BY-NC-ND 3.0 license Open Access Published by De Gruyter June 24, 2016

Capillary electrophoresis-integrated immobilized enzyme reactors

Xiaoxia Liu, Jiqing Yang and Li Yang

Abstract

Online enzyme assay based on capillary electrophoresis (CE) offers several advantages for the assay, such as low consumption of samples, easy automation of all steps, and less requirement of sample work-up. As a widely used approach for online enzyme assay, CE-integrated immobilized enzyme microreactor (IMER) has been applied in almost all aspects of enzyme assays during the past two decades, including evaluation of the enzymatic activity and kinetics, screening of inhibitor, investigation of enzyme-mediated metabolic pathways, and proteome analysis. In a CE-integrated IMER, enzyme is bound to the capillary surface or a suitable carrier attached to the capillary and substrates/products of the enzymatic reaction are separated and online detected by CE at downstream of the capillary. Enzymatic reactions can be viewed as interaction between the stationary phase (immobilized enzyme) and the mobile phase (substrate(s)/co-enzyme(s) solution), in analogy to the well-known separation technique, capillary electrochromatography. From this point of view, CE-integrated IMERs can be categorized into open tubular capillary IMER, monolithic IMER, and packed capillary IMER. In this review, we have surveyed, analyzed, and discussed advances on fabrication techniques of the three categories of CE-integrated IMERs for online assays involving various enzymes in the past two decades (1992–2015). Some recent studies using microfluidic-based IMERs for enzyme assays have also been reviewed.

Introduction

The metabolic pathways that occur in a cell to maintain life’s activities involve catalysis by particular enzymes. Any malfunction, including mutation, overproduction, underproduction, or deletion, of a single critical enzyme can lead to a severe human disease. In addition, enzymes are important targets in research and development of drugs and play a role in clinical diagnosis of diseases. A rapid, accurate, and efficient method to determine the activity, kinetics, and inhibition of enzymes is therefore of significant importance for understanding the mechanism of enzyme-catalyzed reactions in cell, for drug development, and for treatment of human diseases.

Since Banke et al. (1991) first used it for monitoring alkaline protease activity, capillary electrophoresis (CE) has become a powerful approach for quantitative studies of enzymatic reactions. Among a variety of separation techniques, CE offers several advantages such as high separation efficiency and sensitivity, extremely small sample requirement, rapid analysis, and utilization of various detection modes. During the past 20 years, CE has been widely applied in almost all aspects of enzyme assays, including the evaluation of the enzymatic activity and kinetics (Glatz 2006, Krizek and Kubickova 2012), screening of inhibitors (Liu et al. 2015b), investigation of enzyme-mediated metabolic pathways (Nowak et al. 2013, Nehme and Morin 2015), and proteome analysis (Ma et al. 2009, 2011b, Safdar et al. 2014), etc. Generally, enzyme assays based on CE can be classified into two categories, off-line assay and online assay, with respect to where the IMER is placed/constructed relative to the CE separation capillary. Early studies of CE-integrated enzyme assay were performed off-line, in which the enzyme-catalyzed reaction was carried out in a separate vial before injecting into the CE separation capillary. The capillary was used only for the separation of the substrate(s) and the product(s). The main advantage of the off-line approach is that the conditions for enzyme reaction and CE separation/detection can be optimized separately. There are many reports in the literature using this approach for identification of enzymes, evaluation of the activity of an enzyme towards substrates, investigation of the proteasome, enzyme kinetics and inhibitor screening, and so on.

Perhaps the main disadvantage of off-line enzyme assay is plenty of sample work-up, large amount of enzyme and chemicals, as well as difficulty in automation and miniaturization of the analytical system. Online enzyme assay based on CE, on the other hand, becomes more popular in the recent years. In an online enzyme assay, all the necessary steps for analysis, including initiating and terminating the enzyme reaction, sample injection and CE separation, and detection of the substrate(s) and/or product(s), can be performed automatically either pre-capillary or in one capillary. In this case, CE is applied not only as a separation tool with high performance but also as a versatile platform for enzyme studies. Online enzyme assay shows many advantages, such as low consumption of samples, easily automation of all steps and less requirement of sample work-up, thus will be useful for automation and miniaturization of the enzyme analysis system.

Two approaches are generally applied for online assay on the basis of the status of the enzyme used in the analysis. In one approach, which is referred as homogeneous enzyme assays, all reactants are presented in solution. Usually, this method is accomplished by utilizing different electrophoretic mobilities of enzyme and substrate to initiate the reaction inside the capillary and to separate the components of the reaction mixture for CE quantification, thus is named as Electrophoretically Mediated Microanalysis, EMMA (Bao and Regnier 1992). We refer to a series of reviews by Schepdael and co-workers, which give an overview of recent developments in EMMA (Zhang et al. 2008, Wang et al. 2014). Additionally, free enzyme in solution is also used in the sequential CE assays. Our group has developed an automatic sequential injection method recently (Chen et al. 2012), which allows us to monitor the enzyme activity (and inhibition) from the beginning to the end of the reaction (Fu et al. 2014, Liu et al. 2015c).

The other online approach, which is the subject of this review, is heterogeneous enzyme assay where the enzyme is immobilized on solid supports, such as the capillary inner surface or a suitable carrier attached to the capillary. This set-up for enzyme assay is also named an immobilized enzyme reactor (IMER). After injected into capillary, reactant (substrate(s), co-enzyme) flow first passes through the part of the capillary containing an IMER to initiate enzyme reaction. The resulting product(s) and remaining reactants are electrophoretically separated in the downstream of the capillary for quantitative determination of the enzyme activity. Various methods have been used for enzyme immobilization, including physical adsorption, covalent attachment, cross-linking and encapsulation (see, e.g. Iqbal et al. 2013 and references therein). Upon immobilization, stability and repeatable usability of enzyme can be improved, while experimental cost, sample manipulation and analysis time can be greatly reduced. It is worth noting that CE-integrated IMERs mentioned here is different from capillary-sized IMERs coupled with any type of analysis/separation technique (HPLC, MS). In the latter case, enzymes are immobilized in a capillary, after injecting the reactant into capillary to initiate enzyme reaction, the resulting product(s) and remaining reactants are collected and separated/detected by HPLC/CE/MS, thus such assay is viewed as an off-line approach in this review.

During the past decade, the application of CE-integrated IMER has been attracted increasing research interest in chemical and biological enzyme assays. There are several celebrated reviews reported in the literature to overview CE-integrated IMER. For example, Iqbal et al. (2013) have published a review that is dedicated to IMERs in CE and microchip and their applications in biochemical research. Several other reviews have been published on advances in a broad aspect of CE-integrated enzyme assays, including IMER and other online assays as well as off-line assays (Glatz 2006, Fan and Scriba 2010, Wang et al. 2014, Nehme and Morin 2015, Scriba and Belal 2015). The use of IMERs in proteomics (Ma et al. 2009, 2011b, Safdar et al. 2014), pharmaceutics (Spross and Sinz 2009), and inhibitor screening (Liu et al. 2015b) has also been reviewed. In this review, we focus on the fabrication techniques of the CE-integrated IMERs, which we categorized into open tubular capillary IMER, monolithic IMER and packed capillary IMER, as described below.

In an IMER, enzymatic reactions can be viewed as interaction between the stationary phase (immobilized enzyme) and the mobile phase (substrate(s)/co-enzyme(s)). Preparation of stationary phase in a capillary for capillary electrochromatography (CEC) is mainly achieved by three formats (Mu et al. 2015, Xue et al. 2015), i.e. open tubular capillary, monolith, and packed capillary. Accordingly, fabrication of CE-integrated IMERs is proposed via three different categories: (i) immobilizing enzymes on the surface of a capillary leading to an open tubular capillary IMER; (ii) immobilizing enzyme on continuous beds (monoliths), which are formed in situ in the capillary (monolithic IMER); (iii) immobilizing enzyme on magnetic beads, particles, or membranes that are entrapped in a capillary or placed in a defined area of the capillary network to form a packed capillary IMER. From this point of view, we did a comprehensive overview of advances in CE-integrated IMERs from 1992 to 2015. The advantage and disadvantage of the three categories of CE-integrated IMERs are summarized in Table 1 and will be explained in detail in the review. Table 2 summarizes recent studies using different categories of IMERs for various online enzyme assays based on CE.

Table 1:

Advantage and disadvantages of the three categories of CE-integrated IMERs.

Type of IMERSectional view of capillary IMERAdvantageDisadvantge
Open tubular capillary IMER
Simple, mild condition, easy to carry outLimited enzyme loading
Monolithic IMER
High enzyme loading, better stability, Various matrixTime consuming
Packed capillary IMER
High enzyme loading, robust; easily loaded and removed for magnetic beadsFrit is difficult to prepare,a poor reproducebility

aNo frit is needed with magnetic beads.

Table 2:

Summary of studies using OT-IMER, monolithic IMER, and packed capillary IMER for on-line enzyme assays based CE.

EnzymeImmobilized technique/materialsDetectionApplicationRef.
OT-IMER
 ACEIonic binding/HDBUV 230 nmInhibitor screening(Tang and Kang 2006)
 G6PDHElectrostatic assembly/PDDAUV 340 nmEnzyme activity and inhibitors studies(Camara et al. 2015)
 DAAOIonic binding/HDBUV 214 nmChiral seperation(Qi et al. 2009)
 GOxElectrostatic assembly/PDDAAmperometricSubstrate determination(Hooper and Anderson 2007)
 NTPDase2Electrostatic assembly/HDBUV 260 nmEnzyme kinetic data and inhibition studies(Iqbal et al. 2010)
 APIonic binding /polybreneUV 322 nmInhibition studies, Ki determination(Iqbal 2011)
 AchELayer by layer electrostatic assembly /PDDAUV 230 nmInhibitor screening(Tang et al. 2007)
 Adenosine deaminaseEncapsulation and ionic binding/PEIDAD 254 nmInhibitor screening in natural extracts(Ji et al. 2010)
 Ribonuclease TCross-linking/GAUV 258 nmNucleic acids analysis(Nashabeth and Rassi 1992)
 β-NAD+, ADH and LDHCross-linking/GAUV 340 nmQuantitative determination(Simonet et al. 2004)
 GDH, GPTCross-linking/GAUV 340 nmSubstrate determination(Yang et al. 2009)
 Adenosine deaminaseCross-linking/GAUV 254 nmEnzyme kinetic data(Pei et al. 2011)
 GOxCross-linking/GAUV 220 nmEnzymolysis and enzyme inhibition assay(Wang et al. 2012)
 TrypsinCross-linking/GAUV 214 nmInhibitor screening(Min et al. 2013)
 NeuraminidaseCross-linking/GAUV 320 nmScreening inhibitor from traditional Chinese medicines(Zhao and Chen 2014)
 α-GlucosidaseCross-linking/GADAD 405 nmEnzyme kinetics and inhibition assays(Liu et al. 2015a)
 GOxCross-linking/PAMAM dendrimerUV 200 nmEnzyme kinetic data(Wang et al. 2010a)
 PepsinBiotin-avidin-biotin couplingUV 214 nmInhibitor screening(Licklider and Kuhr 1994)
 TrypsinBiotin-avidin-biotin couplingMSPeptide mapping(Licklider et al. 1995)
 APStreptavidin-biotin couplingUV 405 nmEnzymatic and inhibition assays(Rashkovetsky et al. 1997)
 TrypsinCovalent/GNPs or cross-linking/EDC-NHSMSProtein digestion(Safdar et al. 2013)
 L-asparaginaseCovalent/GNPsLIFEnzyme kinetic data(Qiao et al. 2013)
 HRPCovalently bound on an aldehyde-activated membraneChemiluminescentQuantitative detection(Xie et al. 2013)
Monolithic IMER
 TrypsinSol-gel and electrostatic attraction/TMOS, PDDAMALDI-TOFMSPeptide mapping(Xu et al. 2008)
 TrypsinMetal-ion chelating immobilizationMALDI-TOF-MSEnzyme kinetic data, peptide mapping(Guo et al. 2003)
 TrypsinCovalently linked to a photo polymerization capillaryUV 214 nmPeptide mapping(Bossi et al. 2004)
 α-GlucosidaseCovalent binding to GNPs modified polymer monolithUV 280 nmScreening inhibitors from natural products(Zhang et al. 2013)
 TrypsinImmobilized on GMA/EDMA macroporous columnLIFPeptide mapping(Ye et al. 2004)
 TrypsinCovalently linked to a poly(ethylene glycol)monolith by photopolymerizedUV 214 nmProtein digestion(Dulay et al. 2005)
 PepsinImmobilized on GM A/EDMA macroporous columnMSProtein digestion and peptide analysis(Schoenherr et al. 2007)
 TrypsinCovalently bound to hybrid silica monolithUV 214 nmProtein analysis(Wang et al. 2010b)
 TrypsinEncapsulated in tetramethoxysilane-based hydrogelDAD 214 nmEnzyme kinetic data(Sakai-Kato et al. 2002b)
 UDPGTEncapsulated in TMOS-based silica matricesDAD 340 nmDrug-metabolism(Sakai-Kato et al. 2002a)
 APImmobilized on a macroporous polymer monolithESI-MSPhosphopeptide analysis(Mou et al. 2013)
 L-asparaginaseCovalently bound to poly (GMA-co-EDMA) monolithLIFChiral separation of amino acids, enzyme activity(Qiao et al. 2011)
 PepsinEmbedding on the photopolymerized sol-gel column as a thin filmMSProtein digestion, peptide separation and identification(Kato et al. 2004)
Packed capillary IMERs
 TrypsinSingle particle as a frit to pack enzyme immobilized particlesUV 214 nmInhibition assay and protein hydrolysis(Liu et al. 2014)
 HRPImmobilized onto GA-activated magenetic particlesUV 245 nmDrug biotransformation(Yu et al. 2010)
 APImmobilized onto magenetic beads via EDC activiationMSHosphopeptide characterization(Wojcik et al. 2010)
 TrypsinImmobilized onto magenetic beads via EDC activiationESI-MSProtein digestion(Li et al. 2011b)
 APImmobilized to polystyrene magenetic beadsLIFInhibition assay(Yan and Gilman 2010)
 ADH and LDHImmobilized to magenetic beads via EDC activiationUV 340 nmQuantitative detection(Shi et al. 2012)

β-NAD+, β-nicotinamide adenine dinucleotide; ACE, angiotensin-converting enzyme; AChE, acetylcholinesterase; ADH, alcohol dehydrogenase; AP, alkaline phosphatases; DAAO, D-amino acid oxidase; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; EDMA, ethylene dimethacrylate; G6PDH, glucose-6-phosphate dehydrogenase; GDH, glutamate dehydrogenase; GMA, glycidyl methacrylate; GOx, glucose oxidase; HRP, horseradish peroxidase; GPT, glutamic pyruvic transaminase; LDH, lactate dehydrogenase; NTPDase2, nucleoside triphosphate diphosphohydrolase 2; UDPGT, UDP-glucuronyltransferase.

Open tubular capillary IMER

For any CE-integrated IMER, enzymes as the stationary phase are deposited on the inner wall of a capillary and substrate(s) and co-enzyme(s) as the mobile phase are driven by EOF. After injection of the sample into the capillary, the stationary-mobile phase interaction initiates the enzyme reaction, followed by CE separation and detection in the same capillary. Because of the high surface-to-volume ratio of capillary columns, enzyme concentration can be high enough for performing the enzymatic reaction, despite that enzymes are only immobilized on the inner surface of the capillary. On the other hand, to fabricate CE-integrated IMERs for efficient online assays, enzymes are required to be efficiently immobilized without loss of the activity, and IMERs should be stable for repeatable usages.

Open tubular capillary IMER (OT-IMER) is usually located at the capillary end, and the rest of the capillary is used for CE separation and detection. OT-IMER has advantages of easy preparation and simple instrumental handling. Similar to the OTCEC separation technique, small internal diameters of the capillary are required in OT-IMER to facilitate efficient solute diffusion to the stationary phase surface. This leads to relatively poor phase ratio and limited loading amount of enzyme in an OT-IMER. Thus, for online enzyme assays using OT-IMER, in addition to developing efficient methods to improve the stability of the microreactor, efforts have also been made recently to find a strategy to increase the surface area and to increase the interactions between immobilized enzyme and substrate and therefore to enhance response of OT-IMER.

A majority of the studies reported fabrication of OT-IMERs using ionic binding, covalent binding, cross-linking, or bioaffinity coupling methods. Additionally, modern nanometer-size material has been applied as the support for immobilization of enzyme in order to increase the interaction between the stationary and mobile phases. These advances will be described and discussed in detail in the following.

Tang and Kang (2006) reported an strategy using OT-IMER for screening the enzyme inhibitors from the complex mixtures by CE. The OT-IMER was easily fabricated by ionic binding technique, as shown in Figure 1. The capillary wall was first flushed with polycationic electrolyte hexadimethrine bromide (HDB) solution to obtain positively charged coating of the inner surface. Subsequently, a plug of the enzyme solution (in 10 mmpH 8.0 borate-phosphate buffer) was injected and incubated for 5 min to allow the enzyme to immobilize on the capillary wall via ionic binding. This resulted in a 1.5-cm-long IMER at the inlet of the capillary (50-μm i.d.). The fabricated OT-IMER was successfully applied in screening angiotensin-converting enzyme (ACE) inhibitors. Because of its simple fabrication procedure and the regenerable character, ionic binding method has been widely used in preparation of OT-IMERs for online various enzyme assay and inhibition study (Hooper and Anderson 2007, Qi et al. 2009, Iqbal et al. 2010, Iqbal 2011, Jiang et al. 2013, Camara et al. 2015).

Figure 1: Schematic representation of the on-column immobilized enzyme microreactor prepared by the ionic binding method.This figure was reproduced with permission from Tang and Kang (2006). Copyright © 2006 American Chemical Society.

Figure 1:

Schematic representation of the on-column immobilized enzyme microreactor prepared by the ionic binding method.

This figure was reproduced with permission from Tang and Kang (2006). Copyright © 2006 American Chemical Society.

Because ionic binding of enzyme to capillary surface is weak, the stability of OT-IMER is a concern in online enzyme assay studies. Kang’s group developed a layer-by-layer (LBL) assembly based on ionic binding method to improve the stability of the OT-IMER (Tang et al. 2007). The acetylcholinesterase (AChE) was analyzed in the study. By employing polyelectrolyte polydiallyldimethylammonium (PDDA) for the positively charged coating, the authors prepared an OT-IMER with PDDA-AChE-PDDA sandwich-like structure (0.5-cm long, 50-μm i.d.). With the LBL assembly, the OT-IMER could withstand 100 consecutive assays by only losing 10% activity of the enzyme. The time-to-time, day-to-day, and batch-to-batch reproducibility was measured with RSD% <4.7%. Screening a small compound library containing four known AChE inhibitors and 42 natural extracts was demonstrated, and species with inhibition activity can be straightforwardly identified with the system.

More recently, our group employed the similar ionic binding technique using PDDA coating for fabrication of OT-IMERs (2-cm long, 50-μm i.d.) based on the glucose-6-phosphate dehydrogenase (G6PDH) enzyme (Camara et al. 2015). We further demonstrate that the ionic binding technique is an easy-to-operate and efficient method for fabricating OT-IMERs and coated PDDA is stable enough not only to withstand the flush with running buffer but also to achieve reliable and accurate enzyme immobilization for further enzymatic activity and inhibition studies. In addition, using the electrostatic LBL assembly, it is easy to load multi-layers of enzymes in the capillary, which can enhance the response of OT-IMERs (Camara et al. 2016).

An interesting approach to improve the stability of OT-IMERs via ionic binding technique was performed by Ji et al. (2010), in which enzyme was encapsulated in calcium alginate gel. Adenosine deaminase (ADA) was encapsulated in alginate and then immobilized on the surface of capillary (50-μm i.d.) inlet via the ionic binding approach with polyethylenimine (PEI), resulting in a 1-cm-long IMER. When polyanion alginate, enzyme, and polycation PEI were mixed, polyelectrolyte complexes were formed based on ionic interactions. CaCl2 solution was injected into the capillary successively to form the “egg-box” structure hydrogel, which has a three-dimensional net structure, leading to the reduction of the enzyme leaching and improvement of the stability. Figure 2 is a schematic representation of the immobilized enzyme microreactor profile. Although there was a little decrease in enzyme activity in the presence of alginate, the stability of the OT-IMER was reinforced. Because of the good biocompatibility of alginate, the method could be used to fabricate IMERs for many kinds of enzyme encapsulated in the hydrogel. The method makes it possible to immobilize several enzymes simultaneously.

Figure 2: Schematic representation of the immobilized capillary enzyme microreactor. Enzyme was encapsulated in alginate and then immobilized on the surface of capillary inlet via the ionic binding.This image was reprinted from Ji et al. (2010) with permission from Elsevier.

Figure 2:

Schematic representation of the immobilized capillary enzyme microreactor. Enzyme was encapsulated in alginate and then immobilized on the surface of capillary inlet via the ionic binding.

This image was reprinted from Ji et al. (2010) with permission from Elsevier.

In addition to ionic binding, enzyme can also be immobilized in OT-IMERs via covalent binding or cross-linking (Nashabeth and Rassi 1992, Simonet et al. 2004, Yang et al. 2009, Pei et al. 2011, Wang et al. 2012, Min et al. 2013, Zhao and Chen 2014, Liu et al. 2015a). Such type of enzyme immobilization results in a quite stable IMER. On the other hand, active site inactivation may occur during the immobilization procedure, and the capillary could not be recycled if the activity of immobilized enzyme decreases. By covalently attaching the enzyme to the capillary via silanization of the surface with 3-aminopropyltriethoxysilane and its subsequent reaction with a cross-linker reagent, glutaraldehyde, Zhao and Chen (2014) reported an OT-IMER for online assay of neuraminidase (NA). The immobilized NA microreactor (1-cm long, 50-μm i.d.) could be continuously used for more than 200 runs, and the enzyme activity can remain at 90% after 30 days of storage at 4°C, indicating good stability of the IMER by covalent binding. The preparation protocol is shown in Figure 3.

Figure 3: Procedure for immobilizing NA on capillary inner wall (A) and schematic diagram of on-line enzyme assay (B).This image was reprinted from Zhao and Chen (2014) with permission from Elsevier.

Figure 3:

Procedure for immobilizing NA on capillary inner wall (A) and schematic diagram of on-line enzyme assay (B).

This image was reprinted from Zhao and Chen (2014) with permission from Elsevier.

Another example is fabrication of a dual-enzyme co-immobilized OT-IMER using covalent attachment on the silanized capillary surface (Yang et al. 2009). The coupled two enzymatic reactions catalyzed by glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT), respectively, were investigated using the dual-enzyme OT-IMER (30-cm long, 50-μm i.d.). Due to substrate recycling in the coupled enzymatic reactions, the response of online assay is enhanced, leading to a 15.7-fold improvement in sensitivity for substrate glutamate determination. As a result, such dual-enzyme IMER was applied for accurate determination of trace amount of glutamate content in real complex samples, such as rat plasma and serum (Yang et al. 2009).

A new type of glucose oxidase (GOx) IMER was developed based on covalent bonding using polyamidoamine dendrimer (PAMAM)-grafted fused-silica capillaries (Wang et al. 2010a). PAMAM is one of a number of dendritic polymers with precise molecular structure, highly geometric symmetry, and a large number of terminal groups and can be grafted onto silica with microwave assisted protocol (Zhang et al. 2010). GOx was covalently attached to the inner capillary wall (40-cm long, 75-μm i.d.) by development of secondary amine linkages between PAMAM and enzyme. The study indicated that the introduction of PAMAM can enable larger amounts of GOx to be immobilized, markedly improving enzymolysis efficiency with increasing PAMAM generations (Wang et al. 2010a).

Most OT-IMERs were fabricated by coating the enzyme from the inlet end of a capillary, and all the injections were performed from the inlet for online assay. Alternatively, Iqbal et al. (2010) showed that the fabrication procedure of IMER and the online assay can also be performed from the outlet of the capillary. The at-capillary-outlet IMER (1-cm long) was produced by ionic binding positively charged HDB (coated at the outlet of the capillary) to negatively charged phospholipids in the enzyme-containing cell membrane shreds. Enzyme activity of human ectonucleoside triphosphate diphosphohydrolase-2 and its inhibition were monitored. It is indicated that by coupling the OT-IMER with the short-end separation mode, the resulting analysis time was reduced by almost half as compared with the previously applied long-end separation mode.

Because of their unique physical and chemical properties, nanoparticles have attracted increasing research interest in chemical analysis based on CE (see, e.g. Nilsson et al. 2011, Hu et al. 2014). It is demonstrated that separation efficiency is enhanced by using modified nanoparticles coated on a prederivatized fused-silica capillary as stationary phase, which can be attributed to their high surface area and surface-to-volume ratio (Li et al. 2011a, Liu et al. 2013). Such advantages have recently been applied in fabrication of OT-IMERs. Enzyme can be either directly adsorbed on the surface of nanoparticles (i.e. enzyme-modified nanoparticles) prior to attachment to the capillary or can be immobilized to nanoparticle-modified capillary surface. In a recent study, Safdar et al. (2013) showed that the trypsin-immobilized gold-nanoparticle (GNP) microreactors prepared by the two different strategies (see Figure 4) produced comparable results for online assay of trypsin digestion reaction, and the protein digestion was ~150 times faster than in-solution digestion.

Figure 4: (A) Preparation of GNP microreactor with adsorbed trypsin and (B) preparation of GNP microreactor with trypsin immobilized via PEG spacer.This figure was reproduced with permission from Safdar et al. (2013). Copyright © 2013 Wiley.

Figure 4:

(A) Preparation of GNP microreactor with adsorbed trypsin and (B) preparation of GNP microreactor with trypsin immobilized via PEG spacer.

This figure was reproduced with permission from Safdar et al. (2013). Copyright © 2013 Wiley.

GNPs were applied as the support for preparation of OT-IMERs in several recent studies (Zhao et al. 2011, Safdar et al. 2013). Because the thiol groups (-SH) in protein molecules can strongly bind to GNPs, resulting in stable protein-GNP conjugates (Thobhani et al. 2010), the fabricated OT-IMERs thus present high stability, as demonstrated in these studies. The capillary was pretreated with PEI (Zhao et al. 2011, Lin et al. 2013) or (3-mercaptopropyl)-trimethoxysilane (MPTMS) (Qiao et al. 2013) to produce positively charged or -SH group contained in the surface for binding of GNPs or enzyme-modified GNPs. Online enzyme assays for several enzymes, such as L-glutamic dehydrogenase and L-asparaginase enzymes, were studied. These studies proved the efficient method for OT-IMERs using GNPs as the support for high-throughput enzyme assay and inhibition screening.

Graphene oxide (GO) was used as the enzyme support for fabrication of OT-IMERs in our study (Yin et al. 2014). GO is a sheet-based material, which has several advantages such as ease of synthesis, large surface area to mass ratio, and surface functionalities for induced-fit interactions for enzyme binding. Particularly, enriched with oxygen-containing groups, it is possible for GO sheet to immobilize enzymes without any surface modification or any coupling reagents. In our study, we reported the first CE-integrated OT-IMER using GO as an enzyme immobilization support, which was easily fabricated based on LBL electrostatic assembly. The 25-μm-i.d. capillary was first dynamically coated by a 2-cm-long plug of PDDA solution to form positively charged surface, followed by ionic binding of negatively charged GO. Because the pI value of trypsin is about 10.5, trypsin should be positively charged in buffer (pH 8.5) and can be absorbed on the negative-charged GO layer by electrostatic assembly coating. Multi-layer enzyme loading is achieved by repeating the procedure. Online trypsin digestion of BSA using the CE-integrated OT-IMER is comparable with those obtained using free trypsin digestion for 12-h incubation, indicating that immobilization strategy using GO as the enzyme support is reliable and practicable for accurate online enzyme assay and analysis and characterization of peptides and proteins.

Monolithic IMER

Since the beginning of this century, there has been increasing interest in the application of monolith as the support for enzyme immobilization due to their unique properties such as better accessibility of the active site for substrates, stability in most solvents, and versatility of functional group available on the pore surface of monolithic columns. Recently, Vlakh and Tennikova reviewed flow-through immobilized enzyme reactors based on monoliths, including preparation of heterogeneous biocatalysis (Vlakh and Tennikova 2013a) and kinetics study and application (Vlakh and Tennikova 2013b). In addition, application of monoliths in proteomics has been reviewed by several papers (Svec 2006, Ma et al. 2007, Krenkova and Svec 2009, Spross and Sinz 2009, Safdar et al. 2014). In the following, we will mainly overview advances in monolithic IMERs based on CE for online enzyme assays.

Compared to OT-IMERs, monolithic IMERs present better stability but are more complicated in fabrication procedure. Inorganic materials such as sol-gels, organic polymers based on acrylates or acrylamides, and organic-inorganic hybrid materials can be used as the support for preparation of monolithic IMERs. The properties of the support, such as pore size (Bayne et al. 2013), porosity (Magner 2013), and surface chemistry may dramatically affect the characteristics of immobilized enzyme (Kim et al. 2006, Vlakh and Tennikova 2013a). Therefore, the selection of appropriate solid matrix and the preparation of monolithic column, as well as the enzyme immobilization procedure, play a key role and define the activity and applicability of the resulting IMER (Hartmann and Kostrov 2013). In the past two decades, monolithic stationary phases have attracted increased interest in the separation science research community. There are extensive studies regarding the selection of solid matrix as well as the preparation of monolithic columns (see recent review articles, Jonnada et al. 2015, Nischang and Causon 2016, and references therein).

In practice, the methods that are applied to prepare monolithic columns for chromatographic separation can be extended to fabrication of monolithic IMERs for online enzyme assay based on CE. For monolithic IMERs, experimental conditions that affect the formation of network, such as pH, temperature, reagent concentration, reaction time, the rate of hydrolysis, and condensation, can also affect the online CE enzyme assay, thus these should be investigated to optimize the performances of both the column and the assay. Sol-gel encapsulation, which is the hydrolysis of alkoxysilane to form SiO2 network, has been widely used in monolithic IMERs (Sakai-Kato et al. 2002a,b, Kato et al. 2004, Dulay et al. 2005, Schoenherr et al. 2007, Cumana et al. 2014, Hong et al. 2014). The polymerization reaction can be conducted thermally or using a UV-radiation source. The most common monomers used in monolithic IMERs are tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS) because they can be readily hydrolyzed and condensed under mild conditions. The sol-gel reaction usually needs 2–3 days to finish, and the capillary needs to be pretreated with silane reagent to prevent the gel from leaking out of the capillary. Large molecules such as proteins could not be analyzed using the sol-gel monolithic IMERs because of the nano-pores of the hydrogel. Macroporous monolithic columns were reported in several studies for online protein digestion and peptide mapping. Continuous rods of macroporous poly(glycidyl methacrylateco- ethylene dimethacrylate) have high hydrophilicity and easy modification (Frantisek and Huber 2006). The macroporous monolithic IMERs were fabricated by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary (Ye et al. 2004).

There are two approaches to immobilize enzymes in monolithic IMERs (see Figures 5 and 6). In one approach, enzymes were immobilized off-line and reacted to the hydrogel-matrix in solution, then the enzyme-modified hydrogel-matrix solution was injected into a capillary to produce monolithic IMER (Sakai-Kato et al. 2002a,b, Mou et al. 2013, Foo et al. 2015). Because enzymes are attached to the matrix in advance, the activity may be altered in polymerization reaction for preparation of the monolith. In another approach, monolithic column was prepared prior to the injection of enzymes for immobilization (Bossi et al. 2004, Dulay et al. 2005, Wang et al. 2010b, Yao et al. 2010, Qiao et al. 2011, Wu et al. 2013, Li et al. 2014, Han et al. 2015). Thus, preparation of monolithic capillary and immobilization of enzyme can be optimized separately. Enzymes can be immobilized in monolithic IMERs by covalent reaction to functional groups in matrix (such as aldehyde) (Ye et al. 2004, Ma et al. 2008), ionic binding with PDDA-entrapped column (Xu et al. 2008), or metal-chelated adsorption (Guo et al. 2003, Ma et al. 2011a, Wu et al. 2012). High efficiency for online CE enzyme assays was demonstrated due to the large surface area of monolith. Trypsin digestion of proteins and peptide mapping were analyzed in a majority of studies of monolithic IMERs (see Table 2), showing the potential application of monolithic IMERs in proteomic, especially coupled to MS detection. However, direct MS analysis is generally not suitable for adsorbed enzyme IMERs because of potential leaching of the enzyme leading to high background signals. By using CE-UV, CE-LIF, and even CE-MS, immobilization by adsorption is tolerated expanding the approach to immobilize enzyme.

Figure 5: Schematic representation of the monolithic IMER. Enzymes were pre-mixed with polymer, then the enzyme-containing mixture was injected into activated capillary to produce monolithic IMER.

Figure 5:

Schematic representation of the monolithic IMER. Enzymes were pre-mixed with polymer, then the enzyme-containing mixture was injected into activated capillary to produce monolithic IMER.

Figure 6: Strategies for functionalization of the PSG-PEG monolith surface with trimethoxysilylbutyraldehyde linker and attachment of trypsin to a butyraldehyde-activated PSG-PEG monolith.This figure was reproduced with permission from Dulay et al. (2005). Copyright © 2005 American Chemical Society.

Figure 6:

Strategies for functionalization of the PSG-PEG monolith surface with trimethoxysilylbutyraldehyde linker and attachment of trypsin to a butyraldehyde-activated PSG-PEG monolith.

This figure was reproduced with permission from Dulay et al. (2005). Copyright © 2005 American Chemical Society.

Recent reports describe the application of nanoparticles in fabrication of monolithic IMERs, which is an effective way to enhance the stability and operational lifetime (Zhang et al. 2013). In the fabrication strategy, GNPs were first covalently attached to surface of the pores of the porous polymer capillary monolith (20-cm long, 530-μm i.d) via the formation of an Au-S bond, then enzymes simply and stably immobilized onto GNPs through the strong affinity of gold for amino groups of the enzyme. Using a-glucosidase inhibitors (AGIs) as the test example, the study demonstrated accurate and reliable online enzyme assay using GNPs-monolithic IMERs, which could be well suited for assaying complex samples such as natural products extracts.

Packed capillary IMERs

Compared to open-tubular capillary and monolithic capillary, there are less studies to fabricate CE-integrated IMERs using packed capillary. Firstly, high pressure is generally used in traditional procedure for packing particles into capillary, making it difficult for efficient immobilization of enzymes. Secondly, it is also a great challenge to make frits that are compatible with the enzymatic reactions and the followed CE separation as well as have little effect on the enzyme activity. On the other hand, phase ratio and sample capacity in packed capillary are better than those in open tubular capillary, while packing procedure is simpler than fabrication of monolith, thus IMERs using packed capillary (packed-IMERs) could provide an alternative to OT-IMERs and monolithic IMERs.

Packed-IMERs were employed to investigate trypsin digestion of proteins (Bonneil et al. 2000, Dartiguenave et al. 2010). Trypsin was immobilized on controlled pore glass (CPG) beads through diisothiocyanate coupling. The beads were then dry-packed into a fused-silica capillary with i.d. of 530 μm and o.d. of 800 μm. A Swagelok union was fitted with the filter removed from a solid-phase extraction (SPE) cartridge in order to retain the beads during the packing procedure. The resulted immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. However, enzyme assays were performed off-line in the studies; the protein digest was collected at the microreactor capillary outlet and CE separated in a separation capillary.

With rapid development in HPLC grade particulate material in terms of surface chemistry, particle size, and porosity, which could be used as the enzyme support, CE-integrated IMERs using particle packing technique could be applied for online enzyme assays. Using a single 50-μm single particle with “through pores” as a mid-capillary frit, Miller and Lytle (1994) trapped cells in a capillary (75-μm i.d.) followed by CZE-LIF detection for investigation of cell profiling. Because the size of the single particle is smaller than that of the capillary i.d., a restriction section has to be made by heating and pulling the capillary, and the single particle was forced into the restriction by pressure.

Zhang et al. (2007) developed a simple frit-manufacturing method for packing CEC separation by employing single particles with larger size of 110 μm, which was accomplished via the keystone effect without making the restriction section. Based on their study, we reported a novel fabrication approach of CE-integrated packed-IMERs for online analysis of trypsin digestion (Liu et al. 2014). The procedure is rapid and simple, and the length and enzyme loading amount of the packed-IMER can be easily adjusted. Figure 7 shows schematically the fabrication procedure and the online analysis of trypsin digestion. The IMER was fabricated at the inlet of the capillary (100-μm i.d.) by trapping commercial trypsin-immobilized beads between the two single particles (110-μm diameter and 2-μm pore-size), which were used as the outlet and inlet frits for packing. Due to the large pore size of the silica single particle, the back pressure was greatly reduced during packing. Thus, it is not necessary for the use of high-pressure HPLC pump or the de-pressuring step, which is generally needed in the packed capillary technique. Indeed, the trypsin beads were packed in a capillary with only a syringe and a 5-mm-long IMER can be fabricated in only 5 min. Both the inlet and the outlet frits were manufactured without any heat or pressure, which will not affect the enzyme activity. Accurate assay of online trypsin digestion of myoglobin and BSA was achieved with 10-min incubation or even no incubation using the packed-IMER, which shows almost identical results compared to those using free trypsin with 12-h incubation time.

Figure 7: (A) Schematically drawing of the fabrication of the particle-packing CE-IMER and the online analysis of trypsin digestion. (B) SEM images of the trypsin beads and the perfusive silica single particle. (C) An example of the electropherogram of the tryptic peptides. (D) The real picture of the IMER taken by a microscope.This image was reprinted from Liu et al. (2014) with permission from Elsevier.

Figure 7:

(A) Schematically drawing of the fabrication of the particle-packing CE-IMER and the online analysis of trypsin digestion. (B) SEM images of the trypsin beads and the perfusive silica single particle. (C) An example of the electropherogram of the tryptic peptides. (D) The real picture of the IMER taken by a microscope.

This image was reprinted from Liu et al. (2014) with permission from Elsevier.

Magnetic beads (MBs) are another attractive enzyme support material in packed-IMERs. It is unnecessary to use frits because enzyme-immobilized MBs can be fixed by magnetic field at any place of the capillary. Compared to non-magnetic beads, the MBs-based IMERs have some advantages. The beads are replaceable, avoiding regenerating the solid support after each run, and it can be easily loaded or removed from the capillary in an automated CE instrument. Wojcik et al. (2010) and Li et al. (2011b) developed a two-dimensional CE systems incorporating a replaceable enzyme microreactor. The principle is illustrated in Figure 8. The trypsin-modified MBs were captured at the distal end of the first capillary with the help of a pair of magnets. Proteins were separated in the first capillary and metabolized in the IMER, and the resulting products (peptide) were subsequently separated in the second capillary before introduction to a mass spectrometer by an electrospray interface. We reported the use of MBs to form dual-IMERs by simply immobilizing two different enzyme-coated MBs (ADH and LDH) in a single capillary (50-μm i.d) by two pairs of magnets (5 mm) at different position forming two enzyme reaction chambers (Shi et al. 2012). The capability of the microreactor for dual-enzyme assay as well as simultaneous determination of two substrates (acetaldehyde and pyruvate) was demonstrated. The migration time was directly proportional to the position of the IMERs in the capillary. The dual packed-IMERs were successfully applied to simultaneously determine the acetaldehyde and pyruvate contents in beer samples. Compared to other CE-integrated IEMRs, packed-IMERs using MBs present advantages, such as replaceable, controllable, and capable to perform online CE assay for multi-enzymes.

Figure 8: Principle of the two-dimensional CE system incorporating a replaceable enzyme microreactor.This image was reprinted from Li et al. (2011b) with permission from Elsevier.

Figure 8:

Principle of the two-dimensional CE system incorporating a replaceable enzyme microreactor.

This image was reprinted from Li et al. (2011b) with permission from Elsevier.

Microfluidic based IMERs

Microfluidic systems have many advantages, especially their high-throughput, high portability, and automation, thus they are important techniques for the separation of biological samples as well as biosensors based on enzyme and inhibition assay. Besides CE-based IMERs, microfluidic-based IMERs have also been applied in a variety of studies for enzyme and inhibition assays. Enzyme can be immobilized on a support surface, such as electrodes, micro-fluidic channels, paper, thread, or microparticles to fabricate a microfludic-based IMER. Just as in CE-integrated IMERs, there are a variety of methods for immobilization of enzymes in microfluidic-based IMERs, including physical adsorption, covalent binding, and bioaffinity binding. A recent review by Kim and Herr (2013) has presented a comprehensive overview on the methods of protein immobilization for microfluidic assays. More recently, Mross et al. (2015) overviewed microfluidic biosensing systems based on enzyme detection. Typical immobilization methods of enzyme and fabrication, application, and performance of microfluidic biosensing systems were summarized and discussed. The review by Amine et al. (2016) highlighted the research on biosensors based on enzyme inhibition during the past 6 years. In addition, in their broad review on the CE-based enzyme assays, Scriba and Belal (2015) also gave a brief overview on the studies of enzyme assays using microfluidic devices. We refer the readers to these recent reviews for a more detailed insight on the microfluidic-based enzyme and inhibition assays. On the other hand, in the following, we will only collect and discuss some recent studies published in 2015 using microfluidic-based IMERs for enzyme assays.

Küchler et al. (2015a,b) reported an approach of unspecific noncovalent immobilization of enzymes on SiO2 surfaces in microchip channels (30-cm length, 100-μm width, and 40-μm height) and performed the enzyme assay for horseradish peroxidase isoenzyme C (HRP), Aspergillus sp. glucose oxidase (GOD) (Gustafsson et al. 2015), and Engyodontium album proteinase K (ProK) (Küchler et al. 2015a,b). Enzymes were first conjugated to the dendronized polymer (denpol) de-PG2, via stable bis-aryl hydrazine (BAH) bonds. The denpol-enzyme conjugate, which is a several 100 nanometer long macromolecular object, was then simply deposited on the glass surfaces without any detectable desorption because of the many unspecific noncovalent interactions between the large conjugate and the surface. In the case of ProK enzyme, the denpol-enzyme conjugate consisted of about 2000 denpol repeating units and 140 proK molecules. The microfluidic-based IMERs using denpol-enzyme conjugate have remarkable stability, for example, the ProK activity decreased by only 5% under continuous flow for 30 h and very slow loss in activity after storage for as long as 7 weeks (Küchler et al. 2015a,b).

An immunoassay for alkylphenolpolyethoxylates (APE) was investigated by Liu et al. (2015d), using microchips with a compact fluorescence detector from an organic light emitting diode (OLED) as the light source and an organic photodiode (OPD) as the photo-detector. Two types of microchips were used. In the T-shape microchip, anti-APE antibody was immobilized to the surface by physical adsorption, while in the V-shape format, anti-APE antibody was immobilized on the magnetic microbeads. The detection limits that were defined as IC80 were ca. 2 ppb for the antibody immobilized on the surface of magnetic microbeads and ca.1 ppb for physical adsorption of the antibody on the channel surface.

Microfluidic-based IMERs can be applied for multiple enzyme assays and multiplexed bioanalysis. For example, a multiple enzyme-doped thread-based microfluidic system was developed for blood urea nitrogen and glucose detection in human whole blood (Yang and Lin 2015). Two hundred-microliter-diameter polyester threads fixed on the PMMA substrate were used as the liquid routes and the enzymatic reaction sites. Multi-enzymes, including urease, glucose oxidase, and horseradish peroxidase, were directly doped on different sites of the thread, without delicate pretreatment or a surface modification process. To protect enzymes from contamination and to prevent the rapid evaporation of the running buffer due to the Joule heating effect, a thin layer of polyvinylchloride was coated on the enzyme-doped thread. Thus, a novel sealed microfluidic channel embedded with various enzymes was fabricated and was successfully used for detecting the concentrations of blood urea nitrogen and glucose in serum.

Henares et al. (2015) reported the fabrication and simple packaging of a capillary-assembled microchip (CAs-CHIP) for simple and multiplexed bioassays. The CAs-CHIP, which they named as the third generation of CAs-CHIP (CAs-CHIP-3G), has advantages of easy fabrication and fluid handling and assay throughput over their previous CAs-CHIP series (Hisamoto et al. 2004, Henares et al. 2007, 2008, Kimura et al. 2012). The CAs-CHIP-3G was prepared by arraying 12 square glass capillaries [or dry reagent-release capillaries (dRRC)] and glass plates for ease of handling. Enzymes were immobilized with polyethylene glycol (Mn=20,000, PEG-20,000) as soluble coatings. The packaged device was an assembly of a 1-cm-long CAs-CHIP-3G (for biosensor array), paper pad (for absorbent of excess sample solution), and vinyl tape (electrical tape, for adhesive for forming sample introduction microchannel). The authors have successfully attained the multiplexed glycosidase enzyme assay using the device. It was indicated that the CAs-CHIP-3G microdevice could also be used for other bioassays and colorimetric assay with the smartphone camera as the detector.

Conclusion and perspective

During the past two decades, the application of CE-integrated (and microfluidic-based) IMERs has been presented in a variety of research areas. Many publications were focused on the development of IMER methodology, for rapid, accurate, and repeatable determination of enzyme activity and reaction kinetics. It is now widely demonstrated that online enzyme assays with IMERs are more efficient and robust and higher throughput than traditional off-line assays with enzymes in solution. Kinetic constants and mechanisms of various enzyme reactions and inhibitions can be accurately determined with online assays using IMERs. In addition, combination of IMERs with high-performance separation technique CE can be used for quantitative measurement of trace amount substrates in real samples based on enzyme reactions.

The IMERs-based enzymatic assays have advantages for ease to automatize and miniaturize analytical systems. Efforts have also been made for the improvement of the sample throughputs. It is believed that CE- or chip-based IMERs will have valuable application for high-throughput inhibition screening and the development of biosensors and biomarkers, which have already been demonstrated in many previous studies (see Amine et al. 2016 and references therein). Although robustness of IMERs still has room for improvement, the application of IMERs in research and development of pharmaceutical science, as well as in clinical diagnosis will become a topic of increasing interest in the near future.

Another important application of IMERs is proteomics (Ma et al. 2009, 2011b, Spross and Sinz 2009, Yamaguchi and Miyazaki 2013, Safdar et al. 2014). As shown in this review, there are plenty of reported IMERs using protein digestion as the test enzyme reaction. Online integration of trypsin IMERs with high-performance separation methods, as well as the combination with tandem mass spectrometry, provides fast analysis of complex protein mixtures. It is indicated that protein digestion by immobilized trypsin reactors is much faster than that by free trypsin in solution, while high sequence coverages can be still obtained. This is very attractive in proteomics study. Numerous IMERs have been successfully employed for peptide mapping, protein-expression profiling, phospho-/glyco-protein characterization, and protein quantification. IMERs, with further development in automatization, miniaturization, and high-throughput, will play more and more important role in modern proteome research.

Funding source: National Natural Science Foundation of China

Award Identifier / Grant number: 21475019

Award Identifier / Grant number: 21175018

Funding statement: Funding: National Natural Science Foundation of China, (Grant/Award Number: “21475019, 21175018”).

  1. Funding: National Natural Science Foundation of China, (Grant/Award Number: “21475019, 21175018”).

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Received: 2016-1-21
Accepted: 2016-5-3
Published Online: 2016-6-24
Published in Print: 2016-9-1

©2016 Walter de Gruyter GmbH, Berlin/Boston

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