transmembrane potential (Δψ E ) is given by the following equation [36]: Where g(λ) is a complex function of the membrane and buffer conductivities, r is the radius of the cell, E is the electric field intensity and θ is the angle between the normal to the membrane surface and the electric field direction.
When Δψ E exceeds a threshold, the cell membrane can be irreversibly broken down. As a consequence, cell lysis and intracellular content release occur.
The aim of this study was to develop a CE-LIF method for the sensitive determination of amino acids in microalgae extracts. For this, the fluorescent labeling and the electrophoretic separation of amino acids were optimized, and a new extraction procedure based on electroporation at low voltage was introduced. Dunaliella salina (DS) microalgae were used as model algae to evaluate the efficiency of the developed approach.

Solutions
All solutions were prepared with ultra-pure water.
-Separation or BGE: Unless otherwise stated, borate buffer (10 mM or 100 mM, pH 9.4) was used as a separation BGE by dissolving appropriate amount of sodium tetraborate decahydrate in distilled water. 10 mM of alpha-cyclodextrin (α-CD) or beta-cyclodextrin (β-CD) were added to the BGE to improve peak resolution.
-Derivatization buffer (for standard amino acids): a borate buffer at 10 mM and pH 9.4 was used for fluorescent labeling of amino acids.
The different buffers were prepared fresh each day. Their pH was checked with a Meterlab PHM201 Portable pH Meter (Radiometer Analytical, Villeurbanne, France).
-Stock solutions: Amino acid and FITC stock solutions were prepared respectively at 6 mM and at 10 mM in the derivatization buffer and stored at -20°C in the dark. These solutions were diluted appropriately each day in the same buffer.

Dunaliella salina microalgae media and cultivation
Dunaliella salina strain used in this work was isolated from the Sebkha of Sidi El Hani (Tunisia).
-Culture medium: the culture medium used was the F/2 [44] using artificial seawater ASW.

Instrumentation and operating conditions
All experiments were performed with a CE instrument: AB Sciex PA 800+ equipped with a LIF detector (Brea, CA, USA). The excitation was carried out with an Argon ion laser at a wavelength of 488 nm. The band-pass filter 520 nm was used for emission. Data acquisition and instrument control were carried out using 32 Karat acquisition system software.
Separations were carried out using fused-silica capillaries of 60 cm total length (50 cm detection length) purchased from Polymicro Technologies (Phoenix, AZ, USA). The inner diameter was 50 or 75 µm. The capillary was maintained at 25°C and separation was conducted at +25 kV. Hydrodynamic injection mode with 0.5 psi pressure was applied for 5 s.
Each new capillary was conditioned by flushing it with 1 M NaOH (10 min), water (5 min) and BGE (15 min). Between two runs, the capillary was flushed with NaOH 1M (1 min), water (1 min) and BGE (3 min). All rinse cycles were conducted at 20 psi pressure. The buffer vials were renewed every three analyses.

Derivatization procedure
Pre-capillary derivatization of analytes was applied because labeling reaction is slow and thus inadequate for in-capillary derivatization mode. 7 Different ratios of FITC and amino acids were tested to evaluate the efficiency of the derivatization. In a 1 mL total volume, the final amino acid concentration was 1 µM with concentration ratios of FITC to each amino acid of 2:1, 1:1, 1:5 or 1:20. The mixture vials were capped, homogenized and allowed to stand in the dark at 25°C for 5h or 50°C for 16h.
After derivatization, the vials were kept at -20°C. Before CE analysis, the labeling mixture was diluted with the derivatization borate buffer (10 mM, pH 9.5).
The same procedure was conducted for microwave-assisted derivatization using a microwave synthesis labstation (Start Synth, Milestone, Bergamo). Heating was conducted with microwave irradiation under 680 W at constant temperature of 80°C for only 150s.
Blank assays were conducted to evaluate the stability of FITC in the derivatization conditions. For this, FITC was prepared in the derivatization borate buffer and heated at 80°C for 150s by MW or by conventional heating.

CE-LIF was chosen for the analysis of amino acids extracted by electroporation from
Dunaliella salina microalgae. The fluorescent labeling of amino acids by fluorescein isothiocyanante as well as electrophoretic separation conditions were optimized to obtain short labeling time and high sensitivity of the CE-LIF method.

Labeling of amino acids with fluorescein isothiocyanate isomer I 9
All amino acids (Asn, Gln, Arg, Glu, Orn, Ile, Cit and Ala) selected possess a primary amino group, which may react with the isothiocyanate group of FITC to form the fluorescent derivatives. Labeling with FITC is commonly carried out in borate buffer to minimize the hydrolysis of the labeling agent and consequently the number of undesired fluorescent byproducts [45,46]. Moreover, the derivatization reaction must be performed under alkaline conditions [47] to favor the nucleophilic addition of the neutral deprotonated amino group to the double bound of the FITC. Therefore, 10 mM sodium tetraborate at pH 9.4 was used as derivatization buffer for developing the amino acid CE-LIF analysis method.
Different conditions were tested to optimize the yield of the labeling reaction, which was W and 80°C (Figure 1). Comparable results in terms of sensitivity were obtained by conventional convective heating but with longer reaction times (120 times). These results are consistent with literature, demonstrating that microwaves can be very efficient for remarkably accelerating fluorescent tagging [37,48]. As for conventional convective heating, blank assays conducted (section 2.5) confirmed that MW heating had low effect on FITC stability.
For the remaining of this study, microwave-assisted derivatization of amino acids with FITC in alkaline borate buffer was used for CE-LIF analysis.

Analysis of labeled amino acids by CE with LIF detection
Borate buffer was used as the BGE because it provides a stable EOF as reported by several authors [4]. As shown in Figure 2.a, a good separation was obtained for a standard mixture influence on compounds stability. The developed CE-LIF method was validated in terms of linearity. Calibration linear curves were obtained for the different studied amino acids with r² higher than 0.9975. Excellent sensitivity was found with a LOQ of a few nM for the different amino acids (3-9 nM). LOQs were obtained by injecting and analyzing each labeled amino acid at the estimated value for S/N=10. This so-called instrumental LOQ is different from the limit of derivatization which expresses the minimum amount of the analyte that can react with the fluorescent reagent. Repeatability was also satisfactory with RSD on migration times and on peak areas inferior to 1.2 and 1.9% (n=6), respectively.

Analysis of amino acids in microalgae after electroporation extraction
The previously developed CE-LIF method was applied to the analysis of amino acids in  (Table 1). At low voltage 6 V DC, no extraction was obtained due certainly to the incapacity of this electric field to damage the microalgae membrane. For higher direct voltages (12 and 36 V), extraction was successful and three amino acids (D-Arg, L-α-Ala and L-β-Ala) were detected and identified by CE-LIF. To further understand this process, different extraction times were studied. The Figure 3 summarizes extraction results obtained by electroporation of amino acids from Dunaliella salina green algae. As it can be seen, the best extraction results were obtained at 12 V DC for 4 min. When this voltage was applied for a longer time (10 min), the amount of extracted amino acids decreases (-20%) due certainly to their degradation by electrochemical reactions in contact with the electrode surface. Figure 4 shows the electropherograms for the analysis of Dunaliella salina extracts obtained by applying 12 V DC voltage for different times.
Several amino acids have been identified such as D-Arg, L-α-Ala and L-β-Ala. Identification was done by migration time matching and by spiking the samples with labeled amino acids.
On the other hand, good extraction was also obtained when applying pulses of higher voltage i.e.36 V DC during only 20s.
To conclude, best extraction was obtained at lower and long-lasting voltage (12 V DC x 4 min). More precisely, extraction is probably due to iontophoresis phenomenon implicating the motion of ions across the cell membrane under the influence of an electric field. For analytes such as amino acids, both electromigration as well as electroosmosis contribute to the iontophoretic extraction across the damaged membrane, resulting in increased analyte extraction. In other terms, extraction is mainly due to electroporation (increasing cell permeability) as well as to amino acid migration from the cell into the surrounding solvent.
Finally, an alternative voltage (12 V AC, 50 Hz) has been tested applied for 4 or 10 min to perform the extraction by electroporation but did not engender any extraction as confirmed by CE-LIF. Indeed, when using alternative voltage, the iontophoretic extraction was reduced.
Only reversible pores which reclosed rapidly may be obtained in these conditions. The herein developed electroporation-CE-LIF method was then used to study the effect of the cultivation conditions on the amino acid quantity in microalgae ( Figure 5). Indeed, the environmental constrains increase the productivity of biologically active molecules by microalgae [38]. The same amino acids were found in the two cultivations. However, our results showed that the imposed stress conditions induced the production of a higher quantity of amino acids in Dunaliella salina than in normal conditions. For both D-Arg and L-Ala, the concentration is ten times higher in the stressed algal cultivation. Indeed, it has been shown that alanine accumulation in plants and animals in response to exposure to a variety of stress conditions is a general phenomenon. Alanine is a universal first stress signal expressed by cells [50,51].

Concluding remarks
A new CE-LIF method was developed for the determination and the identification of non- In this study, we present a proof-of-concept demonstration of a green approach to extract and analyze amino acids in microalgae. Further optimization will be necessary to identify more than two amino acids in microalgae and to expand this approach for studying varied molecular families in other algae. Several algae products have attracted great interest due to their potential practical application as pharmaceutical agents, cosmetic ingredients, energy sources, valuable food constituents, and future materials for nanotechnology. The derivatization yield 100% corresponds to the maximum peak area obtained using microwave-assisted derivatization.