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Journal of Chromatography A, 1135 (2006) 109–114

Intracellular labeling method for chip-based capillary electrophoresisfluorimetric single cell analysis using liposomes

Yue Sun a, Min Lu b, Xue-Feng Yin a,∗, Xing-Guo Gong b,∗∗a Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310027, China

b Institute of Biologic Macromolecule and Enzymatic Engineering, College of Life Science, Zhejiang University, Hangzhou 310027, China

Received 30 June 2006; received in revised form 4 September 2006; accepted 6 September 2006Available online 26 September 2006

bstract

An intracellular derivatization method mediated by liposome was developed for single cell analysis with chip-based capillary electrophoresis (CE)nd laser-induced fluorescence (LIF) detection. Liposomes with an average diameter of 100 nm were produced from phosphatidylcholine to encap-ulate fluorescent dyes by an ultrasonic method. The encapsulation yield and the vesicle density were determined to be 46 ± 5% and 8.8 × 1014/mL,espectively. The amount of fluorescent dye that entered the cells was dependent on the duration of incubating cells with liposomes, liposome

ensity, and concentration of the dye solution encapsulated in liposomes. The described method introduced cell membrane nonpermeable fluores-ent dyes into living cells without reducing cell viability. Single cell analysis using microfluidic chip-based CE revealed that liposome-membraneusion occurred after entrance of liposomes into the cells, with release of encapsulated fluorescence dyes and labeling of intracellular species.

2006 Elsevier B.V. All rights reserved.

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eywords: Intracellular derivatization; Chip-based CE; LIF; Single cell analysi

. Introduction

Single cell analysis is of significant interest to the biological,edical, and pharmaceutical communities [1]. CE/chip-basedE has many inherent features of its operation suitable for

he analysis of the chemical contents of single cells suchs extremely small sample size, high separation speed, effi-iency, and biocompatible environments [2,3]. In single cellspecies are typically present at femtomole to zeptomole levels10−15 to 10−21 mol) [2], making quantitative detection ofhese compounds extremely challenging. LIF has been anmportant detection technique for detecting contents of singleells and subcellular organelles, owing to its high detectionensitivity.

However, a large number of species in single cells are

atively nonfluorescent, derivatization of these species with auorophore is a key aspect of single cell analysis with LIF4]. In order to avoid diluting the Intracellular analytes that are

∗ Corresponding author. Tel.: +86 571 87991636; fax: +86 571 87952070.∗∗ Corresponding author. Tel.: +86 571 88206549; fax: +86 571 88206549.

E-mail addresses: yinxf@zju.edu.cn (X.-F. Yin),ongxg@zju.edu.cn (X.-G. Gong).

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021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2006.09.020

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lready present at trace levels, minimizing dilution of contentsuring derivatization is critical. Hogan and Yeung [5] describedn intracellular derivatization approach for determination of thi-ls by incubating living cells with a cell membrane–permeableerivatizing reagent, monobromobimane. The cell itself acteds a reaction chamber, so that almost no dilution of the cellu-ar contents occurred during the derivatization. However, onlyerivatizing reagents that penetrate the cell membrane can besed with this approach.

Because cell membranes are relatively impermeable to mostonic and polar compounds of biological and medical interest [6],ilman and Ewing [2] reported an on-capillary derivatization

cheme. In this method, the front end of the separation capillarys used as a derivatization chamber, where the cell and then theysing/derivatizing buffers are introduced by electromigrationnd mixed. After completion of the derivatization reaction in theront end of the capillary, the derivatized analytes are separatednd detected. But the contents of a 20-mm diameter cell areiluted by a factor of approximately 100 during the on-column

erivatization [7].

Recently, Wu et al. [8] fabricated an integrated microfluidicevice for analyzing the chemical contents of single cell. Fluo-escent derivatization was accomplished in a reaction chamber

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10 Y. Sun et al. / J. Chroma

ith a volume of 70 pL by using valves formed by muitilayer softithography to reduced unwanted dilution. Separation of deriva-ized amino acids in individual Jurket T cells was carried outn the integrated microfluidic device. Chen et al. [9] reportedmethod for intracellular fluoresceine isothiocyanate (FITC)-

erivatization mediated by polyethylene glycol (PEG).An alternative approach for introduction of charged or polar

olecules into intracellular domains of cells is electroporation4,7]. This technique is based on the theory of electropermeabi-ization. When cells are exposed to an electrical field of sufficienttrength, small pores will form in their membranes. The analyteo be encapsulated is added to the exterior solution of the cells,nd an electrical field is then applied. The amount of analytehat enters the cells is dependent on the analyte concentrationradient, membrane potential, duration of the applied field, andiffusion rate of the analyte.

It is important to ensure the viability of living cells whilechieving intracellular labeling of constituents for subsequentetection, because some fast enzymatic cellular reactions couldary analyte concentrations by an order of magnitude withinsecond [10]. Therefore, stringent optimization of conditions

or achieving reversible electroporation (i.e. self-healing of theores following electroporation) is required [11], which is a dif-cult procedure [6].

Liposomes are artificial phospholipids vesicles that areormed by the self-assembly of lipids into spherical or qua-ispherical structures, containing an aqueous central cavity12,13]. Because of its similarity to cell membrane structures,iposome has been utilized for drug delivery and targeting [14],or the transfer of genetic materials [15] and fluorescent dyes16] into cells, and in a variety of different analytical and bio-nalytical applications [17].

In the present work, an intracellular labeling approach involv-ng the penetration of liposomes with encapsulated dyes wasdapted and developed for single cell analysis with LIF detec-ion. Nanometer sized-liposomes were produced from phos-hatidylcholine to encapsulate FITC fluorescent dyes and otheraterials of interest using an ultrasonic method. Factors that

ffected the efficiency of intracellular delivery of encapsulatedaterial were studied and the labeling of intracellular species byITC mediated by liposomes was detected by chip-based CE.

. Experimental

.1. Reagents

All chemicals used were of analytical reagent grade, andemineralized water was used throughout. Physiological saltolution (PSS, NaCl, 0.9%, pH 7.4) was used for washing cells.hosphatidylcholines was obtained from the Chemical Factoryf East China Normal University (Shanghai, China). FITC andhodamine B were purchased from Aldrich (St. Louis, MO,SA). Stock solutions of FITC and Rhodamine B were pre-

ared, respectively at a concentration of 0.1 mg/mL in dimethylulfoxide (DMSO) and kept in the dark at 4 ◦C. Hydroxylpropyl-ethal cellulose (HPMC) was purchased from Sigma (St. Louis,O, USA) and a PSS-HPMC solution (0.4% HPMC in PSS) was

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1135 (2006) 109–114

sed as medium for the cell suspension. Ten millimolar sodiumodecyl sulfate (SDS) solution, which was used as the mediumor cell lysis as well as working electrolyte for CE separation,as prepared by dissolving the surfactant in 10 mmol borateuffer (pH 9.2).

.2. Instrumentation

JY98-3D Ultrasonic Homogenizers (Ningbo Xinzhi Sci-nctific Instruments, Ningbo, China) was used for producinganometer sized-liposome. LSM 510 Laser confocal scanningicroscope (Carl Zeiss, Oberkochen,Germany) was used to col-

ect the cellular fluorescence. Mastersizer 2000 particle sizenalyzer (Malvern instruments, Malvern, UK) was used for lipo-ome size measurements.

The home-built confocal microscope LIF system used foretection has been described previously [18]. Briefly, a 488 nmrgon ion laser (model 367, 4 mW, Nanjing Electronic Equip-ent, Nanjing, China) was coupled to an inverted microscope

Jiangnan Optics & Electronics, Nanjing, China), with the nec-ssary optical components. The laser beam was reflected by aichroic mirror and focused to a 20 �m spot on the microchan-el from below the chip. A 0.6 N.A., ×40 long working distanceicroscope objective was used. The fluorescence emission was

ollected by the same objective, passed through the dichroic mir-or and focused with a tube lens onto a pinhole located at theocal point. A photomultiplier tube (PMT, CR 114, Hamamatsu,eijing; bias: 600 V) was mounted on top of the microscope tubeith a 520 nm cutoff filter for spectral filtering and connected to

n amplifier (GD-1, Reike Electronic Equipment, Xi’an, China).ata acquisition and processing were carried out using a model2010 A/D converter (Zheda Instruments, Hangzhou, China)

nd a computer. A laboratory-built multi-terminal high voltageower supply, variable in the range of 0–1500 V, was used forampling and CE separation.

.3. Cell culture

Cells were kindly provided by the Institute of Biologicacromolecule and Enzyme Engineering (College of Life Sci-

nce, Zhejing University, China). Cells were cultured in RPMI-640 medium (GibcoBRL, Gaithersburg, MD, USA) supple-ented with 100 U/mL penicillin, 100 �g/mL streptomycin, and

0% fetal bovine serum in a humidified atmosphere of 95% air,% CO2 at 37 ◦C. All cells were grown from the frozen stocknd were not used for more than five passages to avoid changesn the cell morphology due to mutation. The cells were detachedy trypsin/EDTA solution, washed with PSS several times byentrifugation and then resuspended in PSS to obtain a cell pop-lation of 106/mL.

.4. Liposome preparation and characterization

Preparation of the liposome involved the addition of 100 mghospholipids to a 50 mL beaker containing 20 mL PSS, inhich the encapsulated dyes was dissolved. Once all of the

eagents were combined, the solution was sonicated for 10 min

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Y. Sun et al. / J. Chroma

eriods by placing the 50 mL beaker in an ice-water bath tovoid overheating. Between each 10-min period there was a0-min “rest” interval where the solution was stirred in ice-ater bath to cool. This sonication/stirring cycle was repeated

bout three times or until the solution was clear. The solutionas centrifuged at 10,000 rpm for 30 min at 4 ◦C to precipitate

ny dust particles and aggregated lipids. Non-encapsulated sub-tances was removed by dialysis at 4 ◦C against three changesf 4 L of PSS. Solutions containing liposomes were stored inhe refrigerator and used within 6 days. Liposome size distri-ution was measured by a Mastersizer 2000 particle size ana-yzer. The concentration of non-encapsulated substance (C1) inhe dialysate can be determined by spectrofluorimetry. Assum-ng the original dye concentration is C0, the volumes of theriginal liquid and the dialysate were V0 and V1 (mL), respec-ively. The encapsulation yield were calculated from the formulaC0V0 − C1V1)/C0V0. The numbers of the vesicle (n) were esti-ated from: C0 × 4/3πr3 ×n = (C0V0 − C1V1), where r is the

esicle average radius (cm).

.5. Determination of cell viability

Cells were prepared by seeding 5 × 105 cells/well in a6-well plate and incubated with different densities of FITC-ncapsulated liposomes by diluting the prepared liposomesdensity of 8.8 × 1014/mL) for 2 h in the dark at 37 ◦C. Thenhe cells were cultured again for 24 h in RPMI-1640 medium asescribed in Section 2.3. Fresh cells were used as control cells.fter incubation, the cell populations were determined usinghemocytometer (Shanghai Optical Instrument, Shanghai,

hina).

.6. Microchip fabrication

A multi-depth microfluidic chip was fabricated on a glassubstrate using conventional lithography and a 3-step etchingechnology as described previously [18]. The channel betweenample reservoir (S) and sample waste reservoir (SW) was usedor sampling and channel between buffer reservoir (B) and bufferaste reservoir (BW) was used for separation. The sampling

hannel was 37 �m deep, while the separation channel was2 �m deep. A 1-mm long weir was constructed in the sepa-ation channel, 300 �m down the channel crossing. The channelt the weir section was 6 �m deep. Access holes were drilled intohe etched plate with a 1.2 mm diameter diamond-tipped drill bitt the terminals of the channels. After permanent bonding by ahermal bonding procedure, four 4-mm inner diameter and 6-

m tall micropipet tips were epoxyed on the chip surroundinghe holes, serving as reservoirs.

.7. Intracellular delivery of fluorescent dyes mediated byanometer-liposomes

106 cells were separated by centrifuging 1 mL (10 6/mL) ofhe cell suspension at 1000 rpm for 5 min. After discarding theupernatant, the cells were suspended in 1 mL solution contain-ng liposomes encapsulated dyes and incubated with liposomes

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1135 (2006) 109–114 111

or 2 h in the dark at 37 ◦C. After incubation, the supernatantas discarded and the cells were washed with PSS 3–5 times by

entrifuging to remove the excess liposomes. After discardinghe supernatant, the cells incubated with liposomes were resus-ended in PSS to obtain a cell population of 1.2 × 105 cells/mL.

.8. Procedure for single cell analysis on microchip

One hundred microlitre, 100 and 50 �L working elec-rolyte solutions were added to the reservoirs B, BW andW, respectively. Then 150 �L of the HepG2 cell suspension1.2 × 105 cells/mL) were added to the reservoir S as describedreviously [3,18]. Owing to the differences in liquid levels inhe reservoirs created by the different volumes, the cell suspen-ion flowed from S to SW under hydrostatic pressure. When aingle cell moved within the crossed section of the channels, asbserved visually under the microscope, a set of electrical poten-ials was applied to the four reservoirs, with B at 1200 V, S andW both at +700 V, and BW grounded. The sampled cell was

ransported towards the BW by electroosmotic flow and stoppedy the weir W. After 5 s, the set of potentials was switched off.hen the chip was shifted from the channel-crossing viewingosition to the detection point and the laser beam was re-focused,he set of electrical potentials was resumed. At the same time, theata acquisition and processing system was activated to recordhe electropherograms.

. Results and discussion

.1. The preparation and characterization of liposomes

The method developed for formation of nanometer-liposomesn this work is based on the method described by Lucy’s group19]. The effect of ultrasonic power on the dispersion of lipo-ome size was studied and the results are shown in Fig. 1a.he average diameters of liposomes were 133, 122 and 115 nm

or 30 mL lipid suspension under ultrasonic powers of 200, 400nd 800 W, respectively. By decreasing the lipid suspension vol-me from 30 to 20 mL, the average diameter of liposomes waseduced from 122 to 97 nm under the same ultrasonic powerf 400 W. Therefore, a total volume of 20 mL lipid suspensionnd a ultrasonication power of 400 W were used for preparinghe liposomes for intracellular delivery of florescent dyes. Thencapsulation yield and the numbers of the vesicle were cacu-ated to be 46 ± 5% and 1.76 × 1016, respectively. The vesicleensity was 8.8 × 1014/mL.

Using the particle size analyzer, the stability of liposomes cane determined by measuring the change of the size dispersionf liposome with time. The results are shown in Fig. 1b, whichndicate liposomes began to aggregate after a week, and theggregation was more obvious after 14 days.

.2. Delivery of fluorescent dyes into living cells by

iposomes

Experiments showed that during Intracellular delivery of flu-rescent dyes mediated by nanometer-liposomes, fetal bovine

112 Y. Sun et al. / J. Chromatogr. A

Fig. 1. (a) Effect of ultrasonic power and volume of the lipid suspension onse

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ize distribution of FITC-encapsulated liposomes; (b) size distribution of FITC-ncapsulated liposomes after standing for different periods.

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ig. 2. Images of HepG2 cells before (a) and after incubation with FITC-encapsulatmages of a single HepG2 cell from computer aided three-dimension reconstruction iavelength: 488 nm; emission length wave: 520 nm; pinhole: 215 (d).

1135 (2006) 109–114

erum had a negative effect and should avoid being added into theulture-medium. Nanometer-liposomes can deliver their encap-ulated material into cells when cells were simply suspendedn the PSS solution containing liposomes encapsulated dyesnd incubated with liposomes in the dark at 37 ◦C. Fluorescenticroscopy Images of HepG2 cells before and after incubationith FITC-encapsulated liposomes for 2 h are shown in Fig. 2.reen fluorescence in cells can be seen after incubation withITC-encapsulated liposomes for 2 h (Fig. 2b), while no fluo-escence in cells can be seen after incubation with FITC solutionFig. 2c), because the cell membrane is impermeable to FITC.lice scan images of a single HepG2 cell (Fig. 2d) showed that

he fluorescence in Fig. 2b was emitted from every intracellularocation, implying that nanometer- liposomes could penetratehe cell membrane and release their encapsulated dye within theell.

To demonstrate that different dyes can be delivered into cellsy nanometer-liposomes, cells were suspended in 1 mL solutionontaining liposomes that encapsulated different dyes and sub-ected to incubation for 2 h in the dark at 37 ◦C. As shown in

ig. 3a and b, green and red fluorescence could be observedfter incubation with liposomes that separately encapsulatedITC and Rhodamine B, while cells in Fig. 3c appeared yellowfter incubation with both liposomes, indicating that different

ed liposomes for 2 h (b); incubation with 10−6 M FITC for 2 h (c). Slice scann LSM. Each section was 1.1 �m thick. LSCM parameter was set to excitation

Y. Sun et al. / J. Chromatogr. A 1135 (2006) 109–114 113

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ig. 3. Images showing cells after incubated with liposome encapsulated dyehodamine B-Liposome, Ex = 543 nm, Em = 560 nm; (c) With 10−6 M FITC-lnd 543 nm, Em = 520 and 560 nm.

ncapsulated dyes could enter simultaneously into cells by theuggested method. The differences in distribution of fluores-ence intensity in Fig. 3a and b might be a consequence ofifferent properties of the dyes in that FITC is hydrophilic whilehodamine B is relatively hydrophobic. Rhodamine B couldave a higher affinity for being embedded in the cell membrane20], resulting in higher intensities in the outer shell, and viceersa for FITC.

The biocompatibility of FITC delivered by liposomes afterncubation with cells was investigated by assessing the viabilityf the cells as described in Section 2.5. No significant differencean be seen between control cells and the cells after incubationith 1:100, 1:10, 1:1 and undiluted FITC-encapsulated lipo-

omes, indicating that the suggested method did not reduce theell viability and introducution of fluorescent dyes across theell membrane into living cells is feasible.

Fluorescence micrographs of different cells incubated withITC-encapsulated liposomes for 2 h are shown in Fig. 4. Resultsemonstrate that non-permeable fluorescent dye FITC can bentroduced into various cells by nanometer-lipsomes.

.3. Parameters affecting the transfecting rate

The intracellular fluorescent intensity increased with anncrease in incubation time and remained constant when the

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Fig. 4. Fluorescence micrographs of different cells incubated with F

with 10−6 M FITC-Liposome, Ex = 488 nm, Em = 520 nm; (b) with 10−6 Mme and 10−6 M Rhodamine B-Liposome, dual optical detection, Ex = 488 nm

ncubation time was more than 2 h. Therefore, incubation timef 2 h has been suggested in Section 2.7 for intracellular deliveryf fluorescent dyes mediated by nanometer-liposomes.

The effect of liposome density on the transfection efficiencyas been tested by incubating HepG2 cells in 1:100, 1:10, 1:1nd undiluted liposomes for 2 h in the dark at 37 ◦C. It has beenbserved that the intracellular fluorescent intensity increasedith an increase in liposome density.The effect of concentration of the encapsulated FITC solu-

ion on the intracellular fluorescent intensity has been tested.bservation indicates that the intracellular fluorescent intensity

ncreased with an increase in the concentration of the encapsu-ated FITC solution.

Calcium ion is a strong fusogenic agent, which promotes theusion of cells and vesicles [19]. Results showed that the addi-ion of 20 mM calcium chloride could increase the fluorescencentensity, which apparently resulted from more liposomes pen-trating the cell membranes.

.4. Evaluation of the effect of intracellular derivatizationy chip-based electrophoresis

The occurrence of intracellular liposome-membrane fusionollowing cell-membrane penetration, with subsequent releasef encapsulated fluorescence dyes and labeling of intracellular

ITC-encapsulated liposomes for 2 h. (a) SPC-A-1; (b) U937.

114 Y. Sun et al. / J. Chromatogr. A

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ig. 5. Electropherograms of a single cell with intracellular FITC derivatizationediated by FITC liposomes (10−4 M) for 2 h and a reaction time of 24 h.

pecies, was verified by separation and detection of the deliveredye and its derivatization products with intracellular species athe single-cell level using chip-based CE. Chip-based CE com-ined with high sensitivity detection methods such as LIF, haseen successfully applied in single-cell analysis owing to itsow sampling volume and high separation efficiency. After incu-ating HepG2 cells with FITC-encapsulated liposomes for 2 hnd adding the cell suspension to reservoir S of the microchips described in Section 2.8, the results of single-cell analysishowed that the peak height increased with extending the stand-ng time for the labeling reaction. Without FITC labeling, noeak was obtained in the electropherogram. Incubating cells withITC alone, no signal could be distinguished from the noise level

n the electropherograms, because FITC can not penetrate theell membrane, and most species in single cells such as aminocids and proteins are natively nonfluorescent under the 488 nmxcitation wavelength.

Our experiment results demonstrated that amino acids inntracellular environment can be labeled by FITC after a longtanding time of 24 h, which is consistent with the observationeported before [4,9]. The electropherogram of individual cellsbtained using the suggested method with a standing time of 24 hs shown in Fig. 5. Identification of all these peaks is difficult, asITC is a non-selective labeling agent. However, it is known that

ntracellular amino acids and some proteins can be derivatizedy FITC mediated with liposomes. By the means of comparingith the relative migration times of amino acid standard solution,

wo peaks have been identified as Gly and Asp according to theethod described elsewhere [4]. The other peaks remain uniden-

ified, owing to the lack of standard materials for most of thentracellular proteins at present. The concentration of FITC wasot optimized in Fig. 5. For quantitative determination of speciesn single cells, the amount of introduced labeling agent should

e optimized by varying incubation time, liposome density andoncentration of the labeling agent encapsulated in liposome asescribed in Section 3.3. Because the aim of this paper is toevelop an intracellular labeling method mediated by liposome,

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1135 (2006) 109–114

o effort was made to increasing the detection sensitivity andelectivity. Therefore, the intracellular amino acids in low con-entration could not be identified with coexisted intracellularroteins. By using selective labeling agent, intracellular speciesould be easily identified and determined.

. Conclusions

The method described here proved to be an effective tech-ique for introducing polar compounds into living cells acrosshe cell membrane and for achieving derivatization of intra-ellular species without dilution from extracellular media. Themount of introduced polar compounds can be controlled byhanging incubation time, liposome density and concentrationf the polar compounds encapsulated in liposome. It can be usedn single cell analysis with laser scanning confocal microscopy,E/chip-based CE and flow cytometry. By using selective label-

ng agent, intracellular species could be easily identified andetermined. Other potential applications include the use of sin-le cells as nanoreactors and the use of single cells as a platformor drug testing.

cknowledgement

This work was supported by the National Natural Scienceoundation of China under project No. 20475049.

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