Microchip-Based Nano Chromatographyand Nano Capillary Electrophoresisin Genomics and Proteomics

Imran Ali1, Hassan Y. Aboul-Enein2,&, Vinod K. Gupta3

1 Department of Chemistry, Jamia Millia Islamia, (Central University), New Delhi 110025, India2 Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division,

National Research Centre, Dokki, Cairo 12311, Egypt; E-Mail: enein@gawab.com3 Department of Chemistry, Indian Institute of Technology, Roorkee 247667, India

Received: 2 May 2008 / Revised: 3 July 2008 / Accepted: 21 August 2008Online publication: 24 September 2008

Abstract

The growing interest in proteomics and genomics needs analytical techniques capable ofdetecting proteins and nucleic acids at the ng L-1 levels due to the small amounts of variousproteins and nucleic acids in living cells. During the last decade remarkable development hasoccurred in the evolution of chromatographic and capillary electrophotetic methods onmicrochips. These methods are called nano liquid chromatography (NLC) and nano capillaryelectrophoresis (NCE). The present review article describes the application of NLC and NCEto proteomics and genomics research The analyses of proteins and nucleic acids at low levelsis described together with the optimization and future trends of nano methods in genomicsand proteomics.

Keywords

Nano liquid chromatographyNano capillary electrophoresisMicrochipsGenomicsProteomics

Introduction

Genomics and proteomics have become

newly emerging research areas in the

last 10 years [1–4]. The study of nucleic

acids and proteins is a challenging task

due to their large numbers (millions) in

human beings. Moreover, the concen-

trations of nucleic acids and proteins in

living cells are of low concentration,

often of the order of ng L-1. There-

fore, the separation and identification

of these species are very difficult and

highly sensitive and effective analytical

techniques are required. To over come

this problem, some liquid chromato-

graphic methods and capillary electro-

phoresis with fused silica capillaries

have been tested but found wanting [5–

8]. This has compelled scientists to-

wards new developments in the area of

separation science, resulting in the

evolution of microchip-based LC and

CE [9–11]. We have named these

techniques nano liquid chromatography

(NLC) and nano capillary electropho-

resis (NCE) [12]. Basically, they involve

samples, mobile phase flows and

detections at ng L-1 levels. This sort of

miniaturization provides significantly

reduced solvent consumption together

with fast separation, good combination

with detectors and sample preparation

units and low waste production. In this

article an attempt has been made to

review different aspects of these nano

analytical techniques in genomic and

proteomic researches.

1968–2008: 40 Years of Chromatographia.Reviews of Current Techniques and Devel-opments.

2009, 69, S13–S22

DOI: 10.1365/s10337-008-0813-1� 2008 Vieweg+Teubner | GWV Fachverlage GmbH

Review Chromatographia Supplement Vol. 69, 2009 S13

Nano LiquidChromatography

The most commonly used chip-based

nano analytical technique is nano high

performance liquid chromatography

(N-HPLC). However, nano capillary

electrochromatography (NCEC) and

nano micellar electrokinetic chromatog-

raphy (NMEKC) have also been used.

These sorts of chromatographic instru-

ments are manufactured by fabricating a

micro chip on silicon or glass or quartz or

some other polymer such as poly(dim-

ethylsiloxanes) (PDMS) poly(methyl-

methacrylate) (PMMA), polycarbonate

(PC), polyethyleneteraphthalate (PET),

polystyrene (PS), polypropylene (PP),

luoroethylene, polyimide (PI), poly(tri-

fluoroethylene) (PTFE), poly cyclic olefin

copolymer, polystyrene and polyvinyl

chloride etc. The micro channels are

etched on these chips by laser technology,

lithography, machining and finishing.

The separation channels are packed with

different materials such as reversed phase

silica, monolithic silica gel and polymers.

In fully integrated techniques the sample

preparation, injection, separation and

detection are carried out on the same chip

but such type of devices are rare and un-

der their development stages. The role of

N-HPLC in proteomics and genomic re-

searches is summarized in the following

sections.

Nano High PerformanceLiquid Chromatography

Generally, proteins are digested and

converted into small peptides followed by

analysis. Vollmer et al. [13] reported

analysis of the human proteome by N-

HPLC using cation exchange packing

material with MS detection which iden-

tified 206 proteins. The same group [14]

described a special type of N-HPLC

having enrichment columns for gel-free

proteome studies. A tryptic digest of a

human T-cell proteome was also studied

on cation exchange material and selected

fractions were analyzed by MS-MS. This

system was capable of separating com-

plex peptide mixtures. Hardouin et al.

[15] discussedN-HPLC-MS as useful tool

in biomarker discovery programs, par-

ticularly for identifying low-abundance

proteins and they used MS-MS detection

for identification of autoantigens [16]. A

polymeric polystyrene divinylbenzene

(PS-DVB) monolithic chip column

was used for the analysis of tryptic digest

peptides [17]. Licklider et al. [18]

described a chip-based system on a sili-

con wafer for automated separation

and identification of tryptic peptides.

N-HPLC-ESI-MS has been used for

the analysis of some biological protein

Fig. 1. Nano-scale LC-MS-MS setup with (a) samples at high flow on the trapping column usinga vent connected to a switching valve and (b) the flow through the vent [20]

Fig. 2. Schematic representation of a COMOSS separation column [32]

S14 Chromatographia Supplement Vol. 69, 2009 Review

mixtures in rice seed protein samples [19].

Gaspari et al. [20] described protein

identification at the ng level using an

offline two-step sample cleanup, protein

digestion and separation (Fig. 1). The

mobile phase usedwaswater-acetonitrile-

trifluoroacetic acid (97.95:2:0.05, v/v).

Lanckmans et al. [21] usedN-HPLC-MS-

MS for studying extra-cellular angioten-

sin IV (Ang IV) peptide in rat brain with a

50 pM detection limit. The mobile phase

used was water-acetonitrile-formic acid

(98:2:0.1, v/v) at 300 nL min-1 flow rate.

The analysis of glycoproteins i.e. tur-

key ovalbumin, chicken ovalbumin and

ovomucoid gave detection limits in the pg

range [22]. El Rassi et al. [23, 24] analyzed

iglycoproteins and glycans [24] and Yin

et al. [25] used 2-D N-HPLC for analysis

of BSA proteins. The analysis of serum

proteins from 99 ovarian cancer patients,

87 healthy volunteers and 21 patients

with other ovarian diseases was carried

out with an IMAC3 metal affinity chro-

matography protein chip and a WCX2

cation exchange protein chip [26]. Sepa-

ration efficiencies of 15,000–18,000 plates

per metre were obtained on porous

monolithic silica with a double T-shaped

injector for the separation of catechins

[27]. N-HPLC-TOF-MS was used for

the analysis of complex protein digests

by integrating a 30 nL pre-column

trap. The separation channel was of

45 9 0.075 9 0.050 mm packed with

either Zorbax C18 or C3 of 2.1, 3.5 or

5 lm particles sizes [28]. Huang et al. [29]

described a label-free N-HPLC-MS

method for proteomics profiling of cere-

brospinal fluid (CSF) by addressing

quality control, sample replication steps

and the adaptation of pattern recognition

methods for the detection of experimental

variation and (most importantly) puta-

tive biomarkers. Iimmobilized metal

affinity N-HPLC was used for the analy-

ses of phosphopeptide fragments from

b-casein in peptide mixtures [30]. An

integrated andmodular apparatus for the

rapid analysis of trace level tryptic digests

for proteomics was described. This device

had an auto-sampler and separation

channels with C18 silica as packing

material. The detection limit achieved

was 5 nM [31].

Fig. 3. Representation of PCR product profiles of DNA at zero PCR cycles and 10 PCR cyclesusing a C18 modified PMMAN-CEC. 100, 200, 400, 800, 1,200 and 2,000 bp fragments are sizingladders. The dark lines are primerdimer peak along with the 500-bp (PCR-1) and 1,000-bp(PCR-2) products [38]

Fig. 4. An overview of fabrication process for NCE chip with multiple buried optical fibers, (a)lithography, (b) wet chemical etching, (c) hot embossing, (d) demolding, process (e) upper/lowersubstrate alignment and prebonding, (f) optic fiber insertion, (g) thermal bonding and (h) index-matching fluid filling [44]

Review Chromatographia Supplement Vol. 69, 2009 S15

Nano CapillaryElectrochromatographyand Nano MicellarElectrokineticChromatography

N-CEC and N-MEKC chromatography

have been used in proteomic and genomic

researches. Slentz et al. [32, 33] developed

N-CEC on collocatedmonolithic support

structures (COMOSS) moulded in poly(-

dimethyl siloxane) for analysis of a tryptic

digest of fluorescein isothiocyanate-la-

beled bovine serum albumin (FITC-BSA)

with separation efficiency of 4.0 9 105

theoretical plates meter. The structure of

COMOSS column is shown in Fig. 2

indicating its geometry for fast and effec-

tive separations. The same authors [34]

reported the separation of FITC-labe-

led peptides (FITC–Gly–Phe–Glu–Lys

(FITC)–OH, FITC–Gly–Phe–Glu–Lys–

OH, FITC and FITC–Gly–Tyr–OH.) on

a C18-AMPS modified PDMS COMOSS

microchip. The separation of these pep-

tides was carried out on microchips

with different stationary phases i.e. poly

(vinylsulfonic acid) polyacrylic acid and

poly(styrenesulfonic acid. The best sepa-

ration was achieved on poly(styrenesulf-

onic acid) chip. Jindal and Cramer [35]

reported separation of three peptides on

N-CEC under isocratic conditions. The

optimization of the separation of these

proteins was achieved by selecting suit-

able stationary and mobile phases and

wavelengths of UV detection. N-MEKC

was adopted by Ceriotti et al. [36] for the

separation of low and high density lipo-

proteins (LDL and HDL) respectively.

LDL peak exhibited an apparent effi-

ciency of 2.2 9 107 theoretical plates m-1

by using sodium dodecyl sulfate as

micelle formation agent. According to the

authors, the low concentration of SDS

did not alter lipoprotein particle size;

distribution within the time involved.

Shadpour and Soper [37] described 2-D

separation of a protein mixture on

PMMA microchip N-MEKC with

12 mM TRIS-HCl (0.4% w/v (14 mM)

containing 0.05% w/v SDS (pH 8.5); with

methylhydroxyethylcelullose (MHEC) as

the dynamic EOF suppressor.

N-CEC on a PMMA chip for the

separation of two DNA fragments pro-

duced via the polymerase chain reaction

(PCR) of k-DNA was described by

Galloway and Soper [38]. A DNA sizing

ladder containing six fragments, ranging

in size from 100 to 2,000 base pairs, was

separated. The efficiency was of an order

of 104 theoretical plates for a 3 cm long

column. Figure 3 shows separations of

100, 200, 400, 800, 1,200 and 2,000 bp

fragments as sizing ladders of DNA

indicating good resolution. The same

group reported open channel N-CEC for

the separation of double stranded DNA

ladder in plain PMMA and C18 silica gel

modified chips [39]. 25% Acetonitrile

and 75% aqueous phase containing

50 mM TEAA (ion pairing agent, pH

7.4) was used as mobile phase with

100 v cm-1 as applied voltage.

Nano CapillaryElectrophoresis

Microchip-based capillary electrophore-

sis, (NCE), has been used successfully in

proteomic and genomic research for

identifying proteins and nucleic acids.

The inherent characteristic features of

working at extremely low concentrations

make NCE a technique of choice in

proteomics and genomic programmes.

The analysis of proteins and nucleic

acids by NCE is discussed in the fol-

lowing sections.

Proteomics Research

Colyer et al. [40] described the separa-

tion of human serum proteins (IgG for

mimicking c zone, transferrin b zone,

a-1-antitrypsin and albumin) using a

NCE-laser-induced fluorescence (LIF)

unit. The background electrolyte was

100 mM borate with 2 mM lactate (pH

Fig. 5. A schematic representation of NCEwith (A) sample reservoir, (B) waste reservoir,(C) buffer reservoir, (D) fluidic channel exittip and (E) ball [47]

Fig. 6. Schematic representation of NCE with two orthogonal channels [51]

S16 Chromatographia Supplement Vol. 69, 2009 Review

10.5). Similarly, Rodriguez et al. [41]

reported the separation and identifica-

tion of fluorescein isothiocyanate anti-

human IgG. The applied voltage was

0.268 kV cm-1 on a 6 cm long channel

and analysis was completed within

60 min. Linder et al. [42] reported

poly(dimethylsiloxane)/glass chips for

studying IgG proteins. Forrer et al. [43]

described a chip-based method for

analysis of half-antibody species in IgG4

and their by- and degradation products.

The design and fabrication of micro

chips for NCE, followed by analysis of

various proteins, including bovine serum

albumin and b-casein with detection by

optic fibers was described by Hsuing

et al. [44]. The fabrication process with

multiple buried optical fibers, lithogra-

phy, wet chemical etching, hot emboss-

ing, demoulding process, upper/lower

substrate alignment and prebonding,

optic fiber insertion, thermal bonding

and index-matching fluid filling is shown

in Fig. 4. A flow through sampling

technique was exploited for the analysis

of Cy5-labeled bovine serum albumin

(Cy5-BSA) and anti-BSA [45]. The per-

formance of SU-8 (a epoxy-based pho-

toresist) and glass chips for the

separation of biologically active peptides

with fluorescent detection was compared

by Sikanen et al. [46]; the SU-8 chips

were found to be superior. Different

buffers were used for the separation on

lithography-based patterning of a SU-8

single chip. Yue et al. [30] described

sample preparation and separation in

NCE for proteomics applications

including separation of b-casein in pep-

tide mixtures in less than 1.5 min.

MALDI-TOF-MS was used for the

detection of peptides and peptide frag-

ments produced from a protein digest on

a PMMA chip [47]. The linear separa-

tion channel was 50 lm wide, 100 lmdeep with an 8.0 cm effective separation

channel length (Fig. 5).

Fruetel and co-workers [48] described

a separation of fluorescamine- labeled

protein biotoxins by silica based NCE

with LIF detection. The authors de-

scribed the portability of this instrument

in a laboratory for biotoxin variants.

Hybrid capillary PDMS microchips

integrated with ESI tips were prepared

and used for the separation and identi-

fication of peptides within 2 min [49].

Sun et al. [50] used NCE-LIF to evaluate

a derivatization method mediated by

liposome for single cell analysis. A single

cell analysis revealed that liposome-

membrane fusion occurred after en-

trance of liposomes into the cells. Moh-

anty et al. [51] developed NCE for

separation of proteins by applying a field

of 40 V cm-1. The device was simple to

fabricate and included an on-chip col-

lection scheme that interfaced the macro

world with the micro world (Fig. 6). An

on-line sample pretreatment and analysis

of proteins and peptides on a PMMA

microfluidic device has been described.

The chip contained two hyphenated

electrophoresis channels with integrated

conductivity detectors. The first channel

was used for sample pre-concentration

and sample clean-up and second for

separation [52]. Phillips et al. [53] Two

chip-based systems were made by Phil-

lips et al. [53] for analyzing inflammatory

neuro-peptides in tissue fluids of patients

with neuro-peptide associated muscle

pain. The systems provided a relatively

fast, accurate procedure for studying

inflammatory biomarkers in complex

biological fluids. Vasilyeva et al. [54]

used micro-fluidic technology for quan-

tification of antibody in immunoglobulin

G4 samples. The authors referred this

method as a chip-based capillary gel

electrophoresis in their work. The sepa-

ration was optimized for the effect of

heating time and by varying concentra-

tions of N-ethylmaleimide.

A modular, hand-held unit, called as

l-ChemLab, was designed and fabri-

cated [55] by Renzi et al. [56] it provided

reliability and flexibility for the detec-

tion of proteins and other biomolecules.

An NCF-MS was made on glass wafers

using standard photolithographic/wet

chemical etching methods. The design

integrated with sample inlet ports, the

separation channel, a liquid junction

and a guiding channel for the insertion

of the electrospray chamber of an ion

trap MS. This device was used for

Fig. 8. Electropherogram of gene screening with 1 = IFITM3, 2 = UBB, 3 = CRYAB,4 = IL1B, 5 = G1P2, 6 = G1P3, 7 = APOD, 8 = PLAB, 9 = CYR61, 10 = PDE3A,11 = PEPP2, 12 = IFITM1 [87]

Fig. 7. Electropherograms of 50 bpDNA lad-der under EKS mode on cross microchip [84]

Review Chromatographia Supplement Vol. 69, 2009 S17

analyses of peptides, proteins and pro-

tein tryptic digests. High efficiency and

separations were obtained on an 11 cm

chip with separations within 50 s. Elec-

trokinetic and pressure sampling were

used. The authors reported separation

and identification of 12-angiotensin

peptide mixtures obtained from different

animals on the device with a 4.5 cm

separation channel; with 20 lg L-1

concentration of each peptide. The

background electrolyte was 20 mM

6-aminocaproic acid/acetic acid at pH

4.4 with 650 V cm-1 as applied voltage.

Reviews are also available on capillary

and microchip-based CE for proteomic

programmes [57–60].

Genomics Research

A substantial amount of work has been

carried out in genomics by using NCE.

Generally, DNA is present in very small

amount and, hence, micro-fluidic de-

vices played a crucial role in purification

and separation of DNA from the cell.

Kan et al. [61] and Lin et al. [62] re-

viewed NCE for NDA purification and

sequencing. Shi et al. [63] and Paegel

et al. [64] described a radial micro-

fabricated DNA sequencing unit of 96

channels for high speed, a rotary LIF

scanner and a specialized high pressure

sieving matrix loader for genetic mate-

rials analyses [65–68]. Other NCE

devices comprising 16 and 384 channels

have been fabricated on 6 in. glass wa-

fers [64, 69–72]. In these devices, all the

channels were fabricated on a single

planar substrate providing the necessary

separation length. Chowdhury et al. [73]

described an NCE method to genotype

for common single nucleotide polymor-

phisms in thiopurine S-methyltransfer-

ase gene, which led to serious adverse

drug reactions for patients undergoing

thiopurine therapy. The authors re-

ported complete concordance between

NCE and conventional methods in 80

patients.

Automated parallel DNA sequencing

on multiple sixteen-channel microchips

has been carried out applying high volt-

age for sequencing DNA samples. An

integrated four-color confocal fluores-

cent detector was capable of scanning

more than 450 bases in 15 min in all 16

channels [74]. Hong et al. [75] reported a

micro-fabricated polymer chip of PDMS

for NCE and used it for the separation

of different DNAs. The same authors

[76] studied gene amplification in NCE

on PDMS and glass. An NCE unit

(40.5 mm, 110 and 50 lm as channel

length, width and depth respectively)

was used for the analysis of DNA frag-

ments employing electrokinetic injection

with transient isotachophoretic pre-con-

centration [77]. Liu et al. [78] reported

isotachophoresis nano-chip electropho-

resis (ITP-NCE) for a hepatitis B virus

(HBV) genotyping test in clinical diag-

nosis. Tang et al. [79] discussed an iso-

thermal signal amplification technique

for specific DNA sequences in an inte-

grated NCE.

Genetically modified organisms

(GMOs) were studied with an NCE-LIF

device having glass chips (each

25 9 76 lm), thermally bonded to-

gether to form a closed structure [80].

Similarly, Posedi et al. [81] used NCE

for the differentiation of the closely re-

lated cyathostomin species Cylicocyclus

elongatus and Cylicocyclus insigne from

the horse. These papers may be consid-

ered as the procedures for identifying

closely related strains or species. Dooley

and co-workers [82] described NCE for

determining the mitochondrial cyto-

chrome b genes of fish in UK and a

differentiation of fish species was carried

out. Kataoka et al. [83] reported the

usefulness of NCE (Hitachi SV1100

model microchip) by analyzing non-

standard DNA samples. Separation was

achieved within 4 min with a detection

limit of 1.83 ng lL-1.

Xu et al. [84] used gel NCE for high

sensitivity detection of DNA by com-

bining electrokinetic injection with

transient isotachophoresis pre-concen-

tration. The electropherograms shown

in Fig. 7 indicate a good separation of a

DNA ladder. The effect of buffer flow

and buffer concentration on resolution

of DNA separation was investigated by

Chen et al. [85]; no improvement could

be obtained by increasing the buffer

concentration without increasing the

flow. Sieben et al. [86] described good

labeling capacity and separation of

DNA in NCE based on glass chips.

Hawtin et al. [87] described gene

screening by NCE for reporting work-

flow processes, speed of analysis, data

accuracy and reproducibility and auto-

mated data analysis. Figure 8 indicates

high throughput gene separation of

nucleic acids. Chuang et al. [88] deter-

mined binding of estrogen receptor to

an estrogen response element in geno-

mic pathways by using chip gel elec-

trophoresis. The mobility shift assay on

a microchip bearing neutral surfaces

against the adsorption of acidic DNA

molecules and basic ER proteins was

studied. PMMA and polycarbonate

based-chips for NCE were developed

and tested by separating phiX174

Fig. 9. Typical electropherogram of the optimized separation of HaeIII digested ØX174 DNAmarker (1.25 ng mL-1). 1 = 72, 2 = 118, 3 = 194, 4 = 234, 5 = 271, 6 = 281, 7 = 310, 8 = 603,9 = 872, 10 = 1078, 11 = 1353 [95]

S18 Chromatographia Supplement Vol. 69, 2009 Review

DNA/HaeIII markers [89]. Kim and

Kang [90] described on-channel base

stacking in NCE for analysis of DNA

fragments. Similarly, Chiesl et al. [91]

used NCE for the purification of DNA

on octylacrylamide and dihexylacryla-

mide copolymers.

Polymerase chain reaction (PCR)

microchips have been studied exten-

sively in various fields with special

emphasis on DNA purification and

sequencing. The PCR reaction vessels

increase resolution reducing the overall

size of PCR with the miniaturization of

PCR amplification needed to match

NCE. Dolnik and Liu [11] and Zhang

et al. [92] reviewed the fabrication of

PCR microfluidic devices and their

applications. Nojima et al. [93] isolated

target DNA species from a DNA

mixture generated by a polymerase

chain reaction (PCR), with a starting

material of a ligation mixture of an

insert and an expression vector. The

authors reported total operation in

standard genetic engineering performed

in a cell-free condition. Lutz-Bonengel

et al. [94] reported the use of NCE for

low volume amplification in DNA

analysis. NCE coupled with PCR

restriction fragment length polymor-

phism (RFLP) assay was used for

genotyping A (-6) G single nucleotide

polymorphism in 123 patients [95].

The separation and detection of the

digested PCR amplicons were com-

pleted within 280 s. A typical electro-

pherogram of the optimized separation

of HaeIII-digested ØX174 DNA mar-

ker is given in Fig. 9.

Legendre et al. [96] described on-line

SPE-NCE for DNA isolation from a

nasal swab spiked with anthrax spores

through a polymerase chain reaction

(PCR). Nowadays, the main emphasis is

on functional genomic studies of gene

variants in human beings for disease

diagnosis, prognosis and management.

NCE has also been used to understand

mutation in the tumor susceptibility

genes BRCA1 and BRCA2 [97]. The

separation in a 3 cm long channel took

less than 120 s. Schmalzing et al. [98]

reported NCE combined with (EMD?)

for clinical mutation studies. Russom

et al. [99] described specific extension of

fluorescently labeled nucleotides for

Table 1. The applications of nano chromatographic and nano capillary electrophoretic methods in proteomics and genomic programmes

Species Techniques Chip materials LOD References

ProteomicsActin N-HPLC PMMA 30 nM [37]BSA N-HPLC PDMS, glass, fused silica,

PMMA-CD, polycarbonate100 Fm [37, 103–109]

Carcinoembronic antigen N-HPLC Glass-silica 2 ng mL-1 [110]Chicken ovalbumin N-HPLC Glass 300 pg [22]C-Reactive protein N-HPLC PMMA, Silicon 1 ng mL-1 [111–113]IgG (mouse) N-HPLC PDMS 15.6 ng mL-1 [114]IgG (rabbit) N-HPLC PDMS 244 pg mL-1 [115]Ovamucoid N-HPLC Glass 300 pg [22]Ovalbumin N-HPLC PMMA, quartz, glass 6.4–30 nM [39, 116, 117]Myoglobin N-HPLC PDMS, Silicon 30 ng/mL [111]Myosin N-HPLC PMMA, Glass 100 fM–10 nM [39, 118]Phosphoproteins N-HPLC PMMA-CD 15 fmol [109]Serum albumin N-HPLC PMMA 10 nM [39]Turkey ovalbumin N-HPLC Glass 300 pg [22]a-Fetoprotein N-HPLC Glass, silicon 2 ng mL-1 [110]b-Human choriogonadotropin N-HPLC Glass, silicon 2 ng mL-1 [110]BSA N-CEC PDMS – [32]Peptides N-CEC PDMS – 3434Peptides N-CEC Quartz – [35]Lipoproteins N-MEKC – – [36]Proteins N-MEKC PMMA 30 nM [37]Membrane proteins NCE Silica 3.2–43.5 nM [119]a-Lactalbumin, b-lactoglobulin A,b-lactoglobulin B

NCE Glass <0.5 pg [120]

PSA NCE Silicon nitride 0.2 ng [121]Biotin NCE Silicon 100 fg [122]Theophylline NCE Glass 1.25 ng mL-1 [123]Genomicsk-DNA N-CEC PMMA 10-5 M [38]DNA N-CEC PMMA – [39]DNA polymerase N-CEC Silicon 4.7 ng [124]DNA N-CEC Silicon 10–100 nM [125–127]DNA N-CEC Silicon 75 nM [128]DNA N-CEC Glass 2 fM [113]DNA N-CEC – 1.83 ng lL-1 [83]DNA N-CEC PDMS 1 fmol [129]

BSA bovine albumin serum, DNA deoxyribose nucleic acid, IgG Immunoglobulin G, PDMS poly(dimethylsiloxane), PMMApoly(methylmethacrylate), PSA prostate specific antigen

Review Chromatographia Supplement Vol. 69, 2009 S19

scoring of single-nuleotide-polymor-

phism (SNP) with the help of NCE. Liu

et al. [100] indicated analysis of homo-

duplex and heteroduplex PCR products

by using chip-based temperature gradi-

ent NCE. A 2-DE chip incorporated

with the temperature gradient feature

was used to analyze SNP in a single run.

Zhang and co-workers [101] described

temperature gradient nano capillary

electrophoresis (TG-NCE) for DNA

mutation/SNP analysis. According to

these authors TG-NCE analyses of four

mutant DNA samples; amplified from

plasmid templates; showed that muta-

tions were successfully detected under a

wide temperature gradient of 10 �C. Theeffectiveness of their system was dem-

onstrated by the successful detection of

K-ras gene mutations in 6 colon cancer

cell lines. Similarly, Buch et al. [102]

described TG NGE for the detection of

DNA point mutations. The authors used

the principle of single-strand conforma-

tional polymorphism (SSCP) for DNA

mutation detection. Some other appli-

cations of nano chromatographic and

nano capillary electrophoretic methods

in proteomics and genomic programmes

are summarized in Table 1.

Limitations andComparisonof Nano ChromatographyandCapillaryElectrophoresis

The newly emerging techniques of nano

chromatography and nano capillary

electrophoresis have certain limitations.

They are not yet fully developed and still

need more research to get a complete and

perfect set-up. The most critical chal-

lenges are the creation of devices capable

of performing multiple, complex fluid

handling steps in series on a fully inte-

grated device. The sampling, detection,

chip interfacing, chromatographic and

capillary electrophoretic separations and

total integration are the issues that need to

be addressed. Among these, the integra-

tion of sample preparation into micro-

fluidic devices is one of the main hurdles

towards achieving true nano-analyses.

Nano separations are complimentary

with normal chromatography and capil-

lary electrophoresis with similar working

principles. They differ only in detecting

low amount of samples (ng L-1) that can

not be achieved by normal methods. On

the other hand, normal chromatography

can be used at semi- and preparative

scales. Bothkinds of analytical techniques

have their own merits and demerits.

Conclusion

The role of NLC and NCE in genomic

and proteomic research is remarkable

and they can be used to sequence proteins

and amino acids at nano level concen-

trations. The effectiveness of these tech-

niques lies in the facts that they are very

fast, efficient, selective, reproducible and

inexpensive. Their use will be highly

important in the advancement of geno-

mic and proteomic research by studying

various interactions at molecular levels.

They are still under development. Cer-

tainly, we hope that the future advance-

ment in these techniques will be a boon

especially in genomics and proteomics.

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