Journal of Chromatography A, 1109 (2006) 160–166

Continuous wave-based multiphoton excitation fluorescencefor capillary electrophoresis

Sheng Chen, Bi-Feng Liu ∗, Ling Fu, Tao Xiong, Tiancai Liu, Zhihong Zhang,Zhen-Li Huang, Qiang Lu, Yuan-Di Zhao, Qingming Luo

The Key Laboratory of Biomedical Photonics of Ministry of Education, and Hubei Bioinformatics & Molecular Imaging Key Laboratory,Department of Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

Available online 2 December 2005

Abstract

It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diodelaser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superiorto on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label inbio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated assld(ed©

K

1

[usacpHatoei

(

0d

amples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamicinear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs withivergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescenceLIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellarlectrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of thisetection approach in various micro or nano-column liquid phase separation technologies.

2005 Elsevier B.V. All rights reserved.

eywords: Continuous wave-based multiphoton excitation; Laser-induced fluorescence; Capillary electrophoresis; Diode laser

. Introduction

Over the past two decades, capillary electrophoresis (CE)1], and its miniaturized format microchip CE [2] have beenndergoing a rapid progress. Due to its high performance, lowample requirement and automation, etc. CE has been accepteds a powerful method for applications of a wide range. One ofonvincing examples is DNA sequencing using high through-ut array CE that lays solid foundation for the completion ofuman Genome Project [3]. CE is presently being recognized

s the standard methodology to obtain genome sequences. Ando date, over 100 genomes of virus, prokaryotic and eukary-tic species have been uncovered that lead to current globalndeavors beyond the genomics, where CE continuous to play anmportant role. Recent advancements have showed great poten-

∗ Corresponding author. Tel.: +86 27 87792170; fax: +86 27 87792203.E-mail addresses: bfliu@mail.hust.edu.cn, bifeng liu@yahoo.com

B.-F. Liu).

tials of CE in fast-growing proteomics [4–6] and metabolomics[7–9].

High selectivity is undoubtedly one of decisive reasons forwidespread use of CE. It involves many different separationmodes, such as capillary zone electrophoresis (CZE) [10], micel-lar electrokinetic chromatography (MEKC) [11], capillary gelelectrophoresis [12] and so forth, which make CE a versa-tile approach to profile complex chemical, environmental andbiological systems. Another essential one that will be neveroverstressed is detection development. Various detectors cur-rently are available for CE to meet different requirements onmeasurement, such as UV–vis [10], laser-induced fluorescence(LIF) [13], electrochemical [14,15] and bio/chemiluminescent[16–18] detection, and mass spectroscope [19,20] as well. Theintroduction of LIF is absolutely a milestone to CE. It overcomesa bottleneck on detection, that is, low detectability originatedfrom intrinsic short optical path length of capillary column.At present, LIF has become the most popular detector in CE,with respect to its extremely high sensitivity of down to sin-gle molecules [21]. And with involvement of many other optic

021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2005.11.033

S. Chen et al. / J. Chromatogr. A 1109 (2006) 160–166 161

techniques, for examples, polarization [22,23], total internalreflection [24], correlation spectroscopy [25,26] and multipho-ton excitation (MPE) [27–29], etc. LIF technique for CE hasbeen significantly enhanced.

MPE becomes of great interest in molecular spectroscopysince Goppert-Mayer firstly predicted its possibility in 1931[30]. Two-photon and three-photon excitation fluorescence wereexperimentally proven in 1960 [31] and 1964 [32], respectively.A breakthrough that leaded to wide biological applications ofMPE could be dated back to 1990 when MPE laser scanningmicroscopy was invented [33]. Owing to its high spatial resolu-tion (at sub-fL level), high sensitivity (at single molecules level),deep penetration and less photobleaching, etc. MPE has becomea promising tool for biomedical analyses [34,35]. Recently, MPEhas been introduced into CE as a mode of LIF detection, thanksto a series of excellent contributions by Shear and his colleagues[27–29,36,37]. And more recently, it has been also developed formicrochip based CE [38]. There are two major benefits of usingMPE for CE: (i) ultra-low detection background and extreme-low detection volume. As a result, extremely low mass detectionlimit about zeptomole level can be readily achieved; (ii) capa-bility of simultaneous multi-color excitation in a broad range.With MPE, it is now possible to use only one excitation lineof a laser to detect complex components labeled with divergentfluorescent tags. Nevertheless, high cost of femto-second pulselaser limited its application in CE. With advancement of lasertpiwcl

docmtiSeatags

2

2

cditU

as described in a reference [41]. Recombinant red fluorescentprotein (DsRed) was constructed in laboratory [42] and over-expressed in E. coli. All other reagents were of analytical grade.Water purified by the Milli-Q system (Millipore, USA) was usedfor the preparation of all solutions.

2.2. Experimental procedures

Stock solutions of amino acids were prepared at a concen-tration of 1 × 10−2 M in 10 mM borate buffer (pH 9.0). FITCwas dissolved in acetone at a concentration of 1 × 10−2 M. Forderivatization, amino acids (five-fold excessive in mole) andFTIC solutions were mixed. The mixture was then placed in adark area over night at room temperature.

For new capillary (360 �m O.D. × 50 �m I.D. × 55 cm inlength, Polymicro, USA), column was flushed with 1 M NaOH,1 M HNO3, 0.1 M NaOH, 0.1 M HNO3, and pure water sequen-tially for 30 min, respectively. Before analysis, the capillary wasrinsed and equilibrated with electrophoretic buffer for 20 min.Between runs, the capillary was washed with 0.1 M NaOH, purewater and running buffer in order for 10 min, respectively, toensure the reproducibility.

2.3. E. coli. cell culture

E. coli. DH5� competent cells were transformed withpaafusa

2

pnith4p1ithnt

2

spa

echnology, high or ultra-high energy diode laser (DL) of cheaprice is currently available, and has been extensively employedn analytical chemistry [39,40]. Thus, to investigate continuousave (CW) based MPE fluorescence for CE becomes signifi-

ant interesting. However, no relevant report has been found initeratures.

In this paper, CW based MPE fluorescence detection withiode laser was firstly demonstrated for CE. Different strategiesn MPE configuration, on-column and end-column types wereompared. Three kinds of tags that were most frequently used forolecular labeling, such as small molecular dye, fluorescent pro-

ein and nano particle or also known as quantum dot (QD), werenvestigated in CE with CW based MPE fluorescence detection.imultaneous detection of fluorescent dyestuffs with divergentxcitation and emission wavelengths in a broad range showeddvantage of this scheme over conventional LIF detection. Iden-ification of fluorescein isothiocyanate (FITC) labeled aminocids was further achieved both in CZE and MEKC that revealedood prospect of this detection scheme in various micro-columneparation technologies.

. Experimental

.1. Chemicals

Amino acids (L), calcein, coumarin and fluorescein isothio-yanate were obtained from Sigma–Aldrich (MO, USA). Rho-amine B and sulforhodamine were purchased from TCI Chem-cals (Tokyo, Japan). And 8-aminopyrene-1,3,6-trisulfonic acidrisodium salt (APTS) was bought from Molecular Probes (OR,S). Quantum dots of ZnS/CdSe were synthesized in laboratory

DsRed2 plasmids (Clontech, US) that expressed DsRed andmpicillin resistance genes on the same bicistronic message,nd were grown overnight at 37 ◦C on LB/agar using ampicillinor selection. E. coli cells of high-expression DsRed were pickedp under fluorescence microscope (IX 71, Olympus, Japan). Theelected colony was further grown in LB/ampicilline medium inn incubator at 37 ◦C overnight.

.4. Synthesis of QD

To synthesize CdSe/ZnS [41], cadmium salt in trioctylphos-hine oxide (TOPO) as precursor was heated to 330 ◦C. Sele-ium powder dissolved in trioctylphosphine (TOP) was thennjected. The temperature for nanocrystal growth was main-ained at 270 ◦C for few minutes with subsequent removal of theeat immediately. When the temperature was cooled down to0 ◦C, methanol was added to precipitate the nanocrystal. Afterurifying, the CdSe dots were dispersed in TOPO and TOP at50–230 ◦C. Diethylzine and hexamethyldisilathiane dissolvedn TOP were then injected. The reaction mixture was kept at thisemperature for few minutes and cooled to 100 ◦C for severalours. Finally, methanol was employed again to precipitate theanocrystal. Different sizes of QDs could be obtained accordingo the growth time.

.5. Instruments

Analyses were carried out on a home-built system, con-isting of an inverted fluorescence microscope (CK40, Olym-us, Japan), a laser source, a detection unit and a high volt-ge supply (0–30 kV) for electrophoresis (Shanghai Nuclear

162 S. Chen et al. / J. Chromatogr. A 1109 (2006) 160–166

Research Institute, China). Briefly, a laser beam from a diodelaser (808 nm/1 W; Institute of Semiconductor, Beijing, China)or Ti:Sapphire laser (Spectra Physics, USA) was reflected bya dichroic mirror (E625SP, Chroma, USA), and focused intocapillary column with an oil objective (100×, NA1.25, Olym-pus, Japan). The excited fluorescence was then collected usingthe same objective to a photo multiplier tube (PMT R5070,Hamamatsu, Japan), thru E625SP and a low-pass filter (BG18,Newport, USA). The PMT current was converted and amplifiedinto a voltage, and acquired with an A/D convertor (home-made). Program for data acquisition was locally written in Bor-land C++. The collimator of diode laser was manufactured inlaboratory.

For on-column detection, a detection window was simplymade by burning a specific length of polyimide coating ofcapillary. However, to achieve end-column detection, a specialcathodic reservoir was designed due to the very short work-ing distance of oil objective with ultra high numerical aperture,in which a thin glass chip of 150 �m thickness was employedas the bottom of the reservoir so that the laser could be eas-ily focused into the outlet end of separation capillary. Initialcomparison study of on- and end-column detection schemeswas performed on a commercial MPE microscope (MRC-1024, Bio-Rad, USA), where a 20× (NA 0.45) objective wasemployed.

3

cto(if

φ

wft

awitipt

τ

wsn

standard rectangular shape. For CW laser:

ξ

τf= 1

As a result, a CW laser with about 165-fold higher power canachieve comparative excitation probability. Consequently, usingCW with diode laser of cheap price to accomplish MPE fluores-cence is possible, and will be definitely a rational choice, insteadof femto-second pulse laser of high cost. Thus, it is currently aninteresting topic in biomedical optics. Actually, pioneer workof using CW output laser to achieve MPE for analysis could bedated to three decades ago [44]. Due to potential photobleachingand the damage from high power of employed laser, it wouldbe a challenge, while applying CW-based MPE scheme to invivo investigate biological tissues or cells [45], However, it wasobviously not the same case for capillary electrophoresis, wheremolecules or particles were monitored in vitro at the detectionwindow. For such consideration, here we systematically investi-gated the possibility of CW-based MPE fluorescence detectionfor CE separation.

For laser-induced fluorescence detection in CE, on-capillaryscheme was usually selected due to its simplicity and conve-nience. Post-capillary type with sheath flow cell has proven toachieve much better sensitivity, owing to less light scatteringfrom the capillary. In this study we evaluated on- and end-columnconfigurations for MPE, initially with femto-second pulse laserbweIrwlduacc

BMCiCfdMltacfwnbaD

. Results and discussion

To achieve multi-photon excitation (MPE) fluorescence, arucial issue that should be fundamentally considered was exci-ation probability (φ) that evaluated MPE fluorescence potentialf a molecule. For a two-photon process, the probability of MPEφ2) was determined by several parameters of incident lightncluding average power (P2

av), pulse width (τ) and repeatingrequency (f), etc. as described in Eq. (1) [43]:

2 = ξσ2

(hω)2F2

P2ave

(τf )(1)

here (�ω), F, σ and ξ represent energy of a photon, area ofocus, photon absorption section of molecule and waveform fac-or, respectively.

With femto-second pulse laser, high value of excitation prob-bility could be readily accomplished due to very short pulseidth. However, high cost of such kind of laser bottlenecked

ts widespread use. In theory as show in Eq. (1), equal excita-ion probability could be obtained from continuous wave lasern case that P2

avξ/τf was set to the same value as femto-secondulse laser. For example, the parameters of pulse laser were seto values

= 250fs, f = 82 MHz and ξ = 0.56

hich are the standard parameter of Ti:Sapphire laser in femto-econd mode-lock status. The value of waveform factor ξ isot set to 1, because the waveform of laser pulse is usually not

ased on a commercial MPE microscope. The detector schemesere as described in experimental section. About 10-fold differ-

nce in sensitivity was found (electropherograms not shown).t was reasonable. The incident light was strongly reflected andefracted by capillary wall that lead to a big energy loss. Mean-hile, the capillary wall could be considered as a cylindrical

ens of short focal length that brought optical aberration, and thenecreased excitation intensity. Nevertheless, end-column config-ration significantly reduced such kinds of reflection, refractionnd distortion, and thus, was more suitable for MPE fluores-ence detection. Consequently, end-column configuration washosen for our further investigation.

Fig. 1A showed an electropherogram of two dyes, rhodamineand sulforhodamine, using femto-second pulse laser-basedPE. Switching laser output from pulse mode-lock status toW status, similar electropherogram was obtained as depicted

n Fig. 1B. Although detection sensitivity is relatively lower withW-based MPE, it revealed the feasibility of CW-based MPE

or CE as theoretically predicted. To make a sure that the signalsetected in above electropherograms were really achieved byPE, relationship was evaluated between logarithm of incident

aser power (P) and collected fluorescence intensity (I). As illus-rated in Fig. 1A and B, a two-photon excitation process waspproved. Results in Fig. 1A and B were accomplished withommercial Ti:Sapphire laser. Although successfully appliedor CE, the high cost of such laser undoubtedly would limit itsidespread application. With rapid development of laser tech-ology, diode laser of cheap price with ultra-high power haseen commercially available. Thus, it would be promising tochieve CW-based MPE fluorescence detection for CE usingL. Fig. 2A gave an experimental example that convincingly

S. Chen et al. / J. Chromatogr. A 1109 (2006) 160–166 163

Fig. 1. Electropherograms of two small-molecule dyes using MPE fluorescence detection. (A) By Ti:Sapphire laser in femto-second pulse mode. Peak identity: (1)50.0 nM rhodamine B; (2) 50.0 nM sulforhodamine. (B) By Ti:Sapphire laser CW mode. Peak identity: (1) 0.3 �M rhodamine B. (2) 0.3 �M sulforhodamine. (A′)and (B′) revealed the relationship of fluorescence signal of rhodamine B and time-averaged power of laser. The slopes of linear fits indicated absorption process oftwo-photon. Separations were performed in a 10 mM borate buffer system, with electric field strength of 327 V/cm. Samples were injected by hydrodynamic way(10 s at 8 cm).

Table 1Quantitative data for CW-based MPE in CE

Species Calibrationa Y = A + BX γ Detection limit (S/N = 3)

Concentration (�M) Mass/y mol

Rhodamine B Y = 0.6532 + 0.3248X 0.9951 1.2 54Sulforhodamine Y = −0.2167 + 0.1618X 0.9991 2.0 90

a Y: peak area; X: sample concentration (�M).

proved the possibility of CW-based MPE for CE with DL oflow cost. Relationships between logarithm of laser power (P)and fluorescence intensity (I) shown in Fig. 2B confirmed thatabove signals were surely from two-photon excitation. Calibra-

Fig. 2. (A) Electropherogram of two small-molecule dyes using CW-based MPEfluorescence detection by LD laser. (B) Relationship of fluorescence signal ofrhodamine B and time-averaged power of laser. The slope of linear fit indicatedabsorption process of two-photon. Peak identity: (1) 2.0 �M rhodamine B; (2)2.0 �M sulforhodamine. Other conditions were the same as in Fig. 1.

tion curves were established, which exhibited a dynamic linearrange of over 2 orders of magnitude between peak height andconcentration of dyes ranged from 8 × 10−6 M to 1 × 10−3 M.Detection limits were found to be 1.2 × 10−6 M and 2 × 10−6 Mfor rhodamine B and sulforhodamine, respectively (S/N = 3),which corresponded to be at zeptomole level in mass. The detec-tion volume was calculated to be 45.0 attoliter following themethod as recommended previously [46]. Quantitative data weredetailed in Table 1. To reveal the capability of simultaneousmulti-color excitation in a broad range, further discussion on aseparation and detection of six dyestuffs including rhodamine6G (490/532 nm), rhodamine B (540/625 nm), sulforhodamine(565/585 nm), calcein (495/520 nm), coumarin (445/550 nm)and APTS (424/595 nm) whose excitation and emission spectra(data in parentheses are the maximum excitation/emission wave-length of each species) were totally divergent was performed asdepicted in Fig. 3. In theory, a laser can potentially excite two-photon fluorescence of molecules whose excitation line is largerthan the half value of the laser line. Because the excitation wave-length of MPE is much longer than emission wavelength, we canuse a low-pass filter to simultaneously collect all the fluores-cence of different emissions of different molecules. It is surelybeneficial for investigating complex systems where fluorescentcharacteristics of molecules were different.

Molecular tag or marker plays a key role in LIF, because sam-ples seldom have native or auto fluorescence. There are mainly

164 S. Chen et al. / J. Chromatogr. A 1109 (2006) 160–166

Fig. 3. Electropherogram of small-molecule dyestuffs with divergent excitationand emission wavelength using CW-based MPE fluorescence detection by LDlaser. Peak identity: (1) rhodamine 6G; (2) rhodamine B; (3) sulforhodamine;(4) calcein; (5) coumarin; (6) APTS. Other conditions were the same as in Fig. 1.Concentration for each species was 10.0 �M.

three kinds of fluorescent tags that are currently quite popu-lar in biomedical analysis. Besides small molecular dyestuff asinvestigated above, fluorescent protein and nano particle or alsoreferred to as quantum dot are gaining new precedence. As such,fluorescence protein and QD were subsequently tested to expandCW-based MPE fluorescence detection in CE for more extensiveapplications.

Fluorescence protein [47] that can be easily fused to a targetprotein using transgenic operation, is a category of endogenousproteins extracted from biological species, e.g., green fluores-cence protein (GFP) from the jellyfish Aequorea victoria, basedon which many variants have been developed with divergentabsorbance and emission spectra. In this work, a recently cloned28-kDa fluorescence protein (named DsRed) responsible for thered coloration around the oral disk of a coral of the Discosomagenus, was selected as a representative of this kind. Fig. 4Ashowed an electropherogram of DsRed. For preventing the sam-ple from denaturation in cell lysis, E. coli cells of over-expressedDsRed were directly injected into capillary for electrophoresis.No extraction of DsRed from cultured cells was carried out.Meanwhile, another injection of E. coli cells without DsRedexpression construct was also performed as a control as shownin Fig. 4B. Certainly, no peak was found.

With development of “nano” science, QD rushes into thefield of biomedical analysis [48]. As a high efficient moleculartag, QD attracts a great of attention due to its extreme brilliance(inslwwd

Fig. 4. Electropherogram of red fluorescence protein in E. coli cell using CW-based MPE fluorescence detection by LD laser. Other conditions were the sameas in Fig. 1. (A) Injection of with DsRed over-expressed E. coli cell. (B) Injectionof E. coli cell without DsRed as a control.

because these untreated QD were highly hydrophobic. Fig. 5gave the result.

Based upon above investigations, it confidently proved thatCW-based MPE fluorescence detection with DL of low cost hasbeen successfully achieved for CE. To further validate our idea,well established CW-based MPE fluorescence detection methodwas applied for capillary zone electrophoresis and micellar elec-trokinetic chromatography (MEKC). Fig. 6 showed the elec-tropherograms of CZE and MEKC separation of FITC-labeledamino acids that exhibited great potentials of this novel detec-tion method for micro or nano-column liquid phase separation

Fbasa

usually 1000-fold brighter than small-molecular dye) and glow-ng in a rainbow of colors depending on its size. In this work,anocrystal ZnS/CdSe was synthesized in laboratory withouturface modification that was believed to be necessary whileabeling molecules. Non-aqueous CE in which 50% ethanolas incorporated into 10 mM borate running buffer (pH 9.0),as used and optimized for separating two kinds of quantumots with diameters of about 3.0 nm and 5.0 nm, respectively,

ig. 5. Electropherogram of QD using CW-based MPE fluorescence detectiony LD laser. Peak identity: (1) QD with a diameter of ca. 3.0 nm; (2) QD withdiameter of ca. 5.0 nm. Separation was performed in a 10 mM borate buffer

ystem incorporating with 50% (v/v) ethanol. Other conditions were the sames in Fig. 1.

S. Chen et al. / J. Chromatogr. A 1109 (2006) 160–166 165

Fig. 6. Electropherogram of FITC-labeled amino acids using CW-based MPEfluorescence detection by LD laser. (A) By MEKC mode with 20 mM SDS inrunning buffer. (B) By CZE mode Peak identity: (1) FITC; (2) ornithine; (3)tryptophan; (4) asparagine; (5) serine; (6) cysteine. Separation voltage for (A)24 kV, (B) 18 kV. Other conditions were the same as in Fig. 1.

techniques, e.g., nano/microfluidic chip with submicro or nano-scale channels [49].

4. Conclusion

Continuous wave-based multiphoton excitation fluorescencedetection was firstly attempted for capillary electrophoresis,using cheap diode laser instead of high cost femto-secondpulse laser. On- and end-column configurations were comparedfor optimizing the detection scheme. End-column configura-tion exhibited better detectability, due to less light scattering.Three different types of fluorescence tags that were most fre-quently used as molecular label for bio-analysis were employedto evaluate applicability of this detection method, such as small-molecule dye, fluorescence protein and nano particle or alsoreferred to as quantum dot. Simultaneous detection of fluo-rescent dyestuffs with divergent excitation and emission wave-lengths in a broad range showed advantage of this scheme overconventional laser-induced fluorescence detection. Detection forcapillary zone electrophoresis and micellar electrokinetic chro-matography separations of FITC-tagged amino acids furthervalidate the feasibility of our idea, and realized a promisingfuture in various micro-column separations, where detectionvolume was ultra-small, for instance, nano/microfluidic chipwith sub-micro or nano-scale channels. In those cases, on-lwnbiIpe

photon fluorescence is proportional to the square of the laserpower.

Acknowledgements

The authors gratefully acknowledge financial supports fromNational Natural Science Foundation of China (No. 20405006to B.-F. L., No. 20105004 to Q. L.), and Program for DistinguishYoung Scientist of Hubei Province (2004ABB004 to B.-F. L.).

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