Rapid determination of superoxide free radical in hepatocellular carcinoma cells by MCE with LIF

Research Article

Rapid determination of superoxide freeradical in hepatocellular carcinoma cells byMCE with LIF

A method for determination of superoxide free radical (O��2 ) based on MCE with LIF was

developed. Fluorescent reagent 2-chloro-1, 3-dibenzothiazolinecyclohexene, which was

synthesized in our laboratory, was employed as the labeling reagent, the highest deri-

vatization efficiency was obtained in 20 mM HEPES buffer (pH 7.4) for 10 min at 371C.

Optimal determination of O��2 was achieved on a glass microchip, using 50 mM HEPES

buffer (pH 7.4). Under the optimized conditions, linearity of response was obtained in

the range of 4.0� 10�7–1.0� 10�5 M, the detection limit (S/N 5 3) was 0.15 mM, the

RSDs of migration time and peak area were 2.6 and 3.8%, respectively. Interference

experiment was investigated and the result indicates that 1000-fold molar excess of

hydrogen peroxide does not interfere with the determination of O��2 in complex system.

Finally, the method has been successfully applied to determine O��2 in hepatocellular

carcinoma cells as well as phorbol 12-myristate 13-acetate stimulated RAW264.7

macrophages. The average recoveries were 97.3 and 98.6%, respectively.

Keywords:

Hepatocellular carcinoma cells / Macrophages RAW264.7 / MCE with LIF /Superoxide free radical / 2-Chloro-1,3-dibenzolinecyclohexene

DOI 10.1002/elps.200800421

1 Introduction

Superoxide anion radical (O��2 ) is a short-lived and highly

reactive free radical in biological system. In a normal

cellular environment, it mediates signal transduction and

defenses against viral or bacterial attack [1]. In the case of

overproduction, O��2 can lead to oxidative damage of

proteins, DNA, and lipid peroxidation [2]. It has been

reported that O��2 inhibits enzymes including glutathione

peroxidase, catalase, and creatine kinase [3]. O��2 also serves

as a precursor to other reactive oxygen species (ROS). For

example, O��2 converts to hydrogen peroxide (H2O2)

spontaneously or under action of superoxide dismutase;

O��2 reacts with nitric oxide to form the powerful oxidant

peroxynitrite, which can cause many diseases related to

inflammatory processes and autoimmune diabetes [4, 5].

Therefore, rapid, sensitive detection and quantification of

intracellular O��2 is critically important in understanding its

physiological functions and pathogenesis of various diseases

associated with ROS.

Although several methods to detect O��2 such as electron

spin resonance [6], electrochemistry [7], fluorescence spec-

trometry [8], and HPLC [9] have been developed, there were

some drawbacks for the above-mentioned methods, includ-

ing the large sample volume, the long analyzing time,

inconvenience to operate, or cost. CE also has obtained

some achievements in analyzing total ROS [10] or single

ROS [11, 12]. CE combined with LIF has been introduced to

detect O��2 in rat skeletal muscle mitochondria, after deri-

vatization with fluorescence reagent hydroethidine (HE)

[12]. Unfortunately, HE has low selectivity to O��2 , mmol level

of H2O2 may interfere with the determination of O��2 , while

the content of H2O2 in biological system is far higher than

mmol level due to accumulation in the biological system, and

the long analysis time is unsuitable for rapid trapping of

O��2 . Meanwhile, the injection and separation procedures in

CE analysis were quite tedious and inconvenient using one-

dimensional structure.

Since 1990s, microfluidic chip or lab-on-a-chip provides

a new technology platform for the research in chemistry,

Xin LiuQingling LiXiaocong GongHongmin LiZhenzhen ChenLili TongBo Tang

College of Chemistry, ChemicalEngineering and MaterialsScience, Engineering ResearchCenter of Pesticide and MedicineIntermediate Clean Production,Key Laboratory of Molecular andNano Probes, Ministry ofEducation, Shandong NormalUniversity, Jinan, P. R. China

Received June 30, 2008Revised August 19, 2008Accepted September 4, 2008

Abbreviations: DBZTC, 2-chloro-1, 3-dibenzothiazoline-cyclohexene; HE, hydroethidine; HepG2 cells,

hepatocellular carcinoma cells; H2O2, hydrogen peroxide;

O��2 , superoxide radical; PMA, phorbol 12-myristate13-acetate; ROS, reactive oxygen species; Tiron, 4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt; XA,

xanthine; XO, xanthine oxidase

Correspondence: Professor Bo Tang, College of Chemistry,Chemical Engineering and Materials Science, EngineeringResearch Center of Pesticide and Medicine Intermediate CleanProduction, Key Laboratory of Molecular and Nano Probes,Ministry of Education, Shandong Normal University, Jinan,250014, P. R. ChinaE-mail: [email protected]: 186-531-86180017

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 1077–1083 1077

biology, microengineering, and other related microsystem

fields [13, 14]. MCE offers many attractive benefits such as

reducing sample requirements and reagent volumes, which

can reduce overall cost and shorter analyzing time. Different

functions could be integrated on a single microchip, which is

an important step toward maintaining a completely closed

system, thereby reducing contamination and eliminating

human intervention and error [15, 16]. Among the several

detection techniques employed in microchip analysis, LIF

detection method is most easily adapted to the dimensions of

microchips [17]. The coherence and low divergence of a laser

beam make it easy to focus on very small analyte volumes and

obtain much high irradiation, resulting in one of the most

sensitive and powerful means of any detection systems

[17, 18]. However, much attention has been focused on

detection of total ROS using MCE coupled with LIF [19–22].

As different reactive species always coexist in the reactive

environment and single ROS has its own unique physiolo-

gical activity [23, 24], detection of single ROS is far more

significant for further insight on its action mechanisms in

biological processes. Currently, only Zhu et al. [25] employed

HE as the labeling reagent and achieved O��2 determination

on microchip using LIF detector. Despite the contribution, it

is still urgent to develop an MCE-LIF method with better

selectivity for O��2 determination. In our group, a new

fluorescence reagent 2-chloro-1, 3-dibenzothiazolinecyclo-

hexene (DBZTC) has been synthesized, which is highly

sensitive and selective toward determination of O��2 with no

interference from a 500-fold molar excess of H2O2 [26].

In this work, using fluorescence reagent DBZTC, a

simple, rapid and sensitive O��2 determination was devel-

oped by MCE with LIF detection technique. Interference

experiment was investigated with MCE-LIF to test the

feasibility of the method. After optimizing the derivatization

conditions and electrophoresis parameters, the method was

applied to determine O��2 in hepatocellular carcinoma

(HepG2) cells and RAW264.7 macrophages. Finally, it

reached simple, rapid, and sensitive O��2 detection on

microchip.

2 Materials and methods

2.1 Chemicals and reagents

All the chemicals were of analytical reagent grade or HPLC

reagent grade. All aqueous solutions were prepared with

doubly distilled water (18.2 O cm); DBZTC was synthesized

in-house, and DBZTC oxide was synthesized and purified

according to the literature procedure [26]; HEPES, H2O2

(30% aqueous solution), Xanthine oxidase (XO), and

phorbol 12-myristate 13-acetate (PMA) were obtained from

Sigma (St. Louis, MO, USA); 4,5-Dihydroxy-1,3-benzenedi-

sulfonic acid disodium salt (Tiron) and Xanthine (XA) was

purchased from Shanghai Reagent.

The stock solution (1.00 mM) of DBZTC and DBZTC

oxide were prepared with dimethylsulfoxide and stored at

41C in darkness. These stock solutions were diluted to

5.0� 10�4 M before use. The XA solution (1.00 mM) was

prepared with 1.0� 10�2 M NaOH; The XO solution of

(1.00 U/mL) was prepared in 2.30 mM (NH4)2SO4,

1.0� 10�2 M sodium salicylate biology buffer, stored at

2–81C; PMA was prepared in DMSO at a concentration of

1.0 mg/mL and stored at �201C before use; The stock

solution (100 mL) of H2O2 (0.30 M) was freshly prepared by

diluting H2O2 (30%, 3.4 mL) with water; HEPES, phos-

phate, and borate buffer were prepared with doubly distilled

water, and pH of the solutions were adjusted by the addition

of appropriate amounts of hydrochloric acid or sodium

hydroxide to a desired pH; Before use, all solutions were

filtered through a 0.22 mm polypropylene filter film. All

experiments were performed at room temperature

(25721C).

2.2 Fluorescence spectra

Fluorimetric spectra were measured with an Edinburgh FLS

920 spectrofluorimeter (Edinburgh Instruments, UK), fitted

with a xenon lamp, in a quartz cuvette (1.0 cm optical path)

as the container. Spectrometer slits were set for 3.5 nm

band-pass. For recording the emission spectra, the excitation

wavelength was set at 473 nm, with spectral bandwidth

(10 nm), while the emission wavelength was scanned at a

specified scan rate from 495 to 680 nm. A solution of 10 mM

of DBZTC and 10 mM of DBZTC with 20 mM XA/20 mU

XO, was prepared, respectively, for the fluorescence

analysis.

2.3 Cell culture and sample preparation

HepG2 cells and RAW264.7 macrophages (purchased

from the American Type Culture Collection, Manassas,

USA), were cultured in DMEM containing 10% fetal

bovine serum, 1% penicillin, and 1% streptomycin at

371C in a 5% CO295% air incubator MCO-15AC (SANYO).

Cell viability was determined by the trypan-blue exclusion

assay.

When cells were in a logarithmic growth phase, a

proportion of HepG2 cells were incubated with O��2

scavenger Tiron (100 mM) for 1 h at 37711C, another

proportion of HepG2 cells were not incubated with Tiron.

Similarly, a proportion of the macrophage cells were incu-

bated with Tiron (100 mM) for 1 h prior to probe loading,

another portion of macrophage cells were stimulated with

PMA (2.0 ng/mL) at 37711C for 12 h. Then, each group of

cells were incubated with DBZTC (10 mM) for 10 min at

37711C. After that, all of the cells harvested with the

concentration of 1.0� 106 cells/mL by centrifugation in the

cold were washed twice with 0.9% NaCl solution. Finally,

these cells were resuspended again in a volume of HEPES

(20 mM, pH 7.4) equal to that in DMEM, and were then

disrupted for 10 min in a VC 130 PB ultrasonic disintegrator

Electrophoresis 2009, 30, 1077–10831078 X. Liu et al.


(Sonics & Materials). During sonic disruption, the

temperature was maintained below 41C with circulating ice

water. The broken cell suspensions were centrifuged at

12 000g for 5 min and the suspensions were immediately

analyzed or kept at �201C for up to 2 days.

2.4 Microchip and LIF detector

A schematic diagram of the microfluidic chip

channels design is shown in Fig. 1. The glass microchip

was provided by Dalian Institute of Chemical Physics,

Chinese Academy of Sciences (China). The double-T

channels were 65 mm wide and 15 mm deep, and the

detection occurred 12 mm downstream from the injection

cross in the separation channels. The reservoir positions are

depicted in Fig. 1.

A home-made intelligent eight-path-high-voltage elec-

tric device [27] and MCE-LIF detector were employed. Optics

collection system was confocal optics mode structure [28]. A

473 nm semiconductor double-pumped solid-state laser

(20 mW) was used as the excitation source. The confocal

detection module employed an objective to focus the beam

to the center of the microchannel. The emitted fluorescence

was filtered by a 520710 nm narrow band filter, and

detected by a PMT. The data were collected at 20 Hz using a

CT-22 data acquisition card.

2.5 MCE

Prior to electrophoresis, the channels of microchip were

rinsed with 1.0 M NaOH and doubly distilled water,

respectively, for 15 min and equilibrated with running

buffer for 15 min. Then, 10 mL of running buffer solutions

was filled into the reservoirs of B, BW, and SW respectively,

and 10 mL sample solution was filled into the sample

reservoir, S. After that, the chip was placed on the LIF-

detector worktable. By regulating the three dimensions

manipulator, the laser beam was focused at the detection

point. Sample injection was carried out using a pinch

injection mode [29]. Four electrodes, randomly chosen from

the intelligent eight-path-high-voltage electric device, were

inserted into the reservoirs to apply voltages for electro-

phoresis. Two sets of voltages were applied for sample

loading and electrophoresis separation according to Table 1.

3 Results and discussion

3.1 Fluorescent derivatization reagent and back-

ground experiments

In this experiment, DBZTC was chosen as O��2 labeling

reagent. Upon reaction with O��2 , nonfluorescent DBZTC

was oxidized to yield strongly fluorescent DBZTC oxide,

which owns better rigidity and a larger conjugated system.

The reaction of DBZTC with O��2 is shown in Scheme 1.

Derivatization conditions were optimized in the literature

[26], and the highest derivation efficiency was achieved in

20 mM HEPES (pH 7.4) for 10 min at 37711C with 10 mM

DBZTC. DBZTC reacts with O��2 in a 1:1 molar ratio

and the derivation efficiency is almost up to 100% according

to our previous experiments [26]; hence, the level of O��2

could be expressed by the level of DBZTC oxide in

biological system. DBZTC oxide was used as the

standard analyte for further electrophoresis parameters

optimization.

XA/XO reaction [30], which is a standard procedure for

generating O��2 , was introduced for background experi-

ments. Figure 2A is the emission spectra of DBZTC and

DBZTC with XA/XO. It showed that the maximum emis-

sion was 530 nm for DBZTC with XA/XO, no emission

fluorescence was observed for DBZTC, when excited at

473 nm. As can be seen in Fig. 2B(a), no fluorescence peak

was observed for DBZTC using the MCE with LIF detection.

Figure 2B(b) and (c) represent the electropherograms of the

mixtures of 10 mM DBZTC with 10 mM XA/10 mU XO, and

10 mM DBZTC with 20 mM XA/20 mU XO, respectively. As

expected, fluorescence intensity increased with the incre-

ment of O��2 concentrations. Together with the above

results, no background fluorescence was produced and the

feasibility of the proposed method for O��2 detection can be

ensured.

Figure 1. Schematic diagram of the microchip channels design.S, sample reservoir; B, buffer reservoir; SW, sample wastereservoir; BW, buffer waste reservoir.

Table 1. Typical output voltage program for injection and

separation

Time section Run time/s Applied voltage/V

V01 (S) V02 (SW) V03 (B) V04 (BW)

1st (injection) 20 400 0 200 240

2nd (separation) 60 1000 1000 1800 0

Electrophoresis 2009, 30, 1077–1083 Microfluidics and Miniaturization 1079


3.2 Optimization of the microchip CE conditions

For the optimized determination of DBZTC oxide, three

kinds of buffers (HEPES, phosphate, and borate buffer)

were tested. The DBZTC oxide was determined using one of

the three buffers at pH 7.2–8.6, when other electrophoresis

conditions were the same. Derivatization in HEPES buffer

had higher signal responses and smoother baseline. There-

fore, HEPES buffer was selected as the running buffer

solution. The buffer pH, concentration, and separation

electric field, which are the main factors affecting the

peak shape, have been optimized and the results were as

follows:Buffer pH mainly affects surface characteristics of the

chip channels, thereby affecting the EOF and mobilities of

derivatives. pH also has a large effect on fluorescence

intensity of analyte. According to the previous report [26],

when pH was in range of 7.0–8.6, DBZTC oxide exhibits

relatively strong fluorescence, and thus the effects of pH on

peak height were tested in the range of 7.0–8.6. As shown in

Fig. 3A, peak height reached the maximum at pH 7.4 and at

the same pH, an excellent peak shape and smoother base-

line were also observed. Therefore, pH 7.4 was adopted for

further testing.

The effect of the buffer concentration on the peak

height was studied at pH 7.4, in the range of 10–70 mM. As

shown in Fig. 3B, the peak height increased with buffer

concentration ranging from 10 to 50 mM. As buffer

concentration was above 50 mM, the peak height remained

unchangeable. The optimal concentration was selected as

50 mM, since it renders an excellent peak shape and low

current (12 mA).

The effects of separation electric field on the peak height

and the separation column efficiency over the range of

240–440 V/cm were investigated. In Fig. 3C and D, when

the electric field was 360 V/cm, the peak height and theo-

retical plates were both at the maximum. As a result, the

optimal electric field was selected as 360 V/cm.

Under the above optimized separation conditions, MCE

analysis of 10 mM DBZTC oxide was performed, the repre-

sentative microchip electropherogram for five repetitive

injections is shown in Fig. 4.

3.3 Reproducibilities, linearity, and detection limit

A standard solution of 10 mM DBZTC oxide was used to

investigate the reproducibilities. The electropherogram of

five repetitive injections of DBZTC oxide is shown in Fig. 4,

the RSDs of migration time and peak areas are 2.6 and

3.8%, respectively.

Figure 2. (A) Emission spectra of DBZTC and the mixture ofDBZTC with XA/XO in 20 mM HEPES (pH 7.4); lex 5 473 nm.(B) Electropherograms of (a) 10 mM DBZTC; (b) 10 mM DBZTC,10 mM XA, and 10 mU XO; (c) 10 mM DBZTC, 20 mM XA, and20 mU XO. XA/XO reaction is a standard procedure for generat-ing O��2 . Experimental conditions: injection time, 20 s; injectionelectric field, 400 V/cm; separation electric field, 360 V/cm;effective separation distance, 12 mm; running buffer, 40 mMHEPES (pH 7.4).

Scheme 1. The reaction ofDBZTC with O��2



The calibration curve for the standard sample was

determined covering the concentration range of

1.0� 10�7–2.0� 10�5 M. Nine concentration levels, namely

0.10, 0.40, 1.0, 2.0, 3.0, 4.0, 6.0, 10.0, 20.0 mM, were exam-

ined. For each level, three measurements were taken

and the average signal was used to make the calibration

curve. Figure 5 shows the typical electropherogram for

seven different concentration levels of DBZTC oxide.

Figure 3. Influence of (A) bufferpH, (B) buffer concentration, (C)separation electric field on peakheight, and (D) Effect of separationelectric field on theoretical plates.Each point represents an averageof experiments repeated threetimes. Experimental conditions:(A) 40 mM HEPES buffer; separa-tion electric field, 360 V/cm; injec-tion time, 20 s; injection electricfield, 400 V/cm; effective separa-tion distance, 12 mm. (B) HEPESbuffer, pH 7.4; other conditionswere the same as in (A) exceptfor buffer concentration. (C) Analiquot of 50 mM HEPES buffer,pH 7.4; other conditions were thesame as in (B) except for separa-tion electric field. (D) The condi-tions were the same as in (C).

Figure 4. Electropherogram for five repetitive injections of 10 mMDBZTC oxide. Experimental conditions: running buffer, 50 mMHEPES (pH 7.4); injection time, 20 s; injection electric field, 400V/cm; separation electric field, 360 V/cm; effective separationdistance, 12 mm.

Figure 5. Typical electropherogram for seven different concen-tration levels of DBZTC oxide in the range of 0.40–10 mM. Otherconditions were the same as in Fig. 4. The inset shows the peakheight signals against DBZTC oxide concentration in the0.40–10 mM range.



The standard curve was linear in the range of

4.0� 10�7–1.0� 10�5 M. The calibration equation and

regression coefficient were: y 5 0.7970x13.4440 and

R 5 0.9988 (n 5 7) in terms of peak height enhancement as

a function of DBZTC oxide concentration.

The concentration LOD was calculated on the basis of

an S/N of 3 and was 1.5� 10�7 M. Considering a repeatable

injection volume of 200 pL with double-T design, the

calculated mass LOD was 0.03 fmol (n 5 3).

3.4 Selectivity of the method

In biological samples, O��2 is present in low concentrations,

and other RS and biological compounds may interfere

with the determination of O��2 . To assess the selectivity of

the method toward determination of 10.0 mM O��2 , a

mixture of 10 mM DBZTC with 10.0 mM H2O2 was

analyzed under the optimized electrophoresis conditions,

and no H2O2 peak was observed. Other biological

compounds such as Vc and glutathione were also

investigated and did not make interferences with the

determination of O��2 . These observations revealed that the

present derivatization and separation method provided

relatively high selectivity toward O��2 , especially without

interference from a 1000-fold molar excess of H2O2,

and allowed its quantitative determination in biological

samples.

3.5 Determination of O��2 in HepG2 cells and

RAW264.7 macrophage extracts

The proposed method was applied to the analysis of HepG2

cells and RAW264.7 macrophages. Before electrophoresis

experiments, to exclude the native interference of these

samples, the fluorescence of cell samples without DBZTC

labeling were examined with Edinburgh FLS 920 spectro-

fluorimeter, and no native fluorescence was detected at lex/

lem 5 473/525 nm.

The electropherogram of DBZTC-labeled HepG2 cells

sample is shown in Fig. 6A-a. A peak was clearly detected

and was identified as O��2 peak by comparison of migration

time with standard solution (Fig. 4). To further confirm the

O��2 peak, Tiron, a cell-permeable O��2 scavenger [31],

was added to the HepG2 cells suspension. The corre-

sponding electropherogram is given in Fig. 6A-b, where O��2

peak completely disappeared. The electrophoresis experi-

ments of DBZTC-labeled HepG2 cells extract have been

repeated three times, and the RSD of peak areas was

about 4.4%. The O��2 concentration for HepG2 cells

extract was quantified by the standard curve and the

regression equation and was calculated as 0.7170.03 mM

(mean7SD).

The proposed method was then applied to the analysis

of RAW264.7 macrophages stimulated by 2.0 ng/L PMA.

PMA is a stimulator of cell respiratory burst to give rise to

ROS. Approximately, the O��2 peak was also observed by the

migration time of 30 s and disappeared after 100 mM Tiron

added to suspension (Fig. 6B). The experiments of DBZTC-

labeled RAW264.7 macrophages have also been repeated

three times, and the RSD of peak areas was about 4.6%. The

O��2 level of RAW264.7 macrophages was calculated as

0.8870.04 mM. The result is consistent with that detected

by fluorescence spectrum of our group previously

(0.9270.02 mM) [26].

To validate the method, recovery experiments were

determined by adding known amounts of DBZTC oxide

standard solution to the cell samples. These samples were

performed under the optimal conditions and the results

are shown in Table 2. The results indicate that the method

is reproducible and satisfactory for determining the level of

O��2 in cells.

Figure 6. (A) Electropherogram of HepG2 cells extracts without(a) and with (b) the Tiron treatment. (B) Electropherogram ofRAW264.7 macrophages without (a) and with (b) Tiron treat-ment. Cell concentration is 1.0� 106 cells/mL. Experimentalconditions were the same as in Fig. 4.



4 Concluding remarks

In this study, an MCE with LIF method was developed for

determination of O��2 using DBZTC as the fluorescent

reagent. With the optimized derivatization conditions and

electrophoresis parameters, rapid, sensitive detection and

quantification of O��2 was obtained within 30 s. A mass

detection limit of 0.03 fmol was achieved owing to the

minute sample volume. The method was applied for the

determination of O��2 in HepG2 cells and PMA-stimulated

RAW264.7 macrophages. Experimental results showed that

the developed method was simple, rapid, and sensitive, and

could be applied for determination of O��2 without

interference from a 1000-fold molar excess of H2O2 in

various biological systems. MCE offers favorable potentials

for facilitating investigation of the cellular homeostasis and

the pathogenesis of various diseases associated with O��2 at

the molecular level. Moreover, the method also provides a

new strategy for determination of other ROS in complex

biological matrix.

This work was supported by National Basic Research Programof China (973 Program, 2007CB936000), National NaturalScience Funds for Distinguished Young Scholar (No.20725518),Major Program of National Natural Science Foundation ofChina (No.90713019), National Natural Science Foundationof China (No.20875058), The Science and Technology Develop-ment Programs of Shandong Province of China (No. 2008GG30003012), and Natural Science Foundation of ShandongProvince in China (No.Y2008B15).

The authors have declared no conflict of interest.

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Table 2. Recoveries of O��2 in HepG2 cells and RAW264.7 macrophages samples (n 5 3)

Sample O��2 concentration (ıM) Added (mM) Found (mM) Mean (mM) Average recovery (%) RSD (%)

HepG2 0.7170.03 1.00 1.68, 1.62, 1.75 1.6870.07 97.3 4.8

Macrophages 0.8870.04 1.00 1.80, 1.96, 1.84 1.8670.08 98.6 4.5



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Rapid determination of superoxide free radical in hepatocellular carcinoma cells by MCE with LIF