microscale separation methods for enzyme kinetics assays

REVIEW

Microscale separation methods for enzyme kinetics assays

Tomáš Křížek & Anna Kubíčková

Received: 29 November 2011 /Revised: 10 January 2012 /Accepted: 12 January 2012 /Published online: 3 February 2012# Springer-Verlag 2012

Abstract Miniaturization continues to be one of the leadingtrends in analytical chemistry and one that brings advan-tages that can be particularly beneficial in biochemical re-search. Use of a miniaturized scale enables efficient analysisin a short time and requires very small amounts of samples,solvents, and reagents. This can result in a remarkabledecrease in costs of enzyme kinetics studies, especiallywhen expensive or rare enzymes and/or substrates are in-volved. Free zone electrophoresis is without a doubt themost common microscale separation technique for capillaryand on-chip enzyme assays. Progress and applications inthis field are reviewed frequently whereas other modes ofseparation, although successfully applied, receive only mar-ginal interest in such publications. This review summarizesapplications of less common modes of separation in capil-lary or chip formats, namely micellar electrokinetic chroma-tography, liquid chromatography, gel electrophoresis,isoelectric focusing, and isotachophoresis. Because thesetechniques are based on separation mechanisms differentfrom those of free zone electrophoresis, they can be, andhave been, successfully used in cases where zone electro-phoresis fails. Advantages and drawbacks of these alterna-tive separation techniques are discussed, as also are thedifficulties encountered most often and solutions proposedby different research groups.

Keywords Enzyme assays .Micellar electrokineticchromatography . Capillary gel electrophoresis . Capillaryisoelectric focusing . Capillary liquid chromatography . Chip

AbbreviationsBGE Background electrolyteCE Capillary electrophoresisCEC Capillary electrochromatographyCGE Capillary gel electrophoresis

Tomáš Křížekhas been a researcher at CharlesUniversity in Prague, Departmentof Analytical Chemistry, since2009. His research interests areapplications of capillaryelectrophoresis in the study ofenzyme kinetics and interactionsof biomolecules

Anna Kubíčkováhas been a researcher at CharlesUniversity in Prague, Departmentof Analytical Chemistry, since2009. In 2007 she won the Hlavka’sFoundation prize for the young scien-tists. She is currently investigatingHPLC methods for monitoring phar-maceutically important reactions

Published in the special issue Young Investigators in Analytical andBioanalytical Science with guest editors S. Daunert, J. Bettmer, T.Hasegawa, Q. Wang and Y. Wei.

T. Křížek :A. Kubíčková (*)Faculty of Science, Department of Analytical Chemistry,Charles University in Prague,Hlavova 8,128 43 Prague 2, Czech Republice-mail: [email protected]

Anal Bioanal Chem (2012) 403:2185–2195DOI 10.1007/s00216-012-5744-x

CIEF Capillary isoelectric focusingCLC Capillary liquid chromatographyCTAB Cetyltrimethylammonium bromideCZE Capillary zone electrophoresisEMMA Electrophoretically mediated microanalysisEOF Electro-osmotic flowIMER Immobilized enzyme reactorITP IsotachophoresisLIF Laser-induced fluorescenceLOD Limit of detectionMEKC Micellar electrokinetic chromatographyPA PolyacrylamidePAGE Polyacrylamide gel electrophoresisPAP Adenosine-3′,5′-bisphosphatePAPS Adenosine-3′-phosphate-5′-phosphosulfateSDS Sodium dodecylsulfate

Introduction

Enzymes, highly efficient and selective biocatalysts [1], arecrucial for the growth and reproduction of any organismbecause they catalyze almost all chemical reactions in livingnature. The catalytic activity of individual enzymes must befinely regulated to coordinate all biochemical processes inthe organism. There are numerous such regulation mecha-nisms [2]; the most straightforward examples are regulationof activity by concentration of substrate and competitiveinhibition by product of the reaction. Monitoring of enzy-matic activity is an important source of information in theresearch fields of biochemistry and molecular biology. Be-cause subtle changes in the activity of enzymes can lead toserious errors in metabolism, growth, and reproduction,determination of enzyme activity is also of great importancein medicine [3–5]. Enzymes are usually quantified by theiractivity, because this is the biologically most relevant prop-erty and its value is more readily accessible than the con-centration of enzyme itself. In biological matrices, enzymesare present in very low concentrations whereas other pro-teins occur at levels several orders of magnitude higher.Consequently, monitoring of enzyme activity, i.e. changesin the concentration of a substrate and/or products, is themore practical approach because these compounds are typ-ically present at concentrations measurable without seriousdifficulties.

Absorption or fluorescence spectrometry are routinelyused for enzyme assays because they can readily be auto-mated and enable fast and high-throughput activity determi-nations using commercial plate readers working with 96 to1536-well microtiter plates. However, this approach can beapplied only to substrates and products that significantlydiffer in their spectrometric properties. As an alternative,chromogenic or fluorogenic substrates can be used but the

fact that these are not natural substrates of the enzyme inquestion must be taken into account. Use of electrochemicalsensors is another option well-suited to compounds that canbe oxidized/reduced [6]. A typical example is amperometricdetection of hydrogen peroxide arising from oxidation ofglucose by glucose oxidase [7] or potentiometric detectionof changes in pH or concentration of oxygen caused byenzymatic reaction [8]. In many cases, however, substrateand product of enzyme reaction have similar spectrometricand electrochemical properties. Use of a separation methodfollowed by spectrometric or electrochemical detection maythen be required to perform the enzyme assay.

Enzyme assays using separation methods can be dividedinto twomain groups, off-line and on-line. In off-linemethods,enzymatic reaction is performed outside the analytical system(often in a sample vial) and can be terminated by injection foranalysis. Analytical instrumentation serves as the means forseparation and determination of reaction substrate and prod-uct. The advantage of this experimental setup is in the possi-bility of sequential injections from one reaction mixtureduring the reaction process. The other option is the so calledon-line approach. Here, enzymatic reaction takes place in areactor which is a part of the analytical system. The mainadvantage of this setup is its high degree of automation,because the analytical system incorporates automatic mixingof reagents, enzymatic reaction and its termination, and sepa-ration and detection of reaction products. This approach alsosubstantially reduces manipulation of the reaction mixture andthus minimizes the danger of sample loss or contamination.Enzymatic reaction can occur before, after, or directly on theseparation column and the enzyme can be present in solutionas another zone that is mixed with the substrate zone or can beimmobilized on column walls or stationary phase.

Although conventional HPLC was initially successfullyapplied with pre or post-column immobilized enzyme reactors(IMER) [9, 10], analytical systems miniaturized to capillaryand microchip format soon became more popular. Apart fromshorter analysis times and higher throughput, microscale sep-aration techniques have a remarkable advantage of minimizedconsumption of enzymes and substrates that are often ratherexpensive and/or available in limited amounts. Because of itsversatility, capillary zone electrophoresis (CZE) became awell-recognized technique for enzyme assays. Since pioneer-ing work published in 1991 [11, 12], numerous applicationsemerged during the last two decades which have been thor-oughly reviewed in recent years [13–16]. Capillary electropho-retic methods performed in the on-line setup that wereintroduced one year later [17] are known as electrophoreticallymediated microanalysis (EMMA); these have also beenreviewed [18–22]. This review, on the other hand, focuses onless common microscale separation techniques used for en-zyme assays—methods based on capillary liquid chromatog-raphy (CLC), techniques combining chromatographic and

2186 T. Křížek, A. Kubíčková

electrophoretic separation mechanisms, i.e. capillary electro-chromatography (CEC) andmicellar electrokinetic chromatog-raphy (MEKC), and techniques based on other separationmechanisms, for example capillary gel electrophoresis (CGE)and capillary isoelectric focusing (CIEF). Microchip applica-tions using the above mentioned separation mechanisms willbe considered in a separate section.

Enzyme assays in capillary format

MEKC

As numerous reviews show, most work published on enzymeassays by capillary electrophoresis (CE) successfully uses theCZE separation mode. Although simple and versatile, thismode is not applicable to all cases. Some enzyme assaysrequire separation of neutral compounds or ions of very closecharge-to-size ratio. In such cases, MEKC is a promisingalternative because it conserves the general advantages of CEmethods and, at the same time, incorporates another separationmechanism—partition of analytes between aqueous mobilephase and micellar pseudostationary phase. Thus hydropho-bicity of analytes becomes another important factor in theseparation. There is a substantial number of successful MEKCapplications for enzyme assays. These are summarized inTable 1. Sodium dodecylsulfate (SDS) [23–58] is a typicallyused micelle-forming agent, although cetyltrimethylammo-nium bromide (CTAB) [59–62], sodium cholate [24, 63, 64],and deoxycholate [56, 65, 66] have also been used. Use ofother additives, for example sodium octanesulfonate [67],Tween 20 [26] or Brij 35 [68], has been reported occasionally.

Most MEKC applications have been conducted in off-line mode. The reaction was carried out in test tubes or CEvials in volume of tens to hundreds of microliters. MEKCwas then used to separate and quantify reaction substratesand/or products. Two major detection techniques have beenused. Direct UV detection [23–27, 29, 30, 33, 35–43,46–58, 61–63, 65–67] is highly sensitive, especially forcompounds containing chromophores, for example aromaticrings (nucleotides, drugs, 4-nitrophenol-labeled peptides).Figure 1 illustrates the typical outcome of an off-line MEKC-based enzyme assay using direct UV detection. The otherpopular detection technique is laser-induced fluorescence(LIF) with fluorescently-labeled substrates [28, 31, 32, 34,44, 45, 64]. This technique is particularly advantageous instudies in which very small amounts of substrates or productsare to be detected, because the detection limits of the LIFtechnique are often comparable with those of mass spectro-metric methods.

The number of publications on on-line MEKC enzymeassays is rather limited. The reason is obvious; surfactant-containing running buffer is often incompatible with enzyme

reaction because of the protein-denaturing properties of SDSand other additives. Enzymatic reaction must, therefore, beperformed in a different buffer and the reaction mixture cannotbe in contact with the background electrolyte (BGE) until thereaction is complete. Occasionally the enzyme is compatiblewith MEKC separation buffer and classical EMMA formatscould be used [24, 26, 65]. Van Dyck et al. [23] solved theproblem of incompatibility of amine oxidase with SDS byintroducing the so called “partially filled capillary” concept.The principle of this approach is depicted in Fig. 2. Part of thecapillary at the inlet end is filled with micelle-free buffer. Intothis buffer, zones of enzyme and substrate are injected in twozones separated by a short plug of the buffer. Electrophoreticmixing of zones and enzyme reaction occur inside this micelle-free zone. After the enzyme reaction, resulting product, en-zyme, and remaining substrate migrate into the second part ofcapillary which is filled with MEKC separation buffer. Thisapproach has been adopted by other researchers [59, 60]. Asimilar technique has been reported by Okamoto et al. [25].The main difference is that the plug of enzyme is injectedimmediately after injection of the substrate zone and thus thesetwo zones are in contact from the beginning. Neighboringplugs of enzyme and substrate are “sandwiched” betweentwo micelle-free plugs that separate this “reaction zone” fromthe running buffer. An interesting advantage of on-line enzymeassays was pointed out by Nováková et al. [63]. While devel-oping an assay for determination of phenol sulfotransferaseactivity toward 4-nitrophenol, they encountered a problemwith co-substrate adenosine-3′-phosphate-5′-phosphosulfate(PAPS). PAPS is an unstable compound and commercial PAPSalways contains small amounts of adenosine-3′,5′-bisphos-phate (PAP), which is a strong inhibitor of the enzyme studied.Fortunately, studied electrophoretic mobility of PAP and PAPSwere found to be sufficiently different so that during theelectrophoretic mixing of zones, PAP inhibitor was separatedfrom the PAPS co-substrate before reaching the enzyme zone.Therefore, the resulting reaction zone was free from the PAPinhibitor. Most assays were performed with direct UV detec-tion of substrate and/or product of the enzyme reaction. Twoworks by Telnarová et al. [59, 60] are an exception to this rule.In these cases, monitoring of the concentration of bromide andchloride anions formed during the reaction was used to deter-mine enzymatic activity of haloalkane dehalogenase. The con-centration of chloride was measured by indirect UV detectionin a BGE containing chromate and CTAB.

CLC and CEC

CLC is different from electrophoretic capillary separationtechniques because of the different mechanism of analytemotion inside the capillary. The driving force is hydrodynamicpressure instead of the electro-osmotic flow (EOF) generatedby the applied electric field. Initially slurry-packed fused silica

Microscale separation methods for enzyme kinetics assays 2187

Tab

le1

Sum

maryof

MEKCenzymeassays

Enzym

eSub

strate

Detectio

nOff-line/on

-line

Ref.

Protein

farnesyl

transferase

2′,7′-Difluorofluorescein-5-carbo

xyl-glycinyl-

cysteiny

l-valin

ylisoleuciny

l-alanine

LIF

Off-line

[28]

Laccase

andmanganese

peroxidase

Rem

azol

turquo

iseblue

G13

3,everzol

turquo

iseblue,helig

onblue

S4

UV

254and66

6nm

Off-line

[61]

β-Glucuronidase/arylsulfatase

Pheny

toin,mepheny

toin

UV

192and29

7nm

Off-line

[29]

Sirtuin

17-Dim

ethy

aminocou

marin

N-acetylly

sine,

dansyl

N-acetylly

sine

UV

256nm

Off-line

[30]

Tissuetransglutaminase

Fluorescein-labeled

peptideQLQPFPQPQLPY

LIF

Off-line

[31]

In-vivoenzymatic

reactio

nsin

ratstriatum

Sub

stance

PLIF

Off-line

[64]

μandm-calpain

Fluorescently

labeledα s

andβ-casein

LIF

Off-line

[32]

Trypsin,pepsin,chym

otrypsin

β-Lactoglob

ulin

AandB

UV

214nm

On-lin

e[26]

Ang

iotensin-con

vertingenzyme

Fou

rmedicinally

used

inhibitors

UV

214nm

NAa

[33]

Ang

iotensin-con

vertingenzyme

Eight

inhibitors

UV

214nm

NAa

[67]

Secretory

phosph

olipaseA2

Pho

sphatid

ylcholine

UV

196nm

Off-line

[66]

Cathepsin

DOrego

ngreen-labeledhemog

lobin

LIF

Off-line

[34]

Hum

anliv

erdihy

drod

ioldehy

drog

enase1

Prostagland

inD2

UV

210nm

Off-line

[35]

Adeno

sine

kinase

Eight

adenosinederivativ

esUV

260nm

Off-line

[36]

Purinenu

cleoside

phosph

orylase,adenosinedeam

inase

Adeno

sine,inosine,adenine,hy

poxanthine,xanthine

UV

260nm

Off-line

[37]

Recom

binant

cytochromeP45

02C9

Diclofenac

UV

200nm

Off-line

[38]

Cytochrom

eP45

02C9

Diclofenac

UV

200nm

Off-line

[39]

MgA

TPase

ATP

UV

254nm

Off-line

[62]

Theaninesynthetase

L-G

lutamate,ethy

lamine

indirect

UV

360nm

Off-line

[68]

5-Aminolaevu

linic

acid

dehy

dratase

5-Aminolaevilin

icacid

UV

220nm

Off-line

[40]

Pheno

lsulfotransferase

4-Nitrop

heno

lUV

260and27

4nm

On-lin

e[19]

Alkalineph

osph

atase

Riboflavinph

osph

ate

UV

270nm

On-lin

e[25]

Adeno

sine

deam

inase

Nucleosideprod

rugs,6-am

ino-2′,3′-dideox

yguano

sine,

6-am

ino-2′,3′-dihy

drod

ideoxy

guanosine

UV

254nm

Off-line

[42]

Cytochrom

eP45

0enzymes

(CYP2C

19,CYP2D

6*1,

CYP2C

9*1,

CYP1A

2,CYP3A

4)S-M

epheny

toin

UV

200nm

Off-line

[43]

cAMP-dependent

proteinkinase

Kem

ptidepeptide

LIF

Off-line

[44]

α-Glugo

sidase

IandII,α-1,3-N-

acetylgalactosam

inyltranferase

from

HT29

cell

αGlc(1→

3)αG

lc-TMR;UDP-N

-acetylgalactosamine+

αFuc(1→

2)βG

al(1→

4)βG

lcNAc-TMR

LIF

Off-line

[45]

Glutathione

S-transferase

Styrene

oxide

UV

200nm

Off-line

[46]

Pho

sphatid

ylinosito

l-specific

phosph

olipaseC

Pho

sphatid

ylinosito

lUV

196nm

Off-line

[47]

β-Glucuronidase

Morph

ine-3-β-

D-glucuronide,morph

ine-6-β-

D-

glucuron

ide,α-naph

thyl

sulfate,redu

ced

flun

ixin

glucuron

ide

UV

195nm

Off-line

[48]

Bov

ineplasmaam

ineox

idase

Benzylamine

UV

254nm

On-lin

e[23]


Tab

le1

(con

tinued)

Enzym

eSub

strate

Detectio

nOff-line/on

-line

Ref.

γ-Glutamyltransferase

γ-Glutamyl-p-nitroanilid

eUV

380nm

On-lin

e[65]

Elastasefrom

A.fumigatus,type

IVelastase

from

porcinepancreas,hu

man

neutroph

ilelastase

Suc-A

la-A

la-Pro-Phe-N

A,Suc-A

la-A

la-A

la-N

A,

MeO

Suc-A

la-A

la-Pro-Val-N

AUV

200nm

Off-line

[49]

Ornith

inetranscarbamylase

Ornith

ine,carbam

ylph

osph

ate

UV

200nm

Off-line

[50]

Hum

anneutroph

ilcathepsinG,hu

man

neutroph

ilelastase,

Pseud

omon

asaerugino

saelastase

Peptid

es:Suc-A

la-A

la-A

la-N

A,

Suc-A

la-A

la-Pro-Phe-N

A,

Metho

xySuc-A

la-A

la-Pro-Val-N

A

UV

200nm

Off-line

[51]

γ-Glutamyl

hydrolase

Metho

trexate-Glu4

UV

300nm

Off-line

[52]

Ang

iotensin-con

vertingenzyme

Hippu

ryl-His-Leu-O

HUV

230nm

Off-line

[41]

α-Amylase,glucoamylase

p-Nitrop

heny

l-β-

D-m

altoside

UV

280nm

On-lin

e[24]

Porcine

kidn

eyprolidase

Gly-Pro,Leu-Pro,Ala-Pro

UV

200nm

Off-line

[53]

Cytosolic

5′-nucleotidaseIII(nucleotidaseandtransferaseactiv

ity)

Cytidinemon

opho

sphate,uridinemon

opho

sphate

UV

254nm

Off-line

[54]

Bilirubinox

idase

Bilirubin

UV

450nm

Off-line

[55]

Cathepsin

DHem

oglobin

UV

214nm

Off-line

[56]

Haloalkanedehalogenase

1-Bromob

utane

Indirect

UV

315nm

On-lin

e[59]

Haloalkanedehalogenase

1-Bromob

utane

Indirect

UV

315nm

On-lin

e[60]

Pseud

omon

asaerugino

saelastase,hu

man

leuk

ocyteelastase,cathepsinG

Suc-A

la-A

la-A

la-N

A;Suc-A

la-A

la-Pro-Phe-N

AUV

200nm

Off-line

[57]

Rat

liver

microsomes

S-(−)-Thalid

omide,R-(+)-thalidom

ide

UV

214and23

0nm

Off-line

[58]

aSub

strate

andsetupareno

tmentio

nedbecausekinetic

stud

ywas

notperformed.


capillaries were used [69] but during the last decade they havebeen gradually replaced by monolithic columns based onmodified silica gel or organic polymers [70, 71]. The mainadvantage of monolithic over packed capillaries is the lack ofrequirement for frits, low backpressure, even at high flow-rates, and the large number of theoretical plates, the combina-tion of which leads to fast and highly efficient analyses.Lately, a new sol–gel processing method has been introduced

and used for immobilization of enzymes in a silicate matrix.This novel approach is gentle enough to be used for prepara-tion of bioaffinity columns [72–74]. It has been proved that thestructural properties of such monoliths might strongly affectthe amount of enzyme in the active and accessible form [75,76], and that immobilization of enzyme could be achievedwithout significant loss of its original activity [77].

There are basically two main approaches for enzymeassays by CLC. The first uses CLC with a standard capillarycolumn (often a C18 stationary phase) for separation anddetermination of enzyme and/or products of enzymatic re-action. Kawano et al. [78] used CLC with a packed C18

capillary column and ESI–MS detection for the rapid iden-tification and quantification of proteases produced by Staph-ylococcus aureus. De Jong et al. [79] performed an assay ofacetylcholinesterase (AchE) inhibitors in a natural extract ofNarcissus. The crude extract was first separated by CLC.The eluent was mixed on-line with AchE and acetylcholineand products detected by ESI–MS. A special arrangement ofCLC, micro parallel liquid chromatography, was used byWu et al. [80, 81] for studying protein kinase A. Analysestake place parallel in 24 separation capillaries, which makesthe method rapid with high throughput.

Another approach utilizes reactors with entrapped enzyme.As mentioned previously, in such an arrangement the amountof enzyme necessary for analysis is extremely small. More-over, enzyme assays are performed on-line, which has themany advantages described above. Amankwa et al. [82]immobilized tyrosine phosphatase β on a small piece of fusedsilica capillary and coupled this microreactor directly to theCLC system with ESI–MS detection. Recently, reactors with

Fig. 1 Time course of deacetylation of 7-dimethylaminocoumarin ace-tyllysine (DMAC-K(Ac)-NH2) by sirtuin 1. Experimental conditions: 37/30 cm, 50 μm i.d. fused silica capillary, 25 kV, 25 °C. 50 mmol L−1 Trisbuffer, pH 9.1 containing 180 mmol L−1 SDS. Detection 256 nm.DMAC-K-NH2, 7-dimethylaminocoumarin lysine; NIC, nicotinamide. Reprintedfrom Fan Y et al. (2010) J Pharm Biomed Anal 54:772–778. Copyright(2010) Elsevier

Fig. 2 Illustration of partially filled capillary approach introduced by vanDyck et al. (a) Capillary before application of the potential. (b) The fourphases of the online enzyme assay. (I) a plug of enzyme, followed by anintermediate buffer plug and a substrate plug are introduced hydrody-namically; (II) the potential is applied, fast migrating substrate penetratesthe slowly migrating enzyme plug; (III) when the two plugs have merged

the potential is turned off, the enzyme reaction proceeds; (IV) the potentialis applied and the enzyme, substrate, and product are separated in MEKCmode. 1, reaction buffer; 2, separation buffer; E, enzyme solution; S,substrate solution; P, generated product. Reprinted with permission fromVan Dyck S et al. (2001) Electrophoresis 22:1436–1442. Copyright(2001) John Wiley and Sons


Tab

le2

Sum

maryof

enzymeassays

byCGE,CIEFandCLC

Enzym

eSub

strate

Sep.mod

eDetectio

nOff-line/on

-line

Ref.

Alkalineph

osph

atase,β-galactosidase

p-Nitrop

heny

lpho

sphate,o-nitrop

heny

l-β-galactop

yranoside

CGE

UV

405nm

On-lin

e[90]

Uridine

diph

osph

ateglucuron

osyltransferase

Uridine

diph

osph

ateglucuron

icacid

CGE

UV

310nm

On-lin

e[91]

NADP+-specificform

atedehy

drog

enase

Sod

ium

form

ate

CGE

UV

210and28

0nm

Off-line

[92]

Trypsin

Syn

thetic

nonapeptide

CIEF

LIF

Off-line

[88]

Cellobioh

ydrolase

I–

CIEF

UV

280nm

NA

a[89]

Prostagland

inD

synthase

–CIEF

UV

280nm

NA

a[87]

Trypsin

Syn

thetic

decapeptideC-m

acCLC

UV

214nm

On-lin

e[77]

γ-Glutamyl

transpeptid

ase

Glutamic

acid

p-nitroanilid

eCLC

UV

410nm

On-lin

e[83]

Tyrosineph

osph

atase

Syn

thetic

phosph

opeptid

ePDGF-R

CLC

ESI–MS

On-lin

e[82]

Staph

ylococcalexop

roteins

–CLC

ESI–MS

NA

a[78]

Acetylcho

lineesterase

Acetylcho

line

CLC

ESI–MS

On-lin

e[79]

aSub

strate

andsetupareno

tmentio

nedbecausekinetic

stud

ywas

notperformed

Tab

le3

Sum

maryof

enzymeassays

inmicrochip

form

at

Enzym

eSubstrate

Sep.mode

Detectio

nOff-line/on-line

Ref.

Cathepsin

BZ-Phe–Arg–AMC

CLC

ESI–MS

On-lin

e[95]

GalactosyltransferaseI

p-Nitrop

heny

lβ-

D-xylop

yranoside

CLC

ESI–MS–MS

On-lin

e[96]

Throm

bin

–ITP

LIF

NA

a[99]

Calfintestinal

alkalin

eph

osph

atase,ho

rseradishperoxidase

6,8-Difluoro-4-methy

lumbelliferylph

osph

ate

CGE

Fluorescencemicroscop

yOn-lin

e[98]

Pho

spho

lipaseA2

Fluorescently-labeled

phosph

atidylinosito

landph

osph

atidylserine

MEKC

LIF

Off-line

[97]

L-A

sparginase

L-A

sparigine

MEKC

LIF

On-lin

e[100

]

aSub

strate

andsetupareno

tmentio

nedbecausekinetic

stud

ywas

notperformed.


enzymes immobilized in monoliths have been extensivelyused. Besanger et al. evaluated the effect of morphology andapplied flow-rate on Km of γ-glutamyl transpeptidase entrap-ped in a capillary by the sol–gel method [83]. Ma et al. [77]prepared an immobilized trypsin reactor based on an organic–inorganic hybrid silica monolith and coupled it on-line toCLC–MS–MS. Generally, great emphasis is put on stabilityof the reactors and on high enzymatic activity. Although CLCis not the prevalent technique in enzyme assays, it couldsurpass classic CZE in several aspects, especially in terms ofreproducibility. A summary of CLC methods used for enzymeassays is presented in Table 2.

Attempts to use CEC—a technique combining chromato-graphic and electrophoretic separation mechanisms—for en-zyme assays are surprisingly rare. A paper mentioning thistopic was published by Siskova et al. [46]. Here, CEC with asol–gel C18 stationary phase was unsuccessfully tested and theoff-line assay of glutathione S-transferase was finally based onMEKC separation. The only successful application wasreported by Redman et al. [84]. In this case, chiral CEC–ESI–MS was used for determination of S and R-warfarin tocompare the function of the cytochrome P450 enzyme systemin patients treated with different doses of warfarin.

CIEF and CGE

These techniques (summarized in Table 2) are capillaryadaptations of more common slab gel variants (IEF andPAGE) [85]. CIEF combines the high resolving power ofconventional IEF with the advantages of capillary instru-mentation, and enables rapid and highly efficient separationof analytes of different pI. It has been reported that proteinsdiffering by 0.05 pI units only could be resolved. Comparedwith CZE, CIEF can accommodate larger volumes of sample,because of the formation of well-focused bands, and thus theLOD values are enhanced [86].

CIEF can be performed in a one or two-step process. Intwo-step CIEF, analytes are first focused according to their pIvalues and then mobilized hydrodynamically or chemicallytoward a detector. Capillaries coated either covalently or dy-namically, are used for two-step CIEF because of the need forEOF suppression. In contrast, focusing and mobilization areperformed simultaneously during one-step CIEF. Generally,the one-step procedure enables faster analysis than two-stepCIEF. Hiraoka et al. [87] successfully used one-step CIEF forrapid determination of microheterogenities of the β-trace pro-tein. Two-step CIEF was used by Shimura et al. [88] fortrypsin assay and by Hui et al.[89] for characterization ofcellobiohydrolase I.

CGE is mostly performed in capillaries filled with replace-able gel, which acts as a molecular sieving medium. Thisenables separation of molecules having similar charge-to-sizeratio on the basis of their different size. However, the amountof cross-linking monomer used for gel preparation stronglyaffects pore size. Thus, “normal” CZE, i.e. separation basedsolely on charge without contribution of sieving effect mightbe also obtained in a gel-filled capillary, when the gel concen-tration is low. The main advantage of CGE compared withCZE is minimization of diffusion and consequent band spread-ing of resulting peaks, because of the high viscosity of the gelmatrix. On the other hand, stability of gel-filled capillaries issometimes rather poor, which results in worse reproducibilityof CGE measurements. Wu et al. [90] performed an enzymeassay of alkaline phosphatase and β-galactosidase using CGEwith polyacrylamide (PA) gel-filled capillaries and compared itwith classic CZE with a C18-PF108-modified capillary. Asexpected, CGE enabled more sensitive detection. The maindrawback of the gel-filled capillary was its lifetime—it wasnecessary to prepare a new capillary after every ten analyses.Recently, Kim et al. [91] successfully used CGE with PA gelfor on-line monitoring of glucuronidation. CGE could be alsoused to follow enzyme expression and purity. Klyushnichenko

Fig. 3 Schematic diagram oftwo-step pore limit electrophore-sis with enzyme assay fordetermination of CIP molecularweight and activity in oneseparation channel. PLE separa-tion of a protein mixture contain-ing CIP (a). Enzyme activityassay performed by introductionof weakly fluorescent agentDiFMUP, which is conversed byCIP to strongly fluorescentDiFMU product (b). Reprintedfrom Hughes AJ et al. (2010)Anal Chem 82:3803–3811.Copyright (2010) AmericanChemical Society


et al. [92] used SDS-CGE for this purpose. Compared withtraditional SDS-PAGE, SDS-CGE enables more sensitiveanalysis in a shorter time. Despite these promising resultsobtained with gel-filled capillaries, CGE has never becomewidely used for enzyme assays.

Enzyme assays in microchip format

Miniaturization of separation methods has moved from capil-lary systems to chip-based systems during the last ten years.These are very powerful tools for high-throughput analysiswith broad application potential [93]. Microchips have severaladvantages over traditional capillary methods. The most im-portant are extremely low consumption of sample and reagents,analysis time shorter than 1 min, compactness of the system,and great variability of materials and design available. How-ever, utilization of chip-based systems also has disadvantages.The main drawback is the need for powerful detection, forexample LIF or MS. This is because of the very small amountof analyzed sample, because of the low chip capacity whichresults from its proportions (the size of a chip is typically up to5 cm with channels of maximum depth 100 μm). Despite useof LIF or MS detection, concentration LODs obtained withmicrochip devices are usually higher than those obtained byuse of macro-scale systems. On the other hand, lower absoluteLOD values are often achieved by use of chip-based methods.This means that smaller amounts of analyzed samples arerequired for screening. For this reason chip-based systems areof growing interest in enzyme assays also [94].

Although most microchips were developed for CZE, theyhave also been successfully used in other techniques, forexample CLC, MEKC, CGE, and ITP (Table 3). De Boeret al. [95] used a system comprising on-line CLC for separa-tion of ligands, and a microchip device for monitoring prote-ase cathepsin B and its inhibition. Ono et al. [96] used a CLCchip for screening of multiple enzyme reactions and evaluatedkinetic data for glycosyltransferase. Lin et al. [97] used anMEKC chip to monitor the activity of lipid-modifyingenzymes. Among less common methods one should mentiontwo-step microfluidic gradient-gel zymography, which wasused by Hughes et al. [98] for assay of calf intestinal phos-phatase (CIP) and horseradish peroxidase. During the firststep, pore limit electrophoresis (PLE), enzymes are resolvedaccording to their molecular weight and pseudoimmobilizedin a gel matrix. Second, substrates are electrophoreticallytransported along the separation channel with immobilizedenzymes and products are formed and detected. The wholeprocess is conducted on a single microchannel chip (Fig. 3).This method enables enzyme assays with exceptional sensi-tivity (zeptomole level). Recently, Wang et al. [99] used chip-based ITP followed by on-chip electrophoresis separation fordetermination of thrombin. In this arrangement, concentration

LODs were extremely low (attomolar level). The last twopublications clearly demonstrate the great progress in on-chip enzyme assays made in the last two years. It is evidentthat the drawback of lower concentration LODs is beingovercome. Therefore, a growing number of on-chip enzymeassays overtaking macroscale methods even in terms of theconcentration LODs can be expected [100].

Conclusion

Despite the major importance of CZE inmicroscale separationenzyme assays, other techniques based on complementaryseparation mechanisms have proved their high potential. Thecomplementarity was found particularly useful for MEKC.This mode was frequently used when the CZE approachfailed. Although application of CGE and IEF can furtherimprove separation efficiency, CLC is characterized by in-creased reproducibility and compatibility with highly sensi-tive and selective mass spectrometric detection. The potentialof CLC, MEKC, and CGE has been further demonstrated byuse of these separation mechanisms in the rapidly developingfield of chip-based enzyme assays. Although the techniquesconsidered in this paper are on the sidelines of mainstream inmicroscale enzyme assays, their alternative separation mech-anisms and other specific advantages of individual separationmodes make them an essential complement to the most com-monly used CZE and their further development can thus beexpected.

Acknowledgement Financial support by the Ministry of Education,Youth, and Sports of the Czech Republic, projects MSM0021620857and RP 14/63, and by the Grant Agency of Charles University in Prague,projects 710, SVV 2012-265201 and UNCE 2012/44, is gratefullyacknowledged.

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