Review Jana Kr ˇenková* Frantis ˇ ek Foret Institute of Analytical Chemistry, Brno, Czech Republic Immobilized microfluidic enzymatic reactors The use of enzymes for cleavage, synthesis or chemical modification represents one of the most common processes used in biochemical and molecular biology laboratories. The continuing progress in medical research, genomics, proteomics, and related emerging biotechnology fields leads to exponential growth of the applications of enzymes and the development of modified or new enzymes with specific activities. Concurrently, new technologies are being developed to improve reaction rates and specificity or perform the reaction in a specific environment. Besides large-scale industrial applications, where typically a large processing capacity is required, there are other, much lower-scale applications, benefiting form the new developments in enzymology. One such technology is microfluidics with the potential to revolutionize analytical instrumentation for the analyses of very small sample amounts, single cells or even subcellular assemblies. This article aims at reviewing the current status of the development of the immobilized microfluidic enzymatic reactors (IMERs) technology. Keywords: Immobilized enzymes / Mass spectrometry / Microfluidics / Miniaturization / Review DOI 10.1002/elps.200406096 Contents 1 Introduction .......................... 3550 2 Immobilization techniques .............. 3551 2.1 Covalent immobilization ................ 3551 2.2 Noncovalent immobilization ............. 3551 3 Applications of immobilized enzymes ..... 3552 3.1 Immobilized enzyme reactors ............ 3552 3.2 Coupling IMER with MS ................ 3555 3.3 Immobilized enzyme biosensors ......... 3556 4 Immunoassays ....................... 3557 5 Affinity chromatography and electrophoresis ....................... 3559 6 Affinity interactions in MS ............... 3560 7 References ........................... 3561 1 Introduction The advances in systems biology and related scientific disciplines keep pushing the limits of the standard ana- lytical techniques. Any incremental improvement in the analysis resolution, sensitivity or throughput seems to be totally insufficient given the analytical needs of current proteomics (and other -omics) efforts. The development of miniaturized (microfabricated, microfluidics-based) analytical devices and their integration to create micro- total analysis systems (mTAS) [1] is one of the directions to address the future analytical needs of increased through- put, lower sample and reagent consumption, smaller size, and lower operating costs. The attempts to miniaturize the analytical instrumentation, which can be traced back to the early sixties, have lead to some remarkable devel- opments, including a number of commercially successful sensors such as the gas chromatograph micromachined on a silicone wafer [2]; however, the steady development seems to be continuing only since the early 1990s. Such microfabricated devices have been under development mainly for chromatography, electrophoresis, isoelectric focusing, and other separation methods [3, 4]. It is also worth mentioning, that certain microfabricated elements achieved significant commercial applications – e.g., per- sonal glucose monitors or clinical ion analyzers – http:// www.i-stat.com. The complexity of biological materials, especially when dealing with protein analysis, mostly requires the use of multidimensional analysis for identification and quantifi- cation of individual components. Enzymatic digestion of Correspondence: Dr. Frantis ˇ ek Foret, Institute of Analytical Chemistry, Veveri 97, CZ-61142 Brno, Czech Republic E-mail: [email protected]Fax: 1420-532290242 Abbreviations: Ab, antibody; Ag, antigen; IgG, immunoglobulin G; IMER, immobilized microfluidic enzymatic reactor; PDMS, poly(dimethylsiloxane); S/V , surface to volume 3550 Electrophoresis 2004, 25, 3550–3563 * On leave from the Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Czech Re- public 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Transcript
Review
Jana Krenková*Frantisek Foret
Institute of Analytical Chemistry,Brno, Czech Republic
Immobilized microfluidic enzymatic reactors
The use of enzymes for cleavage, synthesis or chemical modification represents one ofthe most common processes used in biochemical and molecular biology laboratories.The continuing progress in medical research, genomics, proteomics, and relatedemerging biotechnology fields leads to exponential growth of the applications ofenzymes and the development of modified or new enzymes with specific activities.Concurrently, new technologies are being developed to improve reaction rates andspecificity or perform the reaction in a specific environment. Besides large-scaleindustrial applications, where typically a large processing capacity is required, thereare other, much lower-scale applications, benefiting form the new developments inenzymology. One such technology is microfluidics with the potential to revolutionizeanalytical instrumentation for the analyses of very small sample amounts, single cellsor even subcellular assemblies. This article aims at reviewing the current status of thedevelopment of the immobilized microfluidic enzymatic reactors (IMERs) technology.
The advances in systems biology and related scientificdisciplines keep pushing the limits of the standard ana-lytical techniques. Any incremental improvement in theanalysis resolution, sensitivity or throughput seems to be
totally insufficient given the analytical needs of currentproteomics (and other -omics) efforts. The developmentof miniaturized (microfabricated, microfluidics-based)analytical devices and their integration to create micro-total analysis systems (mTAS) [1] is one of the directions toaddress the future analytical needs of increased through-put, lower sample and reagent consumption, smaller size,and lower operating costs. The attempts to miniaturizethe analytical instrumentation, which can be traced backto the early sixties, have lead to some remarkable devel-opments, including a number of commercially successfulsensors such as the gas chromatograph micromachinedon a silicone wafer [2]; however, the steady developmentseems to be continuing only since the early 1990s. Suchmicrofabricated devices have been under developmentmainly for chromatography, electrophoresis, isoelectricfocusing, and other separation methods [3, 4]. It is alsoworth mentioning, that certain microfabricated elementsachieved significant commercial applications – e.g., per-sonal glucose monitors or clinical ion analyzers – http://www.i-stat.com.
The complexity of biological materials, especially whendealing with protein analysis, mostly requires the use ofmultidimensional analysis for identification and quantifi-cation of individual components. Enzymatic digestion of
Correspondence: Dr. Frantisek Foret, Institute of AnalyticalChemistry, Veveri 97, CZ-61142 Brno, Czech RepublicE-mail: [email protected]: 1420-532290242
the analyzed proteins and coupling with mass spectrom-etry are the most common steps in most analyticalprotocols. Additionally, incorporating affinity selectionincreases the system flexibility for selective analyte iden-tification. The analysis of proteins includes enzymaticdigestion for detection of pathological changes of pro-teins occurring in physiological fluids in small amounts,detection of post-translational protein modification, iden-tification and localization of genetic variants.
Typically, enzymes have high efficiency under mild condi-tions and are highly selective, but are not stable forextended time in solution and during storage their activitygradually decreases. Significant improvements in boththe reaction rates (much higher substrate/enzyme ratiocan be achieved) and storage stability, not to mention thedecreased autolysis and the ease of use, can be achievedwith enzymes immobilized on the surface of a suitablecarrier material. Typically, resin beads can be used forsuch a purpose and some immobilized enzymes areavailable commercially. Alternatively, the enzyme sub-strate or affinity ligands can also be immobilized fordetermination of the sample enzymatic activity, selectivesample enrichment or other purposes. All advantages ofimmobilization of enzymes apply also to the miniaturizedenzymatic reactors in capillary of microfluidic forms.
2 Immobilization techniques
A variety of methods are now available for the immobili-zation of proteins onto the surface of the fused-silicacapillary or the channel of a microfluidic chip. Covalentbinding to the activated support, physical adsorption ofthe protein onto a solid matrix and copolymerization of theprotein with the polymers are just few examples. Thespecific immobilization chemistry depends on a variety offactors, character of the support, activation methods andcoupling procedure. Several types of supports (magneticor nonmagnetic beads, monolithic chromatographic sup-ports, etc.) are now commercially available or have beenspecifically developed for immobilization processes. Theideal matrix for immobilization process should have fol-lowing characteristics, (i) large surface area, (ii) perme-ability, (iii) hydrophilic character, (iv) insolubility, (v) chemi-cal, mechanical and thermal stability, (vi) high rigidity,(vii) chemical reactivity for coupling of the ligands, and(viii) resistance to microbial and enzymatic attack.
2.1 Covalent immobilization
The most frequently studied immobilization technique isthe formation of covalent bonds between the protein andthe support matrix [5], e.g., immobilization in the presence
of carbodiimides, cross-linking by glutaraldehyde or cya-nogen bromide activation of the support material. Themain advantage of covalent binding to the activated sup-port relates to minimization of the leakage of the immobi-lized substance. Proteins usually have a number ofpotential immobilizing sites, corresponding to particularfunctionalities on the molecules. The functional groups ofthe proteins suitable for covalent binding include (i) thea-amino groups of the chain and the e-amino groups oflysine and arginine, (ii) the a-carboxyl groups of the chainend and the b- and g-carboxyl groups of aspartic andglutamic acids, (iii) the phenol ring of tyrosine, (iv) the thiolgroup of cysteine, (v) the hydroxyl groups of serine andthreonine, (vi) the imidazole group of histidine, and (vii) theindole group of tryptophan. To prevent modification of theenzymatic activity or complete inactivation of the immo-bilized protein it is important that the catalytic functionalgroups of the enzyme are not involved in the covalentlinkage to the support. Unfortunately, many of the reactivegroups suitable for immobilization are often situated in theactive center of the enzyme. This problem can be some-times eliminated by immobilization in the presence of thesubstrate [6] or competitive inhibitor [7] of the enzyme.This also helps to stabilize the tertiary structure of theenzyme during immobilization. While immobilization ofsmall molecules is typically easier, the active center oflarger proteins may no longer be accessible after immo-bilization. In these cases, improvement can be achievedby introducing a spacer molecule [8]. Good steric acces-sibility of active sites can be obtained by oriented immo-bilization of glycoprotein enzymes through their carbohy-drate moieties [9].
At the end of immobilization (alternatively during this pro-cess) unreacted active groups of solid support must beblocked by reaction with inert moieties providing that thesame group was used for ligand immobilization. Thisblocking reaction is necessary to prevent further non-specific reactions between support and ligand that coulddecrease its stability or specificity. The schemes of themost common covalent immobilization procedures aresummarized in Fig 1. Although immobilization may resultin some changes in enzymatic activity, optimum pH oraffinity for the substrate, the elimination of the enzymefrom the reaction mixture, enhanced stability, reducedmixing and dilution-related problems and the possibilityto reuse the reactors outweigh these changes in practice.
2.2 Noncovalent immobilization
Physical adsorption of the protein onto solid support isprobably the simplest way of preparing immobilizedligand molecule [10, 11]. The method is based on non-
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Figure 1. Schemes of immobilization procedures forcovalent attachment of proteins: (a) immobilization aftersupport activation using cyanogen bromide; (b) immobili-zation after support activation using trichlorotriazine;(c) immobilization of glycoprotein via their carbohydratemoieties; (d) immobilization after support activation usingglutaraldehyde; (e) immobilization via epoxy group;(f) immobilization via azlactone group; and (g) immobili-zation using carbodiimide as “zero linker”.
specific physical adsorption between the ligand and thesurface of the matrix. Binding forces involve ionic inter-actions, hydrogen bonds, van der Waals forces, hydro-phobic interactions, etc. Because no reactive species areinvolved, the conformation changes which might result inthe change of the biological activity are less significant.An advantage of adsorption is that usually no reagentsand only a minimum of activation steps are required.
Unfortunately, the stability of the adsorbed layer is typi-cally much weaker than in the case of covalent bond anddesorption of the ligand resulting from changes in tem-perature, pH or ionic strength, is often observed.
Compared to nonspecific adsorption much better resultscan be obtained by using bio-specific (affinity) adsorp-tion, e.g., the biotin-avidin or streptavidin technique [8,12]. The bonds between the water-soluble vitamin, biotin,and the egg white protein, avidin, or its bacterial counter-part, streptavidin, are among the strongest known andcan be used for oriented immobilization. The main ad-vantage of the oriented immobilization is good stericaccessibility of the active binding site; however, key stepimportant for the binding efficiency and resulting enzymeactivity is the site-specific biotinylation.
Another way of immobilization based on bioaffinity inter-action is based on protein A. Protein A, a coat proteinextracted from the bacterium Staphylococcus aureus,has the unique capacity to bind mammalian immunoglo-bulins G (IgG), especially Fc constant region of IgG. Onecan also use other bacterial Fc binding proteins, insteadof protein A, such as streptococcal protein G, which hasthe advantage of binding to a wider range of IgG speciesand subclasses [9].
Additional approaches for immobilization may involve aphysical step, e.g., by using the low-temperature sol-geltechnologies for protein encapsulation [13, 14]. The reac-tion involves the hydrolysis and polycondensation ofalkoxysilane monomers. During this process, the proteinsare incorporated into a gel matrix. Because encapsulatingoccurs under mild conditions, the biomolecules retaintheir structure and biological activity. There is no chemicalbond formation between protein and polymer matrix, butproteins can be chemically modified without loss of theiractivity by conjugation to one of the monomers of thepolymerization mixture prior to polymerization leading tocovalent bond to the matrix [15].
3 Applications of immobilized enzymes
3.1 Immobilized enzyme reactors
Immobilized or encapsulated enzymes are commonlyused in medical diagnostics and therapy [16], biosensorsor enzyme-based electrodes [17–20], organic synthesis[21, 22], and many other applications such as removal ofwaste metabolites, blood detoxification and/or correc-tions of inborn metabolic deficiency [23]. Most of theimmobilized microfluidic enzymatic reactor (IMER) appli-cations are currently aimed at protein analysis by peptidemapping [24] and for determination of post-translational
modifications (PTMs) such as phosphorylation, glycosy-lation and lipidylation, which are essential in modulatingbiological functions of cells and can be associated with anumber of diseases. More than 200 different types ofprotein modifications have been reported so far [25].
Peptide mapping is typically performed using enzymaticcleavage of the protein and the peptide fragments in theresulting mixture are identified using electrospray ioniza-tion-mass spectrometry (ESI-MS) or matrix-assisted laserdesorption/ionization-mass spectrometry (MALDI-MS). Ineither case, separation of the peptide mixture, e.g., bymicro-high performance liquid chromatography (m-HPLC)or capillary electrophoresis (CE), prior to mass spectro-metric analysis minimizes the ionization suppression andimproves the sequence coverage [26].
One of the limiting steps in peptide mapping is the manualsample manipulation and extended reaction times forproteolytic digestion. Traditionally, enzymatic cleavage isperformed in a homogenous solution consisting of a mix-ture of the proteolytic enzyme (Table 1) and the protein[27–29]. Maintaining optimum pH, temperature, protein-to-enzyme ratio, and reaction time is critical to achieveefficient and reproducible results. The variations in size,structure, type, and level of PTM make proteins signifi-cantly different in their susceptibility to enzymatic diges-tion. Depending on the accessibility of the cleavage sites,complete digestion may require times ranging from sev-eral minutes to overnight. It is also worth mentioning thatwith highly glycosylated proteins deglycosylation (e.g.,using PNGase F) may be necessary prior to proteolyticdigestion. This is less critical with immobilized enzymereactors where the relatively high concentration of pro-tease may compensate for the reduced speed of proteo-lysis of highly glycosylated proteins. Proteolysis can beenhanced by denaturating the sample by urea or guani-dine hydrochloride and by reducing the disulfide bridges,e.g., with dithiothreitol to open the protein structure forthe proteolytic enzyme. Subsequent derivatization ofsulfhydryl groups with an alkylating agent, such asiodoacetic acid, is used to prevent protein refolding. Inprinciple, the time of digestion can be reduced using highconcentration of the free enzyme [27]; however, such anapproach has several disadvantages. Besides the addedcost at high concentration, the enzymes often lose theiractivity and specificity and the enzyme autodigestionresults in undesirable formation of additional peptides,which may lead to the ionization suppression in MSanalysis and complicate the interpretation of the data.Immobilizing the enzyme on a solid support eliminatesunwanted autodigestion and an extremely high localconcentration of proteolytic enzyme provides rapid cata-lytic turnover.
Table 1. Some of the most common enzymes used forprotein digestion
Enzymes IUBMBa)
Enzymenomenclature
Site of cleavage
Trypsin EC 3.4.21.4 C-terminus of Arg and LysChymotrypsin EC 3.4.21.1 C-terminus of Phe, Tyr,
Trp, Leu, and MetEndoproteinase Lys-C EC 3.4.21.50 C-terminus of LysEndoproteinase Asp-N EC 3.4.24.33 N-terminus of AspEndoproteinase Glu-C
(S. aureus V8, pH 4)EC 3.4.21.19 C-terminus of Glu
Endoproteinase Glu-C(S. aureus V8, pH 8)
EC 3.4.21.19 C-terminus of Glu and Asp
Endoproteinase Arg-C(clostripain)
EC 3.4.22.8 C-terminus of Arg
Thermolysin EC 3.4.24.27 N-terminus of Leu, Ile,Val, Phe, Met, and Ala
Pepsin EC 3.4.23.1 C-terminus of Phe, Met,Leu, and Trp
a) IUBMB, International Union of Biochemistry andMolecular Biology
Many of the reported microreactors are based on immo-bilization of the enzyme directly onto the surface of afused-silica capillary. Amankwa and Kuhr [12, 30] immo-bilized the enzyme on the inner surface of a 50 mm IDaminoalkylsilane-treated fused-silica capillary via biotin-avidin-biotin coupling. The strong coupling constant ofthis method makes the immobilized enzyme structurallyrobust to any flow rate and enables the use of wide rangeof pH, temperatures and solvent systems. Because theenzyme is coated on the capillary wall, a very low flow rate(e.g., 40 nL/min) was needed to permit time for diffusion ofthe protein sample to the immobilized enzyme. Kuhr’sgroup [31, 32] later found that the proteolysis reaction ratecould be enhanced by applying low-power acousticvibration to the capillary, with digestion carried out in abatch-wise procedure. Efficient tryptic digestions of largeproteins have been carried out in as few as 30 min [33].The capillary microreactors were used for protein char-acterization by trypsin, pepsin and carboxypeptidase Ydigestion. Guo et al. [34] also used a 50 mm ID fused-silicacapillary for preparation of a reusable capillary micro-reactor based on the immobilization of the enzyme (tryp-sin, chymotrypsin) on the capillary wall by metal-ion che-lated adsorption. Metal chelating agent, iminodiaceticacid (IDA), was firstly reacted with glycidoxypropyl-trimethoxysilane (GLYMO) to form GLYMO-IDA-silane,and consequently immobilized onto the inner wall of thefused-silica capillary. The copper ions and subsequentlyenzyme was specifically adsorbed onto the surface to
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form the capillary enzyme microreactor. The advantage ofsuch a protocol microreactor is in the ease with whichdifferent types of enzymes can be immobilized. The pre-pared microreactor should be regenerated after 4–6 timesof usages. Although capillary pretreated with NH4HF2 wasused to increase the surface area for the enzyme immo-bilization, the time necessary to complete the digestion ofcytochrome c (0.5 pmol) and bovine serum albumin (BSA;0.5 pmol) in a 100 cm long capillary IMER was about15 min and 30 min, respectively. These enzymatic micro-reactors were combined with MALDI-MS for mass map-ping analysis of several proteins.
Enzyme immobilization on a glass microchip can beachieved using the standard procedures for enzyme cou-pling to a silica matrix [35, 36]. This is done in three steps:starting with silanization using (3-aminopropyl)triethox-ysilane, followed by glutaraldehyde activation, and finalenzyme coupling. Alternative approaches were alsodescribed by Mao et al. [37] using the immobilization ofstraptavidin-conjugated alkaline phosphatase on a poly-(dimethylsiloxane) (PDMS) michrochip using a lipid bilayerwith biotinylated lipids, and by Holden et al. [38] usingphotochemical attachment for immobilization of alkalinephosphatase inside the microfluidic channel.
Problems related to long diffusion times and rather smallsurface-to-volume ratio of the open-tubular IMER can beeliminated by decreasing the ID of the capillary (microchipchannel) or packing or using channels packed with beadsthat significantly increase the available surface area. Awide variety of beads made from agarose, polystyrene,glass, etc., has been tested. Cobb and Novotny [39]prepared a reactor from thick-walled Pyrex tubing(30 cm61 mm ID) packed with trypsin-immobilizedagarose gel, Wang et al. [40] packed microfluidic chip with40–60 mm diameter beads and Seong and Crooks [41]used 15.5 mm diameter polystyrene microbeads in PDMSchip. The use of polystyrene-encapsulated superpara-magnetic beads (2.8 mm diameter) has been exploredwith commercial CE instrumentation for performingenzymatic and inhibition assays, as well as for analysis ofbiological molecules such as antigens and substrates. Asmall quantity of magnetic beads containing immobilizedbiomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2–3 mm) ofbeads was held fixed by a magnet placed in the cartridgeof the CE system, without the use of frits. The beads couldbe replaced after each run, eliminating the need toregenerate the solid support. Alkaline phosphatase (AP)and human immunodeficiency virus (HIV) protease wereused to demonstrate the procedure for enzymatic andinhibition assays and to quantitate an antigen (mouseanti-human growth hormone antibodies) using antibodies
(sheep anti-mouse IgG) immobilized on beads [42].Porous supports are favored and some conjugates ofenzymes (trypsin) or antibodies and porous polymerbeads (including magnetic) have already been com-mercialized (http://www.appliedbiosystems.com, http://www.piercenet.com, http://www.dynal.no). In an alter-native approach, the enzyme immobilization on a micro-chip was achieved by spotting and drying a drop of dis-solved nitrocellulose (NC) on a glass substrate followedby adsorbing the enzyme on the reconstituted NC mem-brane. This enzyme-immobilized glass plate was attach-ed to a PDMS cover with the separation channel forelectrophoresis. A b-galactosidase activity was demon-strated with fluorescein di-b-D-galactopyranoside usingthis integrated on-column enzyme reactor [43].
In recent years, monolithic phases have emerged as anattractive and increasingly more popular alternative topacked columns due to simplicity of preparation as wellas virtually unlimited choice of chemistries they offer.Additionally, there is no need for retaining frits and veryfast separations can be achieved due to the typicallylower flow resistance even with smaller pore sizes.Preparation, properties and applications of variousmonolithic stationary phases for the analysis of pro-teins, peptides, nucleic acids, and small molecules bym-HPLC and capillary electrochromatography (CEC)were discussed in recent reviews [44–47]. Perhaps themost appealing aspect of monolithic materials is theease of preparation. The simple polymerization processstarts from liquid precursors (polymerization mixture)and is performed directly inside the capillary or amicrofluidic chip. In contrast to packed beds, mono-lithic structures exhibit excellent dimensional stability.The through-pores of monolithic materials can be easilycontrolled allowing high-speed flows at low back-pressures and the surface of the monoliths can beeasily chemically modified. Such flexibility is ideal forthe design and development of the enzymatic reactortailored for specific applications.
Petro et al. [48] described comparative studies in whichtrypsin was immobilized on both macroporous poly-(gly-cidyl methacrylate-co-ethylene dimethacrylate) beadsand on chemically analogous monolith. Monolith andbeads were modified by a multistep process involving themodification of epoxide groups with ethylenediamine fol-lowed by activation using glutaraldehyde and final mod-ification with trypsin. Despite the relatively small size(11 mm) of the monodisperse beads used to minimize thediffusional path length, the processivity of the enzymeimmobilized on the monolithic material was nearly twotimes higher compared to that of the bead-based con-jugates. The same researchers developed poly(2-vinyl-
4,4-dimethylazlactone-co-acrylamide-co-ethylene dime-thacrylate) support that is more hydrophilic than themethacrylate-based material described above, andtherefore better suited for enzyme-based applications. Inaddition, preparation of the immobilized enzyme is sig-nificantly easier with the azlactone support, involving onlya single step to couple amine or thiol groups of the en-zyme with the azlactone moieties. This monolithic supportwas prepared in a capillary, microfluidic chip and nanoe-lectrospray needle [7, 49, 50]. Enzymatic microreactorwith a volume of 470 nL prepared by immobilizing trypsinon a 10 cm long reactive porous polymer located in a100 mm ID fused-silica capillary was used for digestion ofa variety of proteins [51]. This reactor afforded sufficientdegrees of protein digestion even after very short resi-dence time of less than 1 min. The fast reaction rateclearly results from the rapid mass transfer of the sub-strate proteins to the reactive sites of the immobilizedenzyme.
The capillary and plastic microchip IMERs were also pre-pared by sol-gel technologies [13, 14]. Unfortunately, thegel matrix has a disadvantage, stemming from the factthat while smaller peptides can penetrate the gel, largerproteins are not able to move through the nanopores ofthe hydrogel network. Therefore, trypsin-immobilizedreactors were only used for digestion of small molecules.Coating the surface of the highly permeable monolithiccolumn with the protein-containing gel can eliminate thisproblem. Kato et al. [52] prepared a monolithic column ina single step from methacryloxypropyltrimethoxysilane.Monolith composed of a porous rigid solid with largethrough-pores was fabricated in a 75 mm ID fused-silicacapillary with a thin film of pepsin-encapsulated sol-gelon the monolith surface.
A different approach for IMER fabrication was recentlyproposed by Gao et al. [10]. These authors reported theconstruction of a miniaturized membrane reactor by fab-ricating the PDMS microfluidic channel and coupling it toa poly(vinylidene fluoride) (PVDF) membrane, providinglarge internal surface area for protein adsorption. Thereactor was used for trypsin immobilization and digestionof model proteins. Despite the large surface-to-volume(S/V) ratio of porous membrane media, this reactor had ahigh total dead volume due to capillary connections withthe microchannel. This problem was eliminated by plac-ing the hydrophobic and porous PVDF membrane insidethe capillary fitting (Fig. 2) [11]. In the case of membraneclogging, the membrane-based reactor can be regener-ated by simply replacing the old membrane with a newPVDF strip, followed by adsorption of a fresh enzyme so-lution.
Figure 2. Schematic representation of the miniaturizedtrypsin membrane reactor inside the capillary fitting.Photograph on the bottom right side shows the expandedview of the electrospray needle. Reprinted from [11], withpermission.
The digestion by immobilized enzyme can also be per-formed on a bioreactive MALDI probe. As described byNelson et al. [53], enzyme was covalently attached to theMALDI probe via a gold-coated stainless steel sampletarget. Proteins were digested on the enzyme-linkedprobes by depositing the sample directly onto the activesurface. Digestion was terminated by adding the MALDImatrix prior to MS analysis. Houston and Reilly [54] usedthis technique for hemoglobin characterization. Becausethe matrix used to enhance ionization was applied directlyto the probe, the enzyme-linked MALDI probe could notbe reused for consecutive digestion.
One of the most important properties of an immobilizedenzyme is its storage and operational stability. Unless it isstable, it cannot be used over prolonged periods. The mostrealistic way to test storage and operational stability is pe-riodic measurement of the residual activity. Immobilizedenzymes are usually characterized by higher stability thantheir soluble form. Stability of grafted enzyme moleculescan be substantially influenced by the coupling strategy.For example, encapsulated trypsin was used continuouslyduring the tested period and free trypsin was stored in thesame buffer at 257C. In one day, the activity of encapsu-lated trypsin decreased 20% of its initial value. On the otherhand, soluble trypsin almost completely lost its activity oneday. Furthermore, encapsulated trypsin held the enzymaticactivity after more than three months of storage at 47C [13].
3.2 Coupling IMER with MS
Due to the relatively long incubation time the currentworkflow of protein analysis includes protein digestion asan off-line step. With miniaturized IMER, complete protein
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digestion can be performed in less than 1 min and directon-line coupling with further peptide concentration andseparation steps becomes possible. Several researchgroups have described enzyme reactor coupled to bothm-HPLC [39, 55, 56] and CE [13, 30–33, 52] separations.Capillary IMER can be directly coupled on-line with ESI-MS [11, 50, 51] for speedy protein identification or off-linewith MALDI-MS [11, 34, 49, 51]. For high-throughputanalysis it is important that separation and detection iscoupled on-line to proteolysis. This also minimizes therisk of losses during manual handling of the sample. Inmany cases, it is necessary to include a desalting andpreconcentration step, e.g., solid-phase extraction [50].Efficient desalting is especially important when couplingproteolysis to ESI-MS.
Although, less developed, on-line coupling with MALDI-MS can, in principle, be also achieved [26]. For example,Ekström et al. [36] described a device that integrated them-chip IMER with a sample pretreatment robot and amicrofabricated microdispenser to transfer digested pro-tein directly to a MALDI target plate for automated MSanalysis (Fig. 3). These authors used anodic etching in ahydrogen fluoride/ethanol solution to produce a poroussurface on the digestion chip. The use of porous siliconprovided a 170-fold increase in enzymatic activity com-pared to nonporous reactor [57, 58]. This increase in sur-face area resulted in increased digestion efficiency andextremely fast digestions. The m-chip IMER allowed on-line enzymatic digestion of protein samples (1 mL) within1–3 min, about 200–1000 faster than digestion in solution.This integrated system provided a throughput of 100samples in 3.5 h.
The most common strategy for protein analysis is currentlybased on two-dimensional polyacrylamide gel electropho-resis (2-D PAGE). Typically, the identification of a proteinmixture involves separation, extraction, proteolytic diges-tion, and MS analysis of each protein spot. New technologydeveloped by Cooper and Lee [59] involves on-line combi-nation of electrophoretic protein transfer from a polyacryl-amide gel with proteolytic digestion in membrane-basedIMER. After electrokinetic-based protein extraction andstacking, real-time proteolytic cleavage of extracted pro-tein, and direct deposition of protein digest onto the MALDItarget, the peptides were identified by MS analysis (Fig. 4).The sensitivity of the technology was demonstrated bydetection of standard proteins from a gel protein loading aslow as 1 ng as well as by the identification of low-abun-dance proteins in complex yeast cell lysates.
3.3 Immobilized enzyme biosensors
Immobilized enzymes have been used as component ofdetection device in various analytical and biomedicalapplications [19, 20, 60–63] including glucose and peni-cillin. Some of these devices have been associated withminiaturized electrochemical detectors and fluidic sys-tems. L’Hostis et al. [19] tested the enzymatic micro-reactor for glucose detection via glucose oxidase immo-bilized to glass beads. Glucose detection was carried outby electrochemical measurements of hydrogen peroxidegenerated enzymatically with a platinum electrode. IMERwas also coupled with an electrochemiluminiscence(ECL) detector to perform glucose measurements by ECLon carbon electrodes.
Figure 3. Microfluidic systemfor MALDI protein analysis.(A) Automated sample pretreat-ment and injection; (B) m-chipIMER (the photo inset shows aSEM picture of the lamellastructure with the porous layer);(C) microdispenser used to de-posit sample into m-vials; (D)shallow nanovials (3006300620 mm) on the MALDI targetplate; and (E) automatedMALDI-TOF-MS analysis. Re-printed from [36], with permis-sion.
Figure 4. Scheme of combinedprotein electronic transfer andminiaturized trypsin membranedigestion for gel protein identifi-cation using MALDI-MS. (A)Electronic protein transfer fol-lowed by (B) introduction ofextracted proteins into a mem-brane reactor and deposition ofprotein digest onto a MALDI tar-get. Reprinted from [59], withpermission.
4 Immunoassays
Because of extremely high selectivity and sensitivity,immunoassays based on antibody(Ab)-antigen (Ag)interactions, represent one of the most important ana-lytical methods widely used in clinical diagnostics andbiochemical studies [64]. In clinical analysis, a largevariety of immunoassays are used for the identification ofhormones, drugs and other antigens in blood. Tradition-ally, immunoassays are performed in heterogeneous for-mat, where Ab or Ag is immobilized on a surface of, e.g.,a microtiter plate. A sample containing the binding part-ner is incubated on the surface, and after removal of theunbound sample solution the bound fraction is quanti-fied.
Various microfluidic devices reported today in literaturefor Ab or Ag determination were based on either homo-geneous [65–69] or heterogeneous [70–79] immu-noassays. In the former case, the immunoassay com-bined with m-chip based CE was based on the separationof the free form and the complex of Ab and Ag. In the latercase, the Ab or Ag was immobilized either directly on thechannel walls [70–75] or on beads [76–79], which were
then introduced into the microfluidic device. The majoradvantage of heterogeneous assays is the ability to con-centrate molecules on an active surface.
Similar to the previously described applications, the inte-gration of immunoassay-based analytical systems into am-chip should provide enhanced reaction efficiency, sim-plified procedures, and lower consumption of sample andreagents. All immunoassay procedures, includingadsorption of the sample, blocking, Ag-Ab reaction,washing, and detection can be performed in the micro-fluidic device. In the conventional heterogeneous immu-noassay using a microtiter plate, the immunoreactionassay is typically completed after 10–20 h. Figure 5 showsthe comparison of a heterogeneous immunoassay per-formed in the microtiter plate and on a m-chip. Theincreased S/V ratio and reduced diffusion distance on themicrodevice result in much shorter analysis time. Sato etal. [76] have demonstrated that the S/V ratio of the micro-channel was 37 times larger than that of the microtiterplate resulting in the corresponding increase of the reac-tion yield. Integration of heterogeneous immunoassay onthe chip reduced the time necessary for the reaction to 1%of the time needed on the standard microtiter plate.
Figure 5. Schematic illustra-tions of the Ag-Ab reaction: (a)microtiter plate; (b) microchip.Reprinted from [76], with per-mission.
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A variety of different applications of heterogeneousimmunoassays have been described in the past years.For example, bead-bed immunoassay system suitable forsimultaneous assay of multiple samples was constructedon the m-chip [79] and applied to determine interferon(IFNg) (Fig. 6). Polystyrene beads were coated with anti-IFNg Ab and then loaded into the reaction area in themicrofluidic channel. Next, a sample containing IFNg (Ag),the biotinylated anti-IFNg Ab, and the ultrafine colloidalgold particles conjugated with streptavidin were reactedsuccessively. The resulting Ag-Ab complex, fixed on thebeads surface, was quantified using a thermal lensmicroscope after washing out unbound species. Thedetection limit of this method was 0.01 ng/mL. This pro-cedure was used for determination of human secretoryimmunoglobulin A [76] and human carcinoembryonic Ag[78].
Ag or Ab can be also immobilized directly on the micro-channel walls. Dodge et al. [77] introduced a microfluidicchip for an electrokinetically controlled heterogeneousimmunoassay for the determination of rabbit immu-noglobulin G (IgG). PDMS microchannel surfaces havebeen modified with phospholipids for the detection ofanti-dinitrophenyl antibodies [70] or with proteins forsheep immunoglobulin M (IgM) quantification by a het-erogeneous sandwich enzyme-linked immunoassay(ELISA) [71]. Eteshola and Leckband [71] used protein Ato immobilize the capture Ab (rabbit anti-sheep IgM) on
the surface of the microchannels. The scheme of theprotein architecture within the microchannel is shown inFig. 7. Sheep IgM was captured by the primary anti-sheep Ab, and then detected with the secondary Ab,which was labeled with horseradish peroxidase. Thisenzyme catalyzed the conversion of the fluorogenicsubstrate 3-(p-hydroxyphenyl)propionic acid into afluorophore, which was quantified off-chip with a spec-trofluorometer. The measured fluorescence signal wasproportional to the analyte concentration in the testsample.
Figure 7. Scheme of sandwich ELISA assay in a micro-chip. Reprinted from [71], with permission.
Figure 6. Quartz-glass micro-chip for immunosorbent assay:(a) overview; (b) cross section ofthe reaction and detection area;(c) photograph of the microchipand close-up photograph ofbeads in a chip. Reprinted from[79], with permission.
Affinity methods have been employed with great successfor analytical and preparative separations of complexmixtures of biological samples [80–82]. This technique isprimarily intended for isolation and purification of com-plex biological mixtures, it has been shown to be powerfuland predictive for monitoring ligand-protein, substrate-enzyme, inhibitor-enzyme, lectin-sugar, and ligand-receptor interactions. Affinity techniques are adsorptionmethods based on formation of specific, strong, butreversible complex formation between the biologicallyactive molecule, affinity ligand, and substances contain-ing a complementary binding site, target molecule. Forpractical applications, the affinity ligand must have afunctional group for covalent immobilization onto the solidphase and sufficient affinity to the target molecule afterthe immobilization process. Affinity ligands can be immo-bilized on a variety of materials, e.g., beads, monolithicmaterial, or directly on the wall of the capillary or channelof the microfluidic device. When small ligands are immo-bilized on a porous matrix, only a limited number of bind-ing sites contribute to the interaction with the target mol-ecule. One way for better exposition of small ligands onthe porous surface is the introduction of a spacer mole-cule. Another technique is conjugation of the ligand with alarge molecule that acts as a place holder, e.g., a 20 kDapoly(ethylene glycol) molecule during the immobilizationstep [83]. One example of heterogeneous immunoassaysand IMERs (e.g., application of immobilized trypsin forisolation of soybean trypsin inhibitor [48] has already beendescribed in the previous section.
Bioaffinity chromatography is typically performed in threesteps, (i) adsorption of the target molecule to the affinityligand immobilized to a suitable matrix, (ii) washing outthe column to remove excess amounts of salt and otherunwanted analytes, and (iii) desorption of the bound tar-get molecule from the affinity ligand. Desorption is usuallyaccomplished by changing the pH, the salt concentration,or by adding a special modifier to the buffer. Monolithiccapillary columns for affinity chromatography were pre-pared by in situ polymerization using glycidyl methacry-late (GMA) as a monomer and trimethylolpropane tri-methacrylate (TRIM) and ethylene dimethacrylate (EDMA)as cross-linkers, respectively [84]. Protein A was immobi-lized on the poly(GMA-EDMA) and poly(GMA-TRIM)monolith placed in a 100 mm ID fused-silica capillary forpurification of IgG from human serum. The extent of non-specific adsorption was verified by analysis of BSA. Af-finity chromatography experiments were performed in acontinuous adsorption/desorption mode by switching theeluents (Fig. 8). Sample loading was achieved in 20 mM
phosphate buffer containing 0.15 M NaCl at pH 7.2. Theelution buffer was 0.2 M glycine in water with the pHadjusted to 2.3 with HCl solution.
Affinity CE can be viewed as a 2-D analytical technique,where the first dimension is used for the extraction ofselected sample components present in a complex mix-ture. In the next step, CE is used for high-resolution ana-lytical separation of the purified and concentrated affinitytarget material after elution from the affinity device. Thisapproach has been popularized mainly by Guzman et al.[85–88]. In one example, Fab fragments of polyclonal anti-bodies raised against gonadotropin-releasing hormonewere covalently immobilized on glass beads that were heldinside a piece of a fused silica capillary (150 mm610 mm).The beads were retained in place by porous frits created onboth sides of the bead bed with the immobilized Ab. Thisaffinity device was used fordetermination of gonadotropin-releasing hormone in serum and urine [88].
Figure 8. Chromatograms of hIgG and human serum ona protein A-trivinyl-based monolithic column. Experi-mental conditions: (a) standard hIgG solution was injec-ted by applying a pressure of 3 bar for 0.5 min; (b) fivefold-diluted human serum was injected by applying a pressureof 3 bar for 1 min. After injection, the column was flushedwith loading buffer for 2.5 min by applying a pressure of10 bar, then flushed with elution buffer by applying apressure of 10 bar. Detection wavelength was at 239 nm.Peak identification: 1, nonretained solutes; 2 hIgG. Re-printed from [84], with permission.
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For the analysis of the very complex samples, e.g., in pro-teomics, a multidimensional separation is almost alwaysrequired for identification and quantification. Slentz et al.[89] integrated trypsin digestion, copper(II)-immobilizedmetal affinity chromatography (IMAC) selection of histi-dine-containing tryptic peptides, and reversed-phaseCEC on the same PDMS microdevice (Fig. 9). Proteolyticdigestion and affinity chromatography were achieved byusing 5 mm particle-based columns with a microfabricatedfrit. The analytes were transported through the trypsindigestion bed and eluted directly into the Cu(II)-IMAC bed.In the next step, histidine-containing peptides were elutedfrom the IMAC particles with EDTA, separated by CEC anddetected fluorescently. On-chip analysis was tested usingfluorescence detection of the fluorescein isothiocyanate-labeled BSA (Fig. 10). This model system has demon-strated that in spite of technical difficulties a three-dimen-sional chromatography on a chip combining trypsindigestion, affinity selection, and reversed-phase separa-tion is possible.
Figure 9. Microchip design for sample injection (point x),trypsin digestion, IMAC selection of histidine-containingpeptides and CEC; inset shows a micrograph of themicrofabricated frit used to retain trypsin or IMAC parti-cles. Reprinted from [89], with permission.
6 Affinity interactions in MS
While coupling of IMER with MS is typically performed inthe on-line regime – see Section 3.2 – the affinity interac-tions are frequently employed off-line in conjunction withMALDI-TOF-MS. This approach, where the affinity selectoris attached directly to the MALDI sample desorption plate,is frequently termed surface enhanced laser desorption/ionization-time of flight-MS (SELDI-TOF-MS) and wasintroduced and popularized by Hutchens and Yip [90]. Adedicated MS instrument and protein chips aimed at thebiomarker discovery market are already commerciallyavailable – http://www.ciphergen.com/. The principle of
Figure 10. On-chip separation of FITC-BSA digest (a)before and (b) after Cu(II)-IMAC selection. Separationconditions: 1 mM phosphate buffer (pH 7.0), 500 V/m.Reprinted from [89], with permission.
the method is very simple and although SELDI provides aunique sample preparation platform, its operation isidentical to standard MALDI-MS instrumentation with theaffinity-captured material being cocrystallized with a suit-able matrix and laser-desorbed inside the linear TOFmass analyzer. SELDI combines high specificity of affinityinteractions with the high sensitivity and high informationcontent of the MS analysis. In this combination, the affin-ity method serves as a purification and preconcentrationstep. The affinity interaction can utilize, e.g., Ag-Ab inter-action, lectin-sugar interaction, avidin-biotin interaction,and/or interaction between immobilized metal ion andpeptides and proteins containing adjacent histidine resi-dues. This technique has already been demonstrated forthe analysis of proteins from a variety of complex biologi-cal materials, e.g., serum, plasma, blood, tears, cere-brospinal fluid, and cell lysates. SELDI was also used fordiagnosis of diseases such as carcinomas [91] and Alz-heimer’s disease [92].
The initial experiments with attached affinity ligands onbeads for direct MALDI desorption were described underthe term surface-enhanced affinity capture (SEAC) [93–95]. Alternatively, affinity ligands can be immobilized on theMALDI probe via a gold foil [96] or a nitrocellulose film [97].Several groups have also illustrated the combination ofnon-covalent affinity interactions with MALDI-MS analysisfor sample purification, e.g., Hutchens and Yip [90] usedagarose beads with attached single-stranded DNA tocapture lactoferrin from preterm infant urine. After affinityisolation of the analyte of interest and removing nonbind-ing components by washing with binding buffer, the beadswere placed on the MALDI probe tip and incorporated intothe matrix. The acidity and organic nature of the matrix aresufficient to release the affinity-bound analyte from the af-finity beads. The samples were analyzed using conven-tional MALDI-MS procedures. The history and recentadvances in SELDI-TOF-MS technology were recentlyreviewed by Merchant and Weinberger [98].
Because the matrix is applied directly to the chip, thespots on the chip cannot be reused after laser desorp-tion. Additionally, the results of SELDI analysis are typi-cally only qualitative. The possibility of quantitativeanalysis has been demonstrated by combining biomo-lecular interaction analysis with MS (BIA-MS) [99–104].In this case, the affinity ligands (receptor, antibodies,nucleic acid, etc.) were first immobilized on a gold-coated glass sensor. The affinity interaction betweensurface-immobilized ligands and analytes in solutionwas monitored and quantified in real time by surfaceplasmon resonance (SPR) [105, 106]. SPR is a label-freequantification method that utilizes an interaction of lightphotons with free electrons on a gold surface to quantifythe changes in the amount of biomaterial on the surface.SPR detection is nondestructive; therefore, MS and/orMS/MS can further analyze the affinity-bound biomole-cules. Each technology brings a unique dimension toBIA-MS analysis, SPR sensing is employed for proteinquantification, and MS is utilized to qualitative analysisof biomolecules. Because the sensor chip of BIA is con-structed as microfluidic device, it can be combined notonly with MALDI but also with ESI-MS. The major ad-vantage of combining BIA with ESI-MS is the directtransfer of the sample from the BIA device to the ionsource of the mass spectrometer. For identificationusing database searching (http://www.isb-sib.ch/), theproteins bound to the sensor chip must be digested afterthe elution prior to MS analysis. This process could leadto critical sample losses. Thus, the digestion was carriedout on-line in the microfluidic device. After on-chipdigestion, the resulting peptide mixture was trapped andconcentrated in a capillary pre-column by an on-linerecovery technique for subsequent MS/MS analysis
[102]. This technology allowed real-time monitoring ofprotein interactions and the simultaneous identificationof components by MS.
In conclusion, the technologies and applications re-viewed in this text document the growing interest in theintegration of immobilized enzymes into microfluidicsystems. The commercial viability of the technologiescurrently under development will depend on many fac-tors, frequently unrelated to the underlying science;however, one can already find elements of IMER tech-nology in commercial products in a number of compa-nies – see, e.g., www.caliper.com, www.diagnoswiss.com, www.gyros.com, www.micronics.net, www.sequenom.com, www.zeptosens.com to name just a few.
The authors wish to thank Dr. Zuzana Bilkova from theUniversity in Pardubice for help with the preparation ofthis review. This work was supported by the Grant Agencyof the Czech Republic under contract 203/03/0515 andthe Grant agency of the Czech Academy of Sciencesunder contract S4031209.
Received July 26, 2004
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