Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

Hua ZhangWenrui Jin

School of Chemistryand Chemical Engineering,State Key Laboratoryof Microbial Technology,Shandong University,Jinan, China

Analysis of amino acids in individual humanerythrocytes by capillary electrophoresis withelectroporation for intracellular derivatization andlaser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intra-cellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced intoliving cells by electroporation. For an 8 mm erythrocyte, the analytes were diluted bya factor of only 1.6. After completion of the derivatization reaction, a single cell wasinjected into the separation capillary tip and lysed there. The derivatized amino acidswere separated by capillary electrophoresis, followed by laser-induced fluorescencedetection. Nine amino acids were quantitatively determined, with amounts of aminoacids ranging from 3.8–32 amol/single cell.

Keywords: Amino acids / Capillary electrophoresis / Electroporation / Laser-induced fluores-cence / Single-cell analysis DOI 10.1002/elps.200305642

1 Introduction

Single-cell analysis is an interesting and significant areafor the analytical chemist. Capillary electrophoresis (CE)has many inherent features of its operation suitable forthe analysis of the chemical contents of single cells suchas extremely small sample size, high separation speed,efficiency, and biocompatible environments. Ewing [1]and Yeung [2] have summarized the work in the area ofsingle-cell analysis by CE with electrochemical (EC) [1] orlaser-induced fluorescence (LIF) detection [2]. The majorinvestigations focused on direct determination of nativelyelectroactive or fluorescent compounds in individualcells. The analysis of intracellular contents at single-celllevel has also been developed in our laboratory [3–7].However, a large number of species in single cells suchas amino acids are natively electroinactive or nonfluores-cent, and methods must be developed to detect theseanalytes. Covalent labeling with an electroactive tag or afluorophore is a selection for detection of these analytes.Derivatization of the electroinactive or nonfluorescentcontents is a key aspect of the single-cell analysis. Itshould be noted that the critical problem is minimizingdilution of the contents of a single cell during derivatiza-

tion in order to maintain favorable kinetics for the labelingreaction and to avoid diluting the analytes that are alreadypresent at trace levels.

Jorgenson and co-workers [8, 9] described a derivatiza-tion method in single-cell analysis by open-tubular liquidchromatography (OT-LC). One giant neuron with ca.125 mm diameter and 1.0 nL volume from a land snail isplaced in a 200 nL microvial for chemical lysis. Aftermicropipet manipulation of nanoliter volume solutions,20–30% of the lysed cell is derivatized. The final volumepresent after derivatization is ca. 25 nL. In this case, thecontents of a single cell in 1.0 nL volume are diluted by afactor of ca. 100. Then the contents are separated anddetected by OT-LC. If the method is used to derivatizethe contents in normally sized mammalian cells with a di-ameter of ca. 20 mm and a volume of ca. 4.2 pL, the dilu-tion factor is much more than 100.

In order to minimize dilution, Gilman and Ewing [10]reported an on-capillary derivatization scheme. In thismethod, the front end of the separation capillary is usedas a derivatization chamber, where the cell and then thelysing/derivatizing buffers are introduced by electromigra-tion and mixed. After completion of the derivatizationreaction in the front end of the capillary, the derivatizedanalytes are separated and detected. The walls of thecapillary limit dilution of the reagent and the cell contentsduring derivatization by restricting diffusion to the longitu-dinal axis of the capillary. According to their calculation,the contents of a 20 mm diameter cell with a 4.2 pL volumeare diluted by a factor of ca. 100 for 10 min reaction time

Correspondence: Professor Wenrui Jin, School of Chemistryand Chemical Engineering, Shandong University, Jinan 250100,ChinaE-mail: [email protected]: 186-531-8565167

Abbreviation: SSA, sulfosalicylic acid

480 Electrophoresis 2004, 25, 480–486

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2004, 25, 480–486 Analysis of amino acids by CE with electroporation and LIF 481

during the on-capillary derivatization. Obviously, the dilu-tion factor increases with prolonging the reaction time.With this method, they determined amino acids in a giantdopamine neuron of the snail P. corneus (ca. 75 mm insize) by CE with on-capillary naphthalene-2,3-dicarboxal-dehyde (NDA) derivatization and EC detection, in whichfour amino acids were quantitated in the range of 0.386–26.5 fmol [11]. The same derivatization methodology hasbeen used with CE and EC detection to quantitativelydetermine amino acids in single mouse peritoneal macro-phages (ca. 15–25 mm in size) in our laboratory [12].Six amino acids, ranging from 0.17 to 15.2 fmol in theseindividual cells, have been quantitated. The fluorescentproperties of NDA derivatives of amino acids have alsobeen exploited to determine amino acids in single cul-tured pheochromocytoma cells (PC12) with a diameterof 15–25 mm by CE with on-capillary derivatization for10 min and sensitive LIF detection [10], in which fiveamino acids have been quantitated. Average values ofthe five amino acids range from 0.18 to 5.1 fmol/cell.

Can one measure the amount of amino acids in singlecells for smaller cells or lower amounts of amino acidsand longer derivatization reaction time? Obviously, mini-mizing dilution of amino acids in single cells during deri-vatization is a critical step. Hogen and Yeung [13] de-scribed an intracellular derivatization approach for de-termination of thiols. Thiols in single erythrocytes arederivatized by incubating living cells with a derivatizingreagent, monobromobimane, to which the cell membraneis permeable. The cell itself acts as a reaction chamber.There is almost no dilution of the contents in the cell dur-ing the derivatization. Unfortunately, the cell membrane isnot permeable for NDA or FITC, a frequently used deriva-tizing reagent for LIF detection. To determine amino acidsin single cells with the intracellular derivatization, a tech-nique that can introduce these cell-impermeant reagentsinto cells should be selected. The electric-field-inducedpermeabilization (electroporation) technique can intro-duce chemicals into cells [14–16]. The application of anexternal pulsed electric field across cell membranesresults in increased permeability and conductance of thecell membrane. During the effective pore-open time, cell-impermeant substances in the extracellular medium canenter the cell interior by diffusion.

Recently, the modified electroporation method was usedto introduce polar cell-impermeant fluorescent reagentsinto single cells [17–19]. We attempted to analyze aminoacids in single human erythrocytes (the human smallestcells with an average diameter of 8 mm and an averagevolume of 87 fL) by CE with EC detection coupled withthis technique [20]. However, the aim of determiningamino acids in a single erythrocyte was not realized be-

cause the high detection limit of EC detection (. 100 amol).In the present work, we develop the method of intra-cellular derivatization to single-cell analysis. FITC is intro-duced into living erythrocytes by electroporation for deri-vatization. After completion of derivatization reaction in-side the cells, a single cell with FITC is electrokineticallyinjected into the capillary and then lysed with 0.1 mol/LNaOH. Once the individual erythrocyte is lysed, the deri-vatized amino acids are separated and detected by CEwith LIF detection. Using a commercial instrument, nineamino acids at the low attomole level in a single erythro-cyte can be determined.

2 Materials and methods

2.1 Preparation of erythrocytes and theirhemolysate

About 0.4 mL human blood with anticoagulant was cen-trifuged at 1916g for 5 min to separate erythrocytes.Then the supernatant liquid was removed. In order towash the erythrocytes, 6-fold v/v phosphate-buffered sa-line (PBS) was added. The mixture was centrifuged andthe supernatant liquid was removed. This step wasrepeated over five times until the supernatant was clearand transparent, thus obtaining the erythrocyte suspen-sion. The cell number in the erythrocyte solution obtainedwas counted using a hemocytometer (Shanghai MedicalOptical Instrument Plant, Shanghai, China). To obtain itshemolysate, the erythrocyte solution (,30 mL) was mixedwith 1.5 mL 1.2561022 mol/L borax/1.2561022 mol/LNaOH (pH 9.4). In this case, the erythrocytes were lysedand their hemolysate was obtained.

2.2 Derivatization of amino acids

A stock solution of amino acids (1.0061023 mol/L) wasdiluted to 1.0061024 mol/L with a 1.2561022 mol/Lborax/4.361022 mol/L NaOH buffer (pH 10). Then thesolution was mixed with the borax/NaOH buffer (pH 10)containing 2.061024 mol/L FITC with the same volume.After 15 h in the dark, the solution was diluted to2.0061027 mol/L with the borax/NaOH buffer and deter-mined. To determine amino acids in the hemolysate,400 mL of 5% w/v sulfosalicylic acid (SSA) was added to1.5 mL hemolysate to precipitate the protein. The mixturewas centrifuged at 4306g for 15 min. The supernatantliquid and 2.061024 mol/L FITC were mixed with thesame volume and then adjusted to pH 10 with NaOH.After 15 h in the dark, the solution was determined.


CE

and

CE

C

482 H. Zhang and W. Jin Electrophoresis 2004, 25, 480–486

2.3 Cell electroporation and intracellularderivatization

The electroporation equipment used here was the sameas in our previous work [5]. Two mL erythrocyte sus-pension was diluted to 0.5 mL with PBS containing2.061024 mol/L FITC. The colorless erythrocytes sus-pended in the yellow solution can clearly be seen underthe microscope with a magnification of 4006. Then theerythrocytes in the suspension were electroporatedaccording to [5]. Since the erythrocytes electroporatedwere brown, they could been monitored under the in-verted microscope. The erythrocyte suspension (,20 mL)was diluted ca. 25-fold by PBS. The suspension wasallowed to stand for more than 2 h. After the cells sub-sided to the bottom and the supernatant liquid wasremoved. This process was repeated at least four times.

2.4 CE-LIF detection

A P/ACE MDQ capillary electrophoresis system (Beck-man Instruments, Fullerton, CA, USA) equipped with anLIF detector (488 nm Laser Module, Beckman Coulter)was used to perform all separation and detection. Fluo-rescence was excited by an argon ion laser at 488 nm(3 mW). The fluorescence light was detected after passingthrough a 488 nm cutoff and a 520 nm interference filter.The fused-silica capillary with 25 mm ID and 375 mm ODwas supplied from Yongnian Optical Conductive FiberPlant (Yongnian, China). All separations were performedon an untreated capillary of 60.2 cm total length and aneffective length of 50 cm. The length of the coolant tubingwas 42.8 cm. The sample temperature of 257C and cool-ant of 257C were maintained throughout. Before each run,the capillaries were flushed with 0.1 mol/L NaOH, water,and the corresponding running buffer for 5 min, respec-tively, using 20 psi. After the electroosmotic flow reacheda constant value, the pressure injection of the derivatizedstandard solutions or the derivatized hemolysate was car-ried out. Then the capillary was carefully moved from thesolution or the derivatized hemolysate into the CE runningbuffer. The separation voltage of 25 kV was applied andthe electropherogram was recorded.

2.5 Analysis of single erythrocytes

Figure 1 shows the laboratory-made cell injection device.At the bottom of the buffer reservoir there were a Pt wireas the cathode and a metal tubing, into which an endof capillary from the capillary cartridge was inserted. Atthe bottom of the cell reservoir there were a Pt wire asthe anode and a metal tubing, into which the other end

Figure 1. Cell injection device.

(the injection end) of the capillary from the capillary car-tridge was inserted. In order to see the opening of theinjection end, a ,5 mm section of the polyimide-coatingat the injection end of the separation capillary wasremoved by burning. The erythrocyte suspension wastransferred into the cell reservoir. The injection end of theseparation capillary was placed under the field of viewof an inverted biological microscope. As soon as oneerythrocyte was drifting towards the injection end, aninjection voltage of 3.0 kV was applied to draw the wholecell into the capillary tip by electroosmotic flow as ob-served under the microscope. The erythrocyte was ad-sorbed on the wall of the capillary 25–50 mm away fromits tip. Then the two ends of the capillary were gentlymoved out from both metal tubings set in the buffer reser-voir and the cell reservoir, respectively. The capillary car-tridge was set into the CE system. A 0.1 mol/L NaOH so-lution was injected into the capillary around the erythro-cyte with 0.2 psi to lyse it. The individual erythrocyte waslysed within 15 s. Finally, the separation voltage wasapplied and the electropherogram was recorded.

2.6 Reagents and solutions

All amino acids (chromatographic grade) were obtainedfrom Shanghai Biochemical Reagents (Shanghai, China).A 1.0061023 mol/L stock solution of amino acids was



prepared by dissolving appropriate amounts of aminoacids in water. FITC isomer I (content 90%) was pur-chased from Acros Organics (Fairlawn, NJ, USA). A2.0061024 mol/L FITC solution was prepared in a brownflask with 1.2561022 mol/L borax/4.3061022 mol/L NaOH(pH 10) by aid of an ultrasonicator. Spermine was pur-chased from Sigma Chemical (St. Louis, MO, USA). A5% w/v SSA was prepared by dissolving an appropriateamount of SSA in water. PBS consisted of 0.135 mol/LNaCl and 0.02 mol/L NaH2PO4/NaOH (pH 7.4). All re-agents were of analytical grade except for amino acidsand FITC. All solutions were prepared with double-dis-tilled water and stored at 47C.

3 Results and discussion

3.1 Limit of detection, linear range, andreproducibility for determination of aminoacids

Figure 2A shows the electropherogram of FITC in1.2561022 mol/L borax/1.2561022 mol/L NaOH-1.2061024 mol/L spermine (pH 9.45) using a separation voltageof 25 kV. Four large peaks of FITC and seven small peaksof impurities in FITC appear on the electropherogram.This phenomenon has been observed by Dovichi’s group[21, 22]. Under the same conditions, the electrophero-gram of a solution containing 20 amino acids derivatizedby FITC is shown in Fig. 2B. From this electropherogram,it can be found that twelve amino acids (Arg, Lys, Trp, Try,Thr, Asn, Ser, Ala, Cys, Gly, Glu, Asp) can be well sepa-rated in the presence of other amino acids. Leu overlapsIle, Gln overlaps Pro, and His, Val, Met, and Phe overlapin one peak. Arg has the shortest migration time, tm, andAsp has the longest tm. In order to determine the linearrange and the limits of detection (LODs) of every one ofthe 12 amino acids separated, individual amino acidswere measured five times by CE with LIF detection.Their LODs calculated are listed in Table 1, when thesignal-to-noise ratio is 3. The concentration LODs ofthe method range from 2.0610210 mol/L for Trp to9.4610210 mol/L for Asp. The linear relationship existsbetween the peak height, h, and the concentration ofamino acids in the range of LOD of each amino acid toat least 2.061027 mol/L over two orders of magnitude,respectively (Table 1). Using least-squares treatment ofthe relationship between the peak height and the con-centration, the slopes, y-intercepts, and the correlationcoefficients yielded are also listed in Table 1. The relativestandard deviations of the method for a series of fiveinjections of these amino acids of 2.061027 mol/L arebetween 0.50–3.3% for tm and 1.7–5.3% for h, respec-tively.

Figure 2. Electropherograms of (A) 8.061026 mol/LFITC and (B) the standard solution containing 20 aminoacids of 2.0061027 mol/L after derivatized with FITC.1.2561022 mol/L borax/1.2561022 mol/L NaOH 1.2061024 mol/L spermine (pH 9.45); injection, 4 psi for 5 s;separation voltage, 25 kV. Peaks of impurities in FITC aremarked as I.

3.2 Determination of amino acids in hemolysate

A typical electropherogram of the hemolysate after thederivatization with FITC is shown in Fig. 3. The highestpeak of FITC (Fig. 2A) can be used as a marker to identifythe peaks in the electropherogram. These peaks havebeen identified on the basis of their relative migrationtimes and spiking corresponding standard solutions ofamino acids. By comparing the electropherogram withFig. 2B, eleven amino acids (Arg, Lys, Trp, Thr, Asn, Ser,Ala, Cys, Gly, Glu, and Asp) can be found in the hemoly-



Table 1. LODs, linear ranges, and their statistical treatment of 12 amino acids

Aminoacid

Linear range (mol/L) Slope (RFU/nmol?L21)a)

y-intercept(RFU)a)

Correlationcoefficient

LOD(10210 mol/L)

Arg 7.4610210–2.061027 0.0068 5.061026 0.9995 7.4Lys 7.2610210–2.061027 0.0070 24.061025 0.9987 7.2Trp 2.0610210–2.061027 0.0250 4.061025 0.9962 2.0Thr 4.9610210–2.061027 0.0100 22.361026 0.9963 4.9Asn 3.7610210–2.061027 0.0120 6.261024 0.9990 3.7Tyr 2.5610210–2.061027 0.0200 7.561025 0.9976 2.5Ser 4.9610210–2.061027 0.0100 2.061026 0.9992 4.9Ala 5.1610210–2.061027 0.0098 2.861025 0.9989 5.1Cys 6.0610210–2.061027 0.0084 21.061025 0.9994 6.0Gly 3.1610210–2.061027 0.0160 9.061026 0.9998 3.1Glu 5.2610210–2.061027 0.0060 6.261024 0.9995 5.2Asp 9.4610210–2.061027 0.0054 22.961025 0.9995 9.4

a) RFU, relative fluorescence unitConditions as in Fig. 2

sate. Pro and Gln could not be found. Identifying Ile, Leu,His, Val, Met, Phe, Tyr, Asn, and Tyr is difficult, becausetheir peaks cannot be separated under the present con-ditions. The mean concentrations of amino acids in thehemolysate obtained by the calibration curve for fivedeterminations are listed in Table 2. To prove the reliabilityof the method, a certain amount of standard amino acidswas added to the hemolysate and then the hemolysatewas measured. From the detected concentrations in thehemolysate with and without the standard amino acids,the recoveries are calculated and listed in Table 2 with theirstandard deviation (n = 5). The recoveries of the method forthe eleven amino acids are between 92 and 102%. From

the cell concentration of 6.066102 cells/mL in the hemoly-sate and the mean volume of a single erythrocyte (87 fL),the mean concentrations of amino acids in a single erythro-cyte are calculated and listed in Table 2 with their standarddeviation (n = 5). Leighton et al. [23] determined erythrocyteamino acid levels by gas chromatography in a group of 34normal human adults. The concentration range of aminoacids in single erythrocytes obtained by them from thevalues in the hemolysate samples are also listed in Table 2for comparison. It can be found that the mean concentra-tions of the eleven amino acids in a single erythrocytedetermined by the present method are in the range of thevalues reported by them in [23].

Table 2. Concentrations of amino acids in the hemolysate and its recoveries and of amino acids inone erythrocyte calculated from their concentration in the hemolysate

Aminoacids

Mean concentrationin the hemolysate 6 SD(n = 5) (1029 mol/L)

Recovery 6 SD(n = 5) (%)

Mean concentrationin one erythrocyte(1025 mol/L)

Concentrationin [23](1029 mol/L)

Arg 2.8460.010 99.662.9 5.40 1.4–6.0Lys 9.6360.025 92.862.6 18.3 12.1–23.9Trp 3.2660.024 96.063.3 6.19Thr 9.6360.027 10263.7 18.3 6.7–19.6Asn 7.6860.021 98.264.2 14.6 9.4–15.8Ser 9.0560.025 10063.8 17.2 12.3–21.9Ala 15.160.010 97.261.4 28.6 21.3–44.1Cys 10.260.028 92.463.8 19.4Gly 16.260.019 10062.4 30.7 25.8–58.0Glu 10.760.039 99.267.8 20.4Asp 18.760.035 99.466.0 35.5

Conditions as in Fig. 2



Figure 3. Electropherogram of a hemolysate after FITCderivatization. Conditions as in Fig. 2. Peaks of impuritiesin FITC are marked as I and unidentified peaks aremarked as U.

3.3 Analysis of amino acids in singleerythrocytes

Usually, cell lysis is accomplished by injecting a plug oflysis solution around the cell in the separation capillary.Erythrocytes can easily be lysed in CE running buffers,but lysis of the erythrocytes, into which FITC was intro-duced by electroporation, took over 10 min. It was notedthat erythrocytes with FITC could be lysed much easier in0.1 mol/L NaOH. Therefore, after an erythrocyte wasinjected and adsorbed on the wall of the capillary,0.1 mol/L NaOH as the cell lysis solution was injectedinto the capillary for 2 s. The individual erythrocyte waslysed within 15 s. After the cell was lysed, a separationvoltage of 25 kV was applied and the electropherogramof the cell was recorded. Two electropherograms of indi-vidual erythrocytes obtained using intracellular derivatiza-tion with FITC are shown in Fig. 4. For the two electro-pherograms, eight and seven different amino acids weredetected, respectively. Identification of these peaks ispossible through comparison with the electropherogramshown in Fig. 2B on the basis of their relative migrationtimes to the highest peak of FITC, indicating the ninepeaks detected to be Arg, Lys, Trp, Asn, Ser, Ala, Cys,Gly, and Glu. However, the contents of the nine aminoacids identified in single erythrocytes cannot be simulta-neously obtained for every erythrocyte, because the con-tents of some amino acids are lower than their LODs. Thecontent difference of amino acids between erythrocytescan be found only using single-cell analysis. Although itcan be concluded that the peak eluting at 15 min belongsto Leu and Ile, and His, Val, Met and Phe should beresponsible for the peak eluting at 15.5 min, their identifi-cation is difficult, because they have the same relativemigration time.

Figure 4. Electropherograms of amino acids in twosingle human erythrocytes with intracellular FITC deriva-tization. Conditions as in Fig. 2. Peaks of impurities inFITC are marked as I and unidentified peaks are markedas U.

The reproducible peak currents, together with the largelinear dynamic range for standard amino acids made itsuitable to use external standardization for the quantifi-cation of amino acids in an erythrocyte. In order tomeasure the amounts of amino acids in individual ery-throcytes accurately, they are quantified by comparisonof the peak height detected for an erythrocyte againstthose of standard amino acids injected after each cellrun. The amount of nine amino acids detected in five ery-throcytes is shown in Table 3. The determined values ofamino acids in the hemolysate are within the range of thevalues found in the present single-cell analysis. An ery-throcyte has a diameter of 8 mm and a volume of 87 fmol.Its height can be calculated to be 1.73 mm. After deriva-tization, the diameter of an erythrocyte changes to ca.10 mm. In this case, the volume of the erythrocyte is136 fL. The contents of an 8 mm diameter erythrocyteare diluted by a factor of approximately 1.6 during theintracellular derivatization. This method represents a sig-nificant reduction in dilution compared to on-capillaryderivatization with a factor of ca. 100 for the reactiontime of 10 min [10].



Table 3. Contents of amino acids determined in five single human erythrocytes (amol)

Cell Arg Lys Trp Asn Ser Ala Cys Gly Glu

1 ND 11.6 3.84 11.7 12.1 22.1 16.5 23.4 ND2 ND ND ND 9.96 11.6 25.5 18.2 26.3 18.93 ND 14.1 4.67 14.6 14.6 30.4 11.7 22.1 16.24 6.53 ND 4.39 12.3 15.6 22.0 ND 32.4 22.45 9.34 20.1 6.34 11.2 18.8 29.2 ND 31.2 NDMean valuea) 4.70 15.9 5.39 12.7 15.0 24.9 16.9 26.7 17.7

a) The values were calculated from the concentrations of the amino acids in the hemolysate; ND, notdetected

Conditions as in Fig. 2

4 Concluding remarks

Electroporation can reversibly introduce the derivatizationreagents, which cannot naturally permeate cell mem-branes, into the cells, where contents are derivatizedwith the reagents. The intracellular derivatization hardlydilutes the contents of cells. Therefore, by our methodspecies without fluorophore such as amino acids in smallsingle cells in size such as erythrocytes can be deter-mined by CE. In principle, the method of introducingreagents into cells by electroporation should be very use-ful. Other species, which have to react with reagents be-fore detection, can also be measured in cells using thistechnique. For example, one can introduce antibodies(or antigens) labeled by fluorophore into cells by electro-poration to determine antigens (or antibodies).

This project was supported by the National NaturalScience Foundation of China (No. 20235010).

Received April 3, 2003

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Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection