Encapsulation of Enzymes in Layer-by-Layer (LbL) Structures: Latest

Review pubs.acs.org/Biomac

Encapsulation of Enzymes in Layer-by-Layer (LbL) Structures: Latest Advances and Applications Omar. S. Sakr†,‡ and Gerrit Borchard*,‡ †

Capsulution Pharma AG, Volmerstrasse 7b, D-12489 Berlin, Germany School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland

‡

ABSTRACT: Layer-by-Layer (LbL) technology has become an active area of research and is currently considered a hot topic with many potential applications, especially in the pharmaceutical and biopharmaceutical fields. This review is providing an overview of current approaches and applications of LbL designs used to immobilize and/or encapsulate various enzymes. One aim was to show the versatility and the potential of this technique in obtaining different designs and architectures fulfilling a wide range of needs and applications. Another important aim was to shed light on the techniques commonly used to characterize LbL structures and to monitor the process of layer deposition. Special attention was given to LbL structures encapsulating multiple enzymes where the function depends on the sequential activities of encapsulated enzymes.

1. INTRODUCTION Since its introduction in 1992by Decher et al.,1 coating of flat surfaces as well as micro- and nanoparticles by Layer-by-Layer (LbL) technology has become an active area of research and is currently considered a hot topic with many potential applications, especially in drug encapsulation, sustained release dosage forms, protein delivery, and biosensors.2,3 LbL deposition is an established method for the fabrication of multicomposite ultrathin films on solid surfaces.1,4,5 Typically, this technique is based on the use of polyelectrolytes of opposite charges assembled layer-wise on the surface of interest, thereby building up a layered system of tunable characteristics (Figure 1), in terms of composition, nanometer range thickness, surface charge, permeability, and elasticity.2UsUsing this approach, a variety of materials, including charged and uncharged species, have been successfully assembled into nanoscale multilayered structures. Multilayers may be formed by using LbL assembly techniques that rely on electrostatic interactions,4 hydrogenbonding,6 coordination bonding,7 charge transfer,8molecular recognition,9 hydrophobic interactions,10or a combination of these. As a result, such films may be comprised of a diverse range of components, including but not limited to biomacromolecules,11−13nanoparticles,14 dyes,15 dendrimers,16 and conductive polymers.17 More particularly relevant to protein encapsulation, LbL deposition has the advantage of utilizing mild conditions (e.g., aqueous solutions), which are more favorable to preserve fragile protein folding and activity in contrast to organic solvents typically employed in the fabrication of other protein encapsulation systems such as microparticles, microcapsules, microemulsions, and liposomes.18 These, in addition, suffer © XXXX American Chemical Society

Figure 1. Simplified molecular picture of the first two adsorption steps, depicting film deposition starting with a negatively charged surface. Steps 1 and 3 represent the adsorption of a polycation and a polyanion, respectively, and steps 2 and 4 are washing steps.

from some limitations including polydispersity, core solidification, and residual organic solvents.19 It has been proven that entrained water is present in LbL films, in spite of the drying procedures applied, which is important for the preservation of activity of biomolecules.20 Received: February 6, 2013 Revised: May 21, 2013

A

dx.doi.org/10.1021/bm400198p | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

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Table 1. Summary of Main LbL Designs Used to Encapsulate Enzymes Reviewed in This Article enzyme catalase (CAT)

α-chemotrypsin

cholesterol oxidase (COX)

glucose isomerase (GI) β-glucosidase (β-GLS) urease

peroxidase (POD)

layer componentsa

LbL design microspheres: based on sacrificial melamine formaldehyde particles, partially decomposed after layer deposition capsules: based on building LbL multilayers on CAT crystals mixed design: CAT microcrystals were first encapsulated in PSS/PAH, then films of alternating layers of CAT capsules and oppositely charged PEs were deposited on planar surfaces mixed design: CAT microcrystals were first encapsulated in PSS/PAH, then thin films of alternating layers of CAT capsules and oppositely charged PEs were deposited on gold electrodes for biosensing of H2O2 coating films: CAT was first encapsulated in small gold nanoparticles, and then electrostatically assembled with cationic PE on planar surfaces and colloidalparticles capsules: based on building LbL multilayers on α-chemotrypsin crystals hollow capsules: based on sacrificial melamine formaldehyde particles, decomposed after layer deposition coating films: cholesterol biosensor based on immobilization of COX in LbL films deposited on glass substrates. coating films: cholesterol biosensor based on immobilization of COX in LbL films deposited on glass substrates. coating films: cholesterol biosensors based on immobilizing COX in LbL films deposited on electrospun polyaniline nanofibers. coating films: Both on planar surfaces and colloidal particles. coating films: on colloidal particles. hollow capsules: based on sacrificial melamine formaldehyde particles, decomposed after layer deposition. coating films: on colloidal particles. coating films: on colloidal particles. hollow capsules: based on sacrificial melamine formaldehyde particles, decomposed after layer deposition.

glucose oxidase (GOD)

peroxidase (POD) and glucose oxidase (GOD) glucose oxidase (GOD) and glucoamylase (GA) catalase (CAT) and glucose oxidase (GOD) a

coating films: on planar quartz slides. coating films: on colloidal particles

hollow capsules: based on sacrificial MnCO3 particles, decomposed after layer deposition coating films: glucose biosensor based on a pyrolytic graphite electrode with a modified surface by LbL deposition of GOD and PEI capsules: glucose-sensitive multilayer shells made of GOD/PDMAEMA, built on insulin crystals coating films: on planar quartz slides capsule in capsule: based on coprecipitating the 2 enzymes with CaCO3 in 2 successive steps, separated by deposition of a thick multilayer membrane in between, followed by dissolving the CaCO3 coating films: on planar filter surfaces coating films: maltose sensor based gold electrode coated with GOD and GA sandwiched between bipolar quaternary ammonium salt capsules:glucose-sensitive multilayer shells made of GOD/CAT held together via glutaraldehyde crosslinking, built on insulin crystals

ref.

dextran sulfate/protamine

35

PSS/PAH PSS/PAH/Cat-capsules

37 39

PSS/PAH/Cat-capsules

40

PSS/PAH/CAT-AUNPs

41

PSS/PAH PSS/PAH alginate/ protamine PSS/PEI/COX

21,42 23,44 45

COX/PAH

20

COX/PDDA

46

GI/bicationic pyridine salt PAH/PSS/β-GLS PSS/PAH PEI/urease PDDA/urease urease/PSS Fe3O4 PSS/POD PSS/(PODPSS) PSS/PDADMAC dextran sulfate/protamine PSS/PEI/GOD PEI/GOD PSS/PAH/ GOD/Fe3O4 NP3/ PDADMAC diasoresin/PSS/PAH GOD/PEI

52 54 58 60 27 36,70 73 27,76

22 80

GOD/PDMAEMA PSS/POD GOD/PEI PSS/PAH

91 84 86

GA/GOD/PEI/PSS GA/bipolar quaternary ammonium salt/GOD GOD/CAT

85 88 92

Not indicating the order of addition.

restricted film permeability to substrates, and the highly specific nature of the assembly limit the wide applicability of all these methods.27 Recently, basic LbL principles5 and their use to functionalize nanoparticles,2 to control and modify permeability and release kinetics,28−30 to deliver biotherapeutics such as antigens and genes,31 and to fabricate stimuli responsive capsules32,33 were reviewed. The present work is devoted to review different approaches and LbL designs applied to immobilize and/or encapsulate various enzymes. One aim was to show the versatility and the potential of this technique in obtaining different designs and architectures fulfilling a wide range of needs and uses (Table 1). Another important aim was to shed light on the techniques usually applied to characterize LbL structures and to monitor the process of layer deposition. Methods for testing enzyme activity and stability were of higher interest. Moreover, a special part is dedicated to LbL structures encapsulating two or more active proteins where the function depends on the sequential actions of encapsulated enzymes. It is noteworthy to mention

Immobilization of enzymes and active proteins is of great scientific and practical importance.21 Especially when considering microreactor and biosensor development, immobilization and encapsulation of enzymes in LbL structures can provide a means of concentrating and protecting the bioactive molecules in a defined volume, creating a partitioned microenvironment with tunable properties, which could therefore be used to balance the diffusion and reaction as needed for the sensor function.22 Moreover, encapsulation offers a great advantage to these sensitive structures by protecting encapsulated enzymes from proteolytic enzymes and microbes.23 LbL encapsulation of proteins and enzymes is superior to previously used techniques, where proteins have traditionally been immobilized onto solid surfaces by physical adsorption, solvent casting, covalent binding, and electropolymerization.24 However, these methods often produce irregular films at low protein density. Ordered protein multilayer films with a high protein density have been constructed by using Langmuir− Blodgett deposition methods24,25 or by exploiting biospecific interactions.26 Denaturation of the immobilized proteins, B


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the microspheres. The authors reported that electrostatic and hydrophobic interactions were responsible for the interaction of the protein with the microspheres’ gelatinous core. Therefore, encapsulation and release of proteins from the proposed LbL system can be controlled via adjustment of pH, ionic strength, and temperature of the incubation medium.35 A different approach was adopted by Caruso et al.,37 where CAT crystals themselves were used as templates to deposit alternating layers of PSS/PAH to form the polymer capsule encapsulating the enzyme in the core at very high efficiency. The method takes advantage of the fact that the enzyme is present as a crystalline suspension in water at pH 5−6 and may therefore be treated as a colloidal particle. It should be noted that enzyme crystal templating, however, presents several challenges that do not apply when templating, e.g., latex particles. First, the crystals are formed only under strictlydefined conditions. Therefore, suitable conditions that facilitate polymer multilayer deposition on the crystal surface and do not destroy the enzyme crystal morphology (i.e., to avoid its solubilization) need to be determined. Second, the permeability of the polymer capsule walls must be such that it permits encapsulation of the enzyme. In addition, since the primary usefulness of enzymes is their biological function, their activity must be preserved during encapsulation. Practically, CAT crystals were separated from solubilized protein by washing and centrifugation several times with potassium acetate buffer of pH 5 at 4 °C. Chilled solutions were used to avoid significant solubilization of the enzyme crystals. CAT crystals exhibited a positive surface charge in water at pH 5 (+20 mV), as determined by electrophoretic mobility measurements. This positive charge at the surface of the crystals in principle makes them suitably charged templates for the deposition of polyelectrolyte layers of PSS and PAH. The successful deposition was proven by the reversal of the surface charge after each cycle of deposition, which is a characteristic of polyelectrolyte multilayer growth on colloidal templates.37 To investigate the effect of the encapsulation process on the activity of CAT, the activity was measured after solubilization of CAT (by changing pH) and release from the polymer capsules. A recovered specific activity of 97% was obtained, compared with 100% for the uncoated CAT. This shows that the polymer multilayer coating of the CAT crystals proceeded without causing any significant loss of enzyme activity. Another important point was the ability of the capsule wall to protect the enzyme against proteolytic activity. As shown in Figure 3, solubilized, uncoated CAT (curves d and e) was inactivated by protease to more than 90% during an incubation time of 100 min. By contrast, no measurable loss in enzyme activity was observed for the polymer-encapsulated (solubilized) CAT within 100 min under the same conditions (curves b and c). These results clearly demonstrate that a thin polymer coating of four layers (thickness of about 8 nm) is sufficient to prevent proteolysis of polymer encapsulated CAT.37 These findings are consistent with the observation that proteins of molecular sizes greater than approximately 5 nm do not penetrate polyelectrolyte multilayer films.38 Compared to traditional LbL methods, where solubilized charged enzyme molecules are deposited among the layers, encapsulated enzyme crystals display an up to 50-fold increased biocatalytic activity, thus making them attractive candidates for various biotechnological applications.39 Due to the interesting advantages of encapsulating enzyme crystals, such as high enzyme loading, preserved bioactivity of

that, due to the great interest LbL encapsulation techniques have gained; considerable research work is published on enzyme encapsulated in LbL structures. This review attempts to cover the different LbL designs and structures supported with selected examples from the literature.

2. CATALASE (CAT) Catalase (CAT) is a common enzyme found in nearly all aerobic organisms. It catalyzes the decomposition of hydrogen peroxide to water and oxygen. 34 CAT was used by Balabushevich et al.35as a model protein of high molecular weight for encapsulation. Polyelectrolyte microspheres were obtained by alternating adsorption of dextran sulfate and protamine on melamine formaldehyde (MF) particles followed by partial hydrolysis of the MF core. In fact, the main difference between the fabrication of hollow capsules and microspheres prepared by this method employing MF cores is the core hydrolysis step (Figure 2). While the complete hydrolysis of the

Figure 2. Scheme of production of microspheres and hollow microcapsules using commercially available melamine formaldehyde particles and alternating adsorption of polyelectrolytes.35 Source: Biochemistry (Mosc) 69, (7), 2004, 763−9, Encapsulation of catalase in polyelectrolyte microspheres composed of melamine formaldehyde, dextran sulfate, and protamine, Balabushevich, N. G.; Zimina, E. P.; Larionova, N. I., Figure 1, Copyright 2004 MAIK “Nauka/Interperiodica”. Reproduced with kind permission from Springer Science and Business Media.

MF core produces hollow capsules, during the slow partial hydrolysis of MF core under mild conditions, the newly formed and positively charged amino groups interact with polyanionic structures of the first layer of the microcapsule shell, resulting in the redistribution of membrane PEs and formation of a homogeneous, weakly cross-linked, charged gelatinous matrix inside the microspheres.36CAT was encapsulated in the microspheres by simple incubation and mixing for sufficient time. The loaded microspheres were collected by centrifugation and washed. Finally, an additional layer of dextran sulfate was added to protect the enzyme adsorbed at the surface of the positive protamine layer. CAT was entrapped in microparticles at high efficiencies (70−100%), depending on its original concentration in the incubation medium. The specific activity observed was dependent on the amount of protein entrapped in C


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CAT, after which a plateau was observed. This was attributed to the increasing difficulty of electron transfer and substrate diffusion limitations. Using immobilized PE-encapsulated enzyme microcrystals for biosensing was shown to provide a versatile method to prepare films of high concentrations and tailored activities of enzymes. More recently, another novel and versatile approach for the preparation of multilayers was introduced, where CAT was first encapsulated in small gold nanoparticles (CAT-AuNPs), and then electrostatically assembled with anionic and cationic PEs on colloidal silica particles.41CAT-AuNPs were synthesized directly from CAT stabilized gold suspensions and the diameter of the obtained particles was about 9 ± 3.5 nm. Since the pI of CAT is 5.6,37 CAT-AuNPs were positively charged at pH 3 and were assembled with the anionic polymer PSS. However, when the pH of the CAT-AuNPs solution was changed from 3 to 9, charge reversal took place, and the anionic CAT-AuNPs were bound electrostatically to cationic PAH. As shown in Figure 4,

Figure 3. Stability of (a,d,e) solution-solubilized catalase and (b,c) polymer-multilayer encapsulated (solubilized) catalase with respect to proteolysis: (a) solution-solubilized catalase crystals, no protease incubation (control); (b) [(PSS/PAH)2]-coated (four layers) catalase, protease incubation; (c) [(PSS/PAH)4]-coated (eight layers) catalase, protease incubation; (d,e) repeat experiments for solubilized catalase, protease incubation. Proteolysis of the catalase was determined by measuring the decrease in the catalase enzyme activity.37 Reprinted with permission from reference 37. Copyright 2000 American Chemical Society.

the encapsulated enzyme, the ability of the semipermeable PE coating to prevent the solubilized enzyme from leakage while simultaneously permitting the diffusion of small (substrate) molecules for enzyme reaction, this idea was taken a step forward by Jin et al.,39 where a mixed approach was adopted. CAT microcrystals were first encapsulated by the alternate adsorption of PSS and PAH on their surface, yielding an extremely high loading of active enzyme in the polyelectrolyte multilayer capsule. Then, multilayer films were constructed on planar surfaces (quartz crystal microbalance (QCM) electrodes or quartz slides) by LbL deposition of the polyelectrolytecoated CAT crystals and oppositely charged polyelectrolyte. Moreover, this mixed approach was adopted by Yu et al.,40 where LbL encapsulated CAT microcrystals were assembled onto gold electrodes by sequential deposition with oppositely charged PEs, utilizing electrostatic interactions to form thin enzyme films for biosensing of H2O2. In addition to the aforementioned advantages of this design, the authors found that the PE layers encapsulating the enzyme effectively increase the surface charge density of the enzyme microcrystals, rendering them suitably charged components for the construction of biofunctional thin films. The PSS/PAH encapsulated CAT was shown to retain biological as well as electrochemical activity. Direct electron transfer between CAT molecules and the gold electrode was achieved without the aid of any electron mediator. As a H2O2 biosensor, films consisting of one layer of the encapsulated CAT displayed considerably higher (∼5-fold) and more stable electrocatalytic responses to the reduction of H2O2 than did corresponding films made of a single layer of nonencapsulated CAT or solubilized CAT. An increase in either the number of “precursor” PE layers between the gold electrodes and the CAT microcrystal layers in the film or the number of PE layers encapsulating the CAT microcrystals was found to decrease the electrocatalytic activity of the electrode. At low precursor PE layer numbers (∼2) and encapsulating PE layers (∼4), the current response was proportional to the H2O2 concentration in the range of 3.0 × 10−6 to 1.0 × 10−2 M. The overall electroactivity of the multilayer film increased for the first two layers of encapsulated

Figure 4. Schematic and TEM images for the preparation of CATAuNP with (a) dispersed, (b) colloidal and (c) network structures.41 Source: S. Kim, J. Park, J. Cho, Layer-by-layer assembled multilayers using catalase-encapsulated gold nanoparticles. Nanotechnology 21(37) (2010): 375702. (http://iopscience.iop.org/0957-4484/21/ 37/375702). Copyright 2010 IOP Publishing Ltd. Reproduced by permission of IOP Publishing. All rights reserved.

an interesting feature of CAT-AuNPs is that the pH-dependent electrostatic properties of CAT-AuNPs can control the structure of the hybrid nanocomposite, transforming them from well dispersed small nanoparticles at pH9 (due to repulsion forces between the similarly charged particles) to agglomerated colloidal particles with a diameter of about 50 nm or D


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the protein and microcapsule suspensions indicated clearly the protein penetration through the open walls into the hollow capsules and the successful encapsulation of the enzyme. Tiourina et al.44 used rhodamine 110 [bis-(succinoyl-Lalanyl-L-alanyl-L-prolyl-L-phenylalanyl amide)] to investigate the activity of the encapsulated enzyme, which was found to retain about 70% of its activity. It is important to mention that the enzymatic reaction inside the microcapsules requires substrate diffusion through the polyelectrolyte membrane while the reaction product is released into the surrounding medium.23 In a different report, 23 the author challenged the encapsulated α-chymotrypsin with one of its inhibitors BPTI. While the native enzyme was completely inhibited at a [BPTI]/ [α-chymotrypsin] ratio of 1:1 after 5 min, the inhibitor did not influence the activity of the encapsulated α-chymotrypsin at a [BPTI]/[ α-chymotrypsin] ratio of 2:1 after 30 min, and the enzyme lost only 40% activity at a [BPTI]/[ α-chymotrypsin] ratio of 50:1 after 30 min. These results indicate that polyelectrolyte shells possess protective properties against high molecular weight inhibitors. The protection is mostly a result of the steric hindrance created by branched polymer molecules because protein−protein interaction requires multidot contacts, which were apparently unavailable in the mentioned cases.

network-structured composites, depending on the initial concentration of gold precursor.41 In addition, this structural transformation had a significant effect on the surface morphology of CAT-Au nanocomposite becoming rougher with fibrillary structures, especially in case of network-structured CAT-AuNP. It was found that the total adsorbed amount of (PE/CAT-AuNP network)5 multilayers was about 5.7 times higher than for (PAH/CAT)5 multilayers at the same solution concentration. Consequently, the higher CAT adsorption led to higher catalytic activity toward H2O2.Considering using these CAT-AuNPs to coat an electrode surface, the rugged and fibrillary structure of PE/CAT-AuNP colloids and PE/CAT-AuNP network multilayers has an increased surface area, and as a result, increases the area of contact between the probe molecules and the CAT as well as the effective electron transfer rate. Therefore, this structural morphology may assist in increasing electrochemical sensitivity, which may be beneficial to a variety of biocatalytic applications.41

3. α-CHYMOTRYPSIN α-Chymotrypsin, a serine proteinase found in the intestinal tract and whose activity can be easily monitored, has been chosen as a model enzyme for the evaluation of the feasibility and possible applications of LbL microcapsules. Two main strategies were followed: in the first method,21,42 α-chymotrypsin was salted out from its acidic solution by mixing with an appropriate volume of a saturated solution of NaCl yielding particles with typical dimensions of 0.1−0.4 mm. Being positively charged at pH 2−3, chymotrypsin (pI:8.5) can be coated with a series of oppositely charged PEs such as PSS/ PAH, with intermediate washing steps to remove excess unattached polymers. Although one can easily accept that the first layer would be the negatively charged polymer PSS, Balabushevitch et al.42found the possibility of starting the deposition with the positively charged polymer PAH. This was attributed to strong hydrophobic forces between PAH and the salted-out protein core. This preparation of microencapsulated α-chymotrypsin retained 73% active site content after storage for 6 days at pH 3.0 at 4 °C, while free enzyme had already lost 57% active site content after 6 days. Basic pancreatic trypsin inhibitor (BPTI, M.W. 6500), one of the known proteinase inhibitors,43 was used to challenge the ability of the LbL film to protect the encapsulated cargo. BPTI was able to suppress 85% of free enzyme activity compared to only 13% of microencapsulated enzyme activity.42 In the second method, hollow polyelectrolyte capsules were fabricated prior to loading with the model enzyme. For this purpose, small micrometer-sized melamine formaldehyde (MF) particles were chosen as sacrificial cores. MF particles were coated with alternating layers of PSS/PAH23 or alginate/ protamine44 at neutral pH, then the cores were dissolved by 0.1 M HCl solution and the empty shells were washed with water to remove MF residues to finally obtain hollow capsules. To load the enzyme α-chymotrypsin into the hollow capsules, a small volume of the empty capsule suspension was centrifuged, and the supernatant removed. Successively, capsules were resuspended in the enzyme solution of appropriate buffers and mixed well. After sufficient incubation of the capsules in the enzyme solution, the mixture was centrifuged, and the capsules were washed three times with water. Confocal fluorescence images of labeled α-chymotrypsin within capsules after mixing

4. CHOLESTEROL OXIDASE (COX) Biosensors employing immobilized COX for the detection of cholesterol may be more advantageous in comparison to standard methods such as spectrophotometry, gas−liquid chromatography, and HPLC, owing to the simplicity and low costs involved. This is relevant to the development of reliable methods of cholesterol detection in blood, which is a fundamental parameter to identify disorders such as hypercholesterolemia, and to control the cholesterol level in foodstuff for human intake.20,45,46 Ram et al.45 reported the formation of a cholesterol biosensor electrode via immobilization of COX in LbL films. All solutions were adjusted at pH = 7.5 as COX (pI between 4.6 and 5.2) can be used as a polyanion. Initially, a layer of polyanion PSS was adsorbed, followed by a layer of polycation PEI. Then, the LbL film was constructed by consecutive adsorption of polycation PEI and negatively charged proteins COX and cholesterol esterase (CE). The assemblies studied can be denoted as PSS/PEI/COX, PSS/PEI/COX/PEI/CE, and PSS/PEI/COX−CE/PEI. To monitor native and esterified cholesterol levels, both COX and CE enzymes were employed simultaneously. Whereas COX catalyzes the oxidation of cholesterol, CE catalyzes the hydrolysis of esterified cholesterol, which is an important factor for the determination of total cholesterol, since about 70% of the cholesterol in blood is found to be esterified cholesterol.47,48 During preliminary studies, UV spectroscopy, QCM, and electrochemical investigation clearly gave evidence for uniform enzyme immobilization on various substrates. The cell used to determine the response current consisted of cholesterol oxidase LbL film as working electrode, a platinum wire as the counter electrode, and Ag/AgCl as the reference electrode in a solution of 100 mM phosphate buffer containing 1% of Triton X-100. Considering the decrease of the activity of cholesterol oxidase at high concentrations of Triton X-100 and low solubility of cholesterol at low concentrations of Triton X-100, 1% of Triton X-100 in the buffer solution was used in this study. E


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Measurements indicated a linear response to cholesterol up to concentrations of 1 mM, and further increase in cholesterol concentration showed a slower rise of the current. The stable current data were used for the calibration curve for actual measurements.45 Moraes et al.20 reported another design of COX-based cholesterol biosensors via the immobilization of COX in LbL films, in alternation with layers of PAH. In preliminary studies, COX was alternately deposited with PAH onto previously cleaned glass substrates using the LbL technique. Before the PAH/COX film fabrication, two bilayers of PAH/polyvinylsulfonic acid (PVS) were deposited onto the solid substrates to reduce substrate effects. Layer growth and film morphology were studied, and the kinetic analysis of the adsorption process showed a two-step process. Impedance spectroscopy measurements were used to detect cholesterol in liposomes (made from egg phosphatidylglycerol) and natural egg yolk. Three electrodes were compared: bare, PAH/PVS-, and PAH/COX-coated gold electrodes. Owing to the specific interaction with COX in the LbL film, only gold electrodes coated with PAH/COX were able to detect cholesterol in aqueous solutions with a significantly higher sensitivity reaching 10−6 M.20 More recently, Shin et al.46 proposed a novel architecture of cholesterol biosensors based on immobilizing COX in LbL films deposited on electrospun polyaniline nanofibers using polydiallyldimethylammonium chloride (PDDA) as a counterion. The nanofibers were composed of polystyrene as a core and polyaniline as a conducting polymer. Nanofibers were produced under a high voltage electrostatic field between a metallic nozzle of a syringe and a metallic collector. The charged polymer solution is jetted from the metal needle to the grounded collector. At working distance, the polymer jet elongates, solidifies, and deposits on the collector. The fibers are deposited in the form of a nonwoven fabric onto the target collector. Such nanofibrous membranes have many attractive features when used as supports for enzyme immobilization. These include a large surface area for the attachment of enzymes, a nanofibrous morphology to improve the masstransfer rate of the substrate, and a membrane-like structure for easy recovery from the reaction media and continuous operations in a bioreactor. The high surface area-to-volume ratio makes electrospun conducting polymer nanofibers particularly interesting for sensing applications.49,50 One layer of TiO2 was coated onto this nanofiber, and five alternate cycles of PDDA and COX adsorption were carried out (Figure 5). A cholesterol biosensor was fabricated using a standard onecompartment three-electrode cell and used for all electrochemical experiments. The sensing electrode was fabricated in 40 mL of 0.1 M phosphate buffer solution at pH 6.3 containing 1% Triton X-100, using the nanofiber mat onto which COX had been immobilized as a working electrode, the reference electrode was Ag/AgCl (3 M KCl), and the counter electrode was a platinum wire (20 cm). Measurements showed a linear electrical response up to a concentration of 0.35 mM cholesterol. Accurate data could not be obtained using the nanofiber mat onto which two layers of COX had been deposited, and best results were obtained when more than five layers of COX had been deposited on the nanofiber mat.46

Figure 5. (a) SEM of nanofibers for the blend of polyaniline and polystyrene before LbL coating (bar represents 5 μm). (b) SEM of nanofibers that COX was immobilized via LbL coating (bar represents 5 μm).46 Reprinted from reference 46, Copyright 2011, with permission of Elsevier.

lized by Kong et al.,52 in ultrathin films both on planar surfaces, and on porous p-trimethylamine-polystyrene (TMPS) beads. Negative charge was induced to GI molecules by adjusting the solution pH to above its pI. GI was deposited in alternation with bicationic pyridine salt (Figure 6). The alternating molecular deposition of glucose isomerase and bipolar pyridine salt was followed by means of UV/vis absorption spectra, and the linear increase of the optical density of the films with

Figure 6. Model for alternating the deposition film of GI and bipolar pyridine salt through electrostatic interaction with bicationic pyridine salt.52 Source: Macromol. Rapid Commun., 15, 1994, 405−409. Kong, W.; Zhang, X.; Gao, M. L.; Zhou, H.; Li, W.; Shen, J. C. Copyright 1994 Hüthig & Wepf Verlag, Basel. This material is reproduced with permission of John Wiley & Sons, Inc.

5. GLUCOSE ISOMERASE (GI) Glucose isomerase (GI, MW of ∼160 000, pI: 4.7) converts Dglucose to D-fructose in a reaction that is industrially applied to the production of high-fructose corn syrup.51GI was immobiF


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were then fitted using the Rayleigh−Debye−Gans theory,55 and refractive indices were fitted for the enzyme and polyelectrolyte layers,56 thereby providing a quantitative measure of the layer thickness. Thicknesses were measured for particles with PSS as the outermost layers, since these particles were found to have a higher colloidal stability. Regular multilayer film growth occurred: linear enzyme multilayer growth was observed with an average thickness of 3.5 ± 0.6 nm for a β-GLS/PSS layer pair. Enzymatically catalyzed glucosidation reactions carried out with multilayer-coated particles showed distinct differences in overall yield. Quantitative determination of dodecyl glucoside in the reaction mixtures revealed a regular increase in yield with increasing β-GLS layer number for particles with an outermost PSS layer. Particles with one enzyme layer yielded only traces of the product, which could not be quantified. The increasing amount of dodecyl glucoside observed for particles with two, three, and four β-GLS layers indicated that enzyme in the inner layers also takes part in the catalytic process. This in turn suggests that the substrate can diffuse into the layers to interact with the active sites of immobilized enzyme. It is worth mentioning that particles covered by enzyme as the outermost layer showed a higher tendency to aggregate compared to those with PE as the outermost layer. The colloidal stability of protein/polyelectrolyte coated particles is known to increase when the polyelectrolyte forms the outer layer, the reason most probably being the electrostatic and steric stabilization of the particles conferred by the polyelectrolyte.56 However, catalysts with β-GLS as the outermost layer yielded higher amounts of dodecyl glucoside (ca. 2−5 times more) than those having the enzyme layer coated with PSS. This may be explained by a loss of enzyme from the surface into solution (i.e., the reaction mixture) when the enzyme is not covered by polyelectrolyte, resulting in higher amounts of product.

increasing number of layers proved the process of deposition. The glucose isomerase activity was assayed following Tomas’s cysteine-carbazole method for fructose.53 The glucose isomerase activity was preserved in the film, and the activity increased with the number of enzyme layers. However, the average activity per enzyme layer decreased with the number of enzyme layers, because the more enzyme layers are deposited, the larger is the limitation of the diffusion of the substrate to reach the enzyme, which resulted in a great decrease of the specific activity of the enzyme (Figure 7).

Figure 7. Relationship between activity of glucose isomerase and the number of the enzyme layers. Total activity of enzyme in the slide (circles) and average activity per enzyme layer (squares).52 Source: Macromol. Rapid Commun., 15, 1994, 405−409. Kong, W.; Zhang, X.; Gao, M. L.; Zhou, H.; Li, W.; Shen, J. C. Copyright 1994 Hüthig & Wepf Verlag, Basel. This material is reproduced with permission of John Wiley & Sons, Inc.

6. β-GLUCOSIDASE (β-GLS) β-glucosidase (β-GLS) enzyme layers, each separated by oppositely charged polyelectrolyte PSS, were deposited onto polystyrene (PS) latex particles using the LbL adsorption technique, as a new means to perform enzymatic glucosidation.54The purpose of o-glycosyl hydrolases such as β-GLS is the hydrolysis of glycosidic bonds. Reverse hydrolysis on its part is known to be an elegant method to form glycosidic bonds via enzymatic catalysis, yielding stereoselective glycosidation. This is achieved by performing the glycosidation reaction in organic solvents, thereby keeping the water content of the system low. In this report, β-GLS was applied to form glycosidic bonds between carbohydrates and noncarbohydrate, hydrophobic moieties (long chain alcohol). Four alternating PAH and PSS layers were deposited onto the PS particles (first layer PAH), resulting in negatively charged particles with PSS as the outermost layer. As the pI of the enzyme is at 5.5, β-GLS is positively charged under the experimental conditions employed (pH 4.8). Four enzyme layers were deposited, each separated by one PSS layer. Successful deposition of layers on the particles surface was proven by showing surface charge reversal, and the growth of the PS latex particle diameter with layer deposition as followed by single-particle light scattering (SPLS). By recording the light scattered from many individual particles, histograms of particle number versus scattering intensity (or SPLS intensity distributions) were obtained. These intensity distributions

7. UREASE Urease is a metalloenzyme that catalyzes the hydrolysis of urea to yield ammonia and carbon dioxide. It is found in a wide variety of organisms including plants, fungi, and bacteria.57 Stable hollow polyelectrolyte capsules containing urease (Mw 480 kDa) were produced by means of the LbL assembling of PAH and PSS on melamine formaldehyde microcores followed by the core decomposition at low pH.58 The authors introduced a new method of loading urease enzyme into the particles depending on a finding that capsules were nonpermeable for urease in water and became permeable in a water/ethanol mixture. As shown in Figure 8 (left), confocal microscopy imaging illustrates that FITC-labeled urease was excluded from the polyelectrolyte shells. The interior of the capsules remained dark and outside the capsule the background containing FITC-urease was fluorescent. This observation indicated the closed state of the capsules. Figure 8 (center) shows a confocal fluorescence image of labeled urease with capsules after addition of ethanol (1:1 water/ethanol mixture). In this case, the fluorescence signal localized in the interior of the capsule has the same intensity as the outside background fluorescence. This indicated the penetration of the enzyme into the capsules and requires an open state of the capsule wall. When the capsules were transferred back into water, the polyion shells became closed, and the urease was captured inside, as illustrated in Figure 8 (right). The interior of the capsule is bright and constant over time, and there is no G


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protects encapsulated enzymes from proteases and microbes, and provides better stability.58 In a different LbL design,60 urease was loaded on the surface of submicrometer-sized polystyrene particles. Urease multilayers were assembled with alternating oppositely charged polyelectrolytes in a predetermined order, utilizing electrostatic interactions for layer growth. Urease, which has a pI at pH 5 and is stable and active between pH 4 and 8.5,61,62 was employed in the LbL assembly either as a negative polyelectrolyte at pH 8 deposited in alternation with polycations (PEI or PDDA), or as a positive polyelectrolyte at pH4.5 and consecutively deposited with the polyanion (PSS). As an added feature, prior to enzyme adsorption, the colloid particles were coated with an additional layer of silica or magnetite nanoparticles in order to enhance their total surface area and promote further enzyme deposition, to give shell architectures of the following sequence: [PDDA/PSS/PDDA/ 40-nm silica/PDDA/(urease/PDDA)1−4] or [PDDA/PSS/ PDDA/12-nm magnetite/PDDA/(urease/PDDA)1−4]. Urease multilayers were first constructed on quartz cell microbalance (QCM) electrodes in order to establish the conditions for suitable multilayer growth. The QCM frequency shift, caused by the deposition of material on the electrode surface, can be related to the adsorbed mass and layer thickness of the material via the Sauerbrey equation.63 Figure 10 shows

Figure 8. Permeation and encapsulation of urease-FITC into polyion multilayer capsules. Left, in water; middle, in water/ethanol mixture 1:1; right, the capsule with encapsulated urease again in the water. Top, scheme; bottom, confocal fluorescence images of the capsules.58 Reprinted with permission from reference 58. Copyright 2001 American Chemical Society.

fluorescence signal from the solution. Thus, urease filled the capsules. The images did not change with time, indicating that urease is preserved inside the capsules. The authors mentioned that the mechanism of reversible permeability changes in the polyion multilayers is not fully understood. It may be related to the segregation of the polyion network in water/ethanol media. Such segregation might lead to defects in the shell, and pores forming might be large enough for 5-nm diameter urease globules to penetrate the wall. Returning capsules into pure water causes a relaxation of the polyion walls to a closed structure.58 A colorimetric assay based on the hydrolysis of urea was used to investigate the activity of free and immobilized urease.59 The increase in solution pH due to ammonia production during the enzymatic reaction was monitored by the pH sensitive dye, bromcresol purple. The absorbance of this dye at 588 nm increases linearly with pH in the range of 5.8 to 7.5. Figure 9

Figure 10. QCM monitoring (frequency change vs adsorption steps) of urease/PDDA assembly: [PDDA/PSS/PDDA /(urease/ PDDA)5].60 Reprinted with permission from reference 60. Copyright 2001 American Chemical Society.

the QCM results for the construction of a PDDA/PSS/PDDA/ (urease/PDDA)5 multilayer film on a QCM electrode. The first three polyelectrolyte layers serve as a precursor film to provide a uniform charge and a smooth surface for subsequent urease deposition. A regular stepwise decrease in the QCM frequency was observed (Figure 10).60 The conditions established for the successful assembly of urease multilayers on the planar QCM substrates were subsequently employed to form enzyme multilayer shells on microparticle templates (470 nm PS spheres). The precursor film (PDDA/PSS/PDDA) with an additional outermost silica or magnetite nanoparticle layer provided a better surface for the formation of stable urease multilayer shells. Attempts to deposit urease onto PDDA/PSS/PDDA-modified PS particles yielded a low enzyme amount in the shells. This finding was attributed to the fact that weakly attached enzyme layers can be removed from the substrate surface by the next incoming polyion via the formation of water-soluble polyelectrolyte-enzyme complexes, as was found for histone/DNA interactions.64 In addition, improved stability with respect to adsorption of glucose oxidase

Figure 9. Absorbance at 588 nm by 2.9 mL of enzymatic activity assay solution after addition of 0.1 mL of urease-loaded microcapsule solution and the same test for addition of 0.05 mL of 0.02 mg/mL free urease.58 Reprinted with permission from reference 58. Copyright 2001 American Chemical Society.

gives a comparison of the catalytic activity of the urease loaded into the capsules and free urease. Urease encapsulated inside the LbL shell preserved 13% of its activity as compared to free enzyme. This decrease is mainly due to low substrate diffusion into the capsules. The urease activity inside the capsules was also stable as compared to free urease: after 5 days of storage at 7 °C, encapsulated urease completely preserved its activity, while free urease kept under the same conditions in aqueous solution lost 45% of its activity. The polyelectrolyte shell H


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particles and the subsequent use of these particles as catalysts. Also, the effect of formation of enzyme−polyelectrolyte complex prior to assembly of the LbL layers was investigated. POD (pI: 8.8) was assembled at pH values of 5.2 or 6.8 rendering the enzyme positively charged, with anionic PSS. For the premixing complexation experiments, POD and the oppositely charged polyelectrolyte PSS were added to a vial and then diluted with the appropriate buffer until reaching the required concentration. Zeta potential measurements showed an alternating trend (Figure 11) in the zeta potential for all multilayer films,

multilayers deposited onto gold nanoparticle layers, compared with glucose oxidase deposited on a less rough substrate, was observed. Hence, a primer nanoparticle layer was deposited on the PS spheres to increase the surface roughness and improve the adsorption stability of urease. The growth of the urease multilayers on the PS particles was followed by microelectrophoresis, where the zeta potential of the coated latex particles alternates between negative and positive values, corresponding to the sequential adsorption of cationic and anionic species, respectively.60 The catalytic activity of encapsulated and free urease was measured by monitoring the change in absorbance of the pHsensitive dye bromcresol purple, corresponding to the increase in pH due to the hydrolysis of urea giving ammonium hydroxide. The catalytic activity was found to increase with the increase in the number of urease layers deposited on the particles. Both urease/PSS multilayers and urease/PEI multilayers yielded very low catalytic activities (∼100 times lower) compared to the urease/PDDA multilayers despite being assembled in a similar fashion (according to the shell architectures). This may be due to the differences in compactness of the multilayers resulting from different polymer conformations on the surface (e.g., PDDA is a linear polycation and PEI is a branched polycation), different substrate diffusion rates and different degrees of blocking active sites of the enzyme. Compared to free urease, investigations revealed that the activity of immobilized urease (in a triple-layer shell) was 25% of that of free enzyme. This is a reasonable decrease because of substrate diffusion limitations and difficulties in reaching the active centers of immobilized urease. Another characteristic feature of the urease catalytic reaction is a 10 min dead time, during which no product was detected. A similar dead time was observed for low concentrations of free urease and, probably, is connected to accumulation of the reaction product.60 Similar LbL design and results for urease were also reported by Wang et al.65 Moreover, adding a magnetic functionality to the particles was investigated. Fe3O4 nanoparticles 12 nm in diameter were deposited in the shell architecture to supply the particles with a magnetic function. The shell sequence was [PDDA/PSS/ PDDA/12-nm Fe3O4/PDDA/(urease/PDDA)1−4]. The Fe3O4 nanoparticle distribution on the surface of the latex spheres was found to be less uniform than the silica shell and more than monolayer coverage. Nevertheless, the absolute enzymatic activity of the magnetic catalytic particles was similar to that of the corresponding particles with a layer of 40-nm silica. In addition, approaching a 0.3 T permanent magnet to the tube wall resulted in the collection of all of the modified latex spheres (on the wall region closest to the magnet) in ∼30 s. Immersing the permanent magnet into the solution containing the magnetic/urease-coated particles resulted in their collection on the magnet. This added magnetic function is particularly useful in applications where separation and reuse of such particles is required.60

Figure 11. Zeta potential of multilayers of (a) PSS/POD, (b) multilayers of POD−PSS complexes alternating with PAH.27 Reprinted with permission from reference 27. Copyright 2000 American Chemical Society.

depending on whether the polyelectrolyte or the enzyme formed the outer layer. This is a qualitative evidence for the stepwise deposition of polyelectrolyte and protein and suggests multilayer growth of charged macromolecules on colloid particles. Interestingly, measurement of the zeta potential of POD−polyelectrolyte complexes in solution yielded values close to those observed for colloids coated with the same polyelectrolyte used to form the complex, indicating that the polyelectrolyte charge dominates the observed value. SPLS was employed to quantitatively determine the thickness of the enzyme multilayers.67 SPLS allows the layer thickness of adsorbed macromolecules on particles to be measured with high precision (several nanometer changes in diameter can be discerned).68,69 Although it was expected that a monolayer coverage of POD would yield a thickness of about 3.5 nm (diameter with approximation from protein data bank), the layer thicknesses obtained corresponded to submonolayer coverage only. The answer was obtained by performing SPLS measurements on particles coated with a single outermost enzyme layer, which revealed that up to 80% of the originally adsorbed enzyme was removed as a result of adsorption of a subsequent polyelectrolyte layer. This indicated that, although a moderate amount of enzyme was adsorbed initially, a large fraction of this enzyme was adsorbed through interactions that

8. PEROXIDASE (POD) Peroxidases have conquered a prominent position in biotechnology and associated research areas (enzymology, biochemistry, medicine, genetics, physiology, histo- and cytochemistry). The importance of peroxidases is emphasized by their wide distribution among living organisms and by their multiple physiological roles.66Caruso et al.27 investigated the immobilization of horseradish POD via LbL coating on PS I


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are weaker than those resulting from subsequent association with the polyelectrolyte in solution. However, despite the removal of enzyme caused by polyelectrolyte adsorption, the remaining immobilized enzyme was successfully coated with polyelectrolyte (as confirmed by zeta potential measurements), hence facilitating deposition of the next enzyme layer. The loss of immobilized enzyme from the particle surface with subsequent polyelectrolyte adsorption was overcome by premixing the enzyme with a polyelectrolyte of opposite charge to form an enzyme−polymer complex in solution prior to adsorption. It was found that the complex could be successfully deposited in alternation with oppositely charged polyelectrolyte, leading to multilayer films assembled stepwise. In this method, the enzyme complex was not removed by the next polymer layer (within the sensitivity of SPLS) and the layer thicknesses were found to be larger than those for the films fabricated using noncomplexedenzymes.27Within experimental error, no loss of activity was observed for the immobilized POD enzymes. Moreover, an increase in total activity was observed for POD single-component enzyme systems with increasing layer number, reflecting that enzyme is immobilized in a stepwise manner. However, particles coated with the preformed complexes showed a lower total enzymatic activity than the corresponding enzyme multilayer films fabricated using the uncomplexed enzymes (about 1 order of magnitude less). Substrate diffusion limitation obviously plays an important role as the layers are more thickened in the case of using enzyme complexes compared to uncomplexed enzymes. The substrate binding sites on the enzyme may also become increasingly blocked or less accessible as a result of the additional complexation step.27 In different reports, encapsulation of peroxidase in hollow LbL capsules was also investigated. In one case, the LbL shell was made from PSS/poly(diallyldimethylammonium) chloride (PDADMAC) multilayers,70 and in the second case, the biodegradable polymers dextran sulfate and protamine were used to construct the capsule shell.36 In both cases, the idea was to deposit the polyelectrolyte multilayers on MF microcores followed by core decomposition at low pH. In such cases, the choice of polymers should be based on two main criteria: First, these polymers should not affect the enzyme activity. Second, the capsules composed of these polymers should be fairly stable, which is not always the case because the core dissolution procedure might lead either to shell rupture or even to disassembly of multilayer films.36In addition, the authors mentioned the importance of gradual lowering of pH to dissolve the MF cores, which even had to be tuned for MF cores from different lots. A sudden abrupt lowering of the pH may lead to a rapid dissolution of the capsule wall after core dissolution.36 Then, to load the enzyme into capsules, a suspension of the obtained empty LbL capsules was mixed with enzyme solution in buffered solutions (pH 8), followed by centrifugation and three cycles of washing to remove an excess of free enzyme molecules. To confirm the localization of the active enzyme into microcapsules, the reagent Amplex Red (10-acetyl-3,7dihydroxyphenoxazine) was used. In the presence of peroxidase, the Amplex Red reagent reacts with H2O2 to produce the red-fluorescent oxidation product, resorufin. Figure 12 shows that an increase of the fluorescence emission of resorufin was observed inside microcapsules but not in a surrounding solution.36 Apparently, peroxidase was embedded in a gel-like structure in the microcapsule interior formed by MF residues in

Figure 12. Imaging of peroxidase activity inside dextran sulfate/ protamine microcapsules: (A) transmission; (B−E) fluorescence image of resorufin after 1, 11, 12, and 13 min, respectively.36 Reprinted with permission from reference 36. Copyright 2003 American Chemical Society.

the complex with polyelectrolytes, which penetrated inside the capsules after the core was decomposed. Other experimental results proved that both loading and release processes were pH dependent.36

9. GLUCOSE OXIDASE (GOD) Glucoseoxidase (GOD) catalyzes the oxidation of β-D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide.71 GOD is the most widely employed enzyme as an analytical reagent for the selective determination of glucose, an analyte of clinical as well as of industrial interest.72 One of the practical aspects of assembling proteins in LbL films is their increased stability and retained activity.73 GOD was chosen as a model enzyme by Onda et al. to investigate the effect of immobilization of enzymes on catalytic activity, storage stability, thermostability, and pH dependency.73 GOD was assembled under conditions where it was negatively charged (pI = 4.2). On a quartz slide, four precursor bilayers of PEI/PSS were assembled by alternate deposition, followed by the deposition of two bilayers of PEI/GOD plus one more PEI layer at the top, so that the whole assembly may be denoted as (PEI/PSS)4 + (PEI/GOD)2 + PEI. The activity of immobilized GOD was assayed depending on the coupled enzymatic reaction of GOD and POD as illustrated in Figure 13. In principal, GOD converts D-glucose and O2 to nglucono-δ-lactone and hydrogen peroxide. Then, POD oxidizes DA67 (indicator) using H2O2 as oxidant. The reaction is easily followed by monitoring the absorbance at a wavelength of 665 nm using a spectrophotometer. The film was immersed in the upper space of a cuvette containing D-glucose and indicator

Figure 13. Sequential enzymatic process based on GOD and POD. J


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solution. The separation between the film and light beam was sufficient not to interfere with the light path.74 To study the long-term storage stability of immobilized GOD in LbL films, 45 samples were prepared on quartz slides. Among them, 5 films were stored in water at 25 °C, 20 films were stored in PIPES buffer (pH 7) at 4 °C, and the others were exposed to air at 4 °C. After given periods of time, each sample was washed with water, and their activity was measured using the procedure mentioned previously. As depicted in Figure 14, the film samples stored in water at 25 °C showed

Figure 15. Thermostability of GOD immobilized in LbL film and in aqueous solution. The samples were incubated at given temperatures for 10 min and their enzymatic activity was measured immediately after the incubation period and at 30−40 min after remaining in air at room temperature (approximately 22 °C). (a) Relative activity of aqueous GOD immediately after the incubation at given temperatures. (b) Relative activity of aqueous GOD 30 min after the incubation. (c) Relative activity of GOD in the film immediately after the incubation. (d) Relative activity of GOD in the film 30 min after the incubation. The activity relative to that at 22 °C is plotted against incubation temperature.73 Reprinted from reference 73, Copyright 1999, with permission from Elsevier.

Figure 14. Storage stability of GOD immobilized in LbL films: (a) storage in pure water at 25 °C; (b) storage in air at 4 °C; (c) storage in PIPES buffer (PH 7) at 4 °C.73 Reprinted from reference 73, Copyright 1999, with permission from Elsevier.

tional changes, and permanent denaturation due to chemical changes.75 The recovered activity in the present case may be related to partial unfolding. On the other hand, GOD in alternately assembled films showed a remarkable improvement in thermostability (Figure 15c). Significant reduction in the activity was not noted even after incubation at 50 °C, although incubation at above this temperature caused a rapid loss of activity. Interestingly, recovery such as found for aqueous GOD was not observed in the film sample at any incubation temperature (Figure 15d). The enhanced enzymatic activity and the absence of reversible changes (room-temperature recovery) were attributed to suppression of conformational mobility of GOD by surrounding polymer chains.73 Nevertheless, deposition of enzyme multilayers on planar surfaces has a major drawback. When more enzyme multilayers are deposited to increase the total catalytic activity of the system, eventually the overall catalytic activity per enzyme layer decreases. This is mainly attributed to the hindered substrate diffusion due to the thickness of the deposited film. Schüler et al. used GOD LbL films to coat colloidal submicrometer polystyrene spheres rather than quartz slides, where GOD multilayers were deposited alternately with PEI. Microelectrophoresis and SPLS measurements revealed regular and stepwise assembly of the multilayers on the colloids. The enzymatic activity was found to increase regularly with an increasing number of GOD layers (i.e., GOD amount) immobilized, indicating that the enzyme multilayer films were sufficiently permeable for substrate diffusion. Thus, unlike multilayer films on planar surfaces, substrate diffusion effects can be avoided with enzyme multilayer-coated colloids since thick or dense protein films are not necessarily required to increase the enzymatic activity: the high surface area afforded by the particles, and control of the total number of particles in

drastic decreases in activity, and approximately 70% of the activity was lost after 4 weeks. The authors mentioned that the reason for this deterioration was unclear; nevertheless, they speculated that bacterial growth was the cause of deterioration. In contrast, the films kept in the buffer at 4 °C did not show a significant decrease in enzymatic activity over 14 weeks. The films kept in air at 4 °C showed 10% decrease in the first week, but the activity was maintained during the following 13 weeks. The initial activity loss was probably due to air-drying of the film. It was clearly shown that GOD activity was unaltered in the assembled films.73 Proteins lose their activity upon denaturation by heat, thus investigating thermostabilization offered by LbL structures was of high interest. Immobilization may suppress structural deformations of enzymes, thus enhancing their stability. Before measuring the enzyme activity, the extent of release of GOD from LbL films caused by heat was examined and no GOD was released into water even after the film was immersed in hot water for 2 h. The GOD LbL films on quartz plates were incubated in water at a given temperature for 10 min. Immediately after the incubation period, measurement of enzymatic activity was performed at 25 °C. The second measurement was carried out after keeping the film in air at room temperature for 30−40 min. Separate samples were used for different temperature conditions. Aqueous GOD rapidly lost activity on incubation at 30−40 °C (Figure 15a), becoming totally inactive at 50 °C. However, this loss in activity was partially recovered on returning the solution to room temperature (Figure 15b). The recovered activity decreased with the incubation temperature, and totally disappeared at 70 °C. It is reported that enzymes undergo destabilization by two different mechanisms: reversible unfolding due to conformaK


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introduced by Zhu et al.,22 based on an interesting feature of the photosensitive material diazoresin, where the weak ionic interactions between diazoresin and PSS are converted to stable covalent bonds upon irradiation with UV. The diazoresin attracted attention because it can improve the stability of the multilayer films by converting weak interactions between neighboring layers into covalent bonds. Furthermore, it may also be applied to a variety of materials containing sulfonate groups, carboxylic acid groups, or phenol groups.77−79 The procedure is simple and involves only two steps: first, ionic (for sulfonate groups) or H-bonding (for carboxylic acid or phenol groups) self-assembly, which forms the initial multilayer; second, UV irradiation, which converts the weak interaction between the neighboring layers to a covalent one (Figure 17).

solution (wt %), allowed for the activity to be conveniently optimized.76 In a different study,27 GOD was used as a model enzyme to investigate the effect of adding layers above the enzyme layer on enzymatic activity. Thus, one GOD layer was immobilized in the interior with multiple PAH/PSS polyelectrolyte layers deposited on top. The GOD activity was measured (as described before) as a function of increasing polyelectrolyte layer number (Figure 16). The decrease in activity most likely

Figure 16. Relative activity of PS particles coated with one layer of GOD as a function of additionally deposited PAH/PSS layers. The activity of the first GOD layer after adsorption of the first polyelectrolyte layer on top of it was normalized to 100% as the polymer adsorption step caused removal of some of the enzyme.27 Reprinted with permission from reference 27. Copyright 2000 American Chemical Society.

Figure 17. Schematic representation of PSS/DAR structure changes upon UV irradiation.22 Reprinted with permission from reference 22. Copyright 2005 American Chemical Society.

reflects diffusion phenomena; the more layers above the enzyme layer, the more it becomes difficult for the substrate molecule to diffuse into the enzyme layer. However, it is also possible that some of the enzyme catalytic centers may be blocked by deposition of additional polyelectrolyte layers. It is well-known that polyelectrolytes within multilayer films interpenetrate one another, and a layer can interpenetrate 3− 4 adjacent polymer layers.4 Hence, immobilized enzyme coated with multiple polyelectrolyte layers may have significantly less enzymatically active sites exposed for catalysis reactions than enzyme that is covered by a single polymer layer.27 In an attempt to add more functionality, the group of Caruso et al.27 also went for adding magnetic properties to the LbL enzyme coated particles. PS particles (200 nm) were precoated with four layers of Fe3O4 nanoparticles and PDADMAC, followed by two additional polyelectrolyte layers (PSS/PAH) and an outer GOD layer. The particles were fabricated using the LbL approach, beginning with the magnetic nanoparticles and followed by the enzyme multilayers. The magnetic functionalized particles were drawn to the bottom of a reaction tube by a magnet. The activity of the GOD layer on the particles was measured, and the particles were then separated with a magnet, after which they were washed several times with water and the cycle was repeated. The measured activity was within 15% for each cycle, showing that it was possible to recover the particles and that the immobilized enzyme remained active after cycling. The above strategy opened a promising pathway for the fabrication of tailored magnetic, biocatalytic, and reusable particles. Moving from surface coating on particles to encapsulation in LbL shells, a novel LbL design for encapsulation of GOD was

Efforts were made to prepare diazoresin-based hollow polyelectrolyte microcapsules encapsulating GOD by LbL assembly on MnCO3 templates as a potential glucose biosensor. MnCO3 particles were coated with one bilayer of (PSS/PAH), and then 1−5 bilayers of (PSS/DAR), followed by one bilayer of (PSS/PAH) as the outer layer. Hollow microcapsules were obtained by decomposing the MnCO3 cores with 0.1 M HCl solution for 20 min. The microcapsule suspension was mixed with rhodamine labeled GOD, for 5 min. While still in the enzyme loading solution, the capsules were irradiated with a UV lamp for 5 min to cross-link the multilayer wall, and then rinsed with deionized water. The inclusion of one (PSS/PAH) bilayer as both the inner and the outer layers was applied to strengthen the microcapsule wall architecture prior to cross-linking (inner layer) and prevent particle aggregation upon UV irradiation (outer layer), which was previously observed to occur in cases where DAR was the terminal layer. UV−vis and zeta potential measurements confirmed the alternate deposition of (PSS/DAR) multilayers on the micrometer-sized dissolvable templates.22 The reaction velocity of the enzyme-catalyzed reaction was determined by an increase in absorbance at 500 nm resulting from the oxidation of o-dianisidine through a peroxidasecoupled system. Glucose oxidase catalyzes the oxidation of glucose to gluconic acid, during which the generation of H2O2 is indirectly measured by oxidation of o-dianisidine in the presence of peroxidase. Encapsulated GOD inside the DARbased microcapsules effectively preserved 52.8% of the total activity per unit mass as compared to the free enzyme. This decrease can be attributed to either partial loss of enzyme structure, transport limitation due to the capsule walls and partitioned enzyme, or a combination of both factors.22 L


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glucose, which showed the good selectivity of the enzyme electrode.80 Based on the direct electron transfer between GOD and electrode surface, this “mediator-free” approach proves to be superior in its stability and convenience of electrode preparation compared to previously reported biosensors having an electron-transfer mediator applied to shuttle the electrons between GOD and the electrode.80

Probably one of the most useful practical applications of immobilizing GOD on solid surfaces is the fabrication of glucose biosensors. GOD is a structurally rigid glycoprotein with two identical polypeptide chains, each containing a flavinadenine dinucleotide (FAD) redox center.80 GOD is reported to be electroactive only in limited cases, and that GOD electron-transfer and enzyme activity are very sensitive to the environment.81,82 The direct electron-transfer behavior of the GOD electrode in the absence of oxygen occurs as follows:

10. SEQUENTIAL ACTION OF MULTIPLE ENCAPSULATED ENZYMES One of the unique features of LbL technology is the ability to encapsulate multiple enzymes in the same polymeric film at precisely controlled distances, which add more functionality to the microreactors and opens the door for many innovative applications.27,84,85 10.1. POD and GOD. Molecular films of POD and GOD were assembled in combination with PSS and PEI, respectively, by means of alternate adsorption through electrostatic interaction on a quartz slide. A precursor film of four layers of PEI/PSS was initially deposited followed by enzyme multilayers, and the whole film assembly can be represented as84

GOD−FAD + 2e− + 2H+ ↔ GOD−FADH 2

In the presence of oxygen, the reduced enzyme is oxidized very quickly at the surface of the electrode: GOD−FADH 2 + O2 → GOD−FAD + H 2O2

The catalytic regeneration of the enzyme in its oxidized form causes the loss of reversibility and the increase in size of the reduction peak. Upon the addition of glucose, a competitive reaction occurs at the vicinity of the electrode surface, which leads to the decrease of the reduction peak and thus the sensitive determination of glucose. Hodak et al.83 reported LbL assembly of GOD with PAHferrocene redox mediator, using a gold electrode modified by a thiol layer with negatively charged end groups on which electrostatically layers of a positively charged redox polymer, PAH with ferrocene redox sites (Fc), attached along the backbone, and polyanionic GOD were built. The main drawback of this design was the need for an electron-transfer mediator to shuttle electrons between the redox centers of GOD and the electrode. However, the redox mediators used in conjunction with redox proteins are in no way selective but rather general, facilitating not only electron transfer between electrode and protein but also various interfering reactions. Therefore, a mediator-free glucose biosensor based on LbL immobilization of GOD was introduced by Zhang et al.80 The measurement cell consisted of a saturated calomel electrode as the reference electrode, and a platinum wire electrode served as the counter electrode and pyrolytic graphite electrode with a modified surface by LbL deposition of GOD and PEI. The immobilized GOD retained its catalytic activity and the electrode coated with a single GOD−PEI bilayer responded linearly to the glucose concentration ranging from 0.5 to 8.9 mM (r2 = 0.998), with a sensitivity of 0.66 μA mM−1 and an estimated detection limit of 0.1 mM. Interestingly, the deposition of a second bilayer of PEI/GOD increased the sensitivity to 0.76 μA mM−1 and lead to a detection limit of 50 μM. This increased sensitivity is believed to arise from higher enzyme loading. Noteworthy, the further deposition of PEI/ GOD layers could not increase the sensitivity, suggesting that the enzymatic activity correlates with the electron-transfer reactivity of GOD. The stability of the two PEI/GOD bilayersmodified PGE was evaluated by examining the response current of the enzyme electrode. After the electrode was kept at 4 °C in phosphate buffer (pH 7.0) for 1 month, 93% of the original current of reduction peak remained, and its activity to glucose remained at about 90%. Thus, this enzyme electrode has good stability and might be used in glucose biosensor fabrications. Possible interference of substances, such as ascorbic acid and acetaminophen, was also tested with no significant changes in reduction peak current observed for ascorbic acid or acetaminophen at a concentration 5-fold higher than that of

(PEI/PSS)4 + (POD/PSS)2 + (PEI/GOD)2

Coupled enzymatic reactions used in this study are shown in Figure 13. GOD converts D-glucose and O2 to D-glucono-δlactone and hydrogen peroxide. Then POD oxidizes DA67 (indicator) by using H2O2 as oxidant. The reaction was followed with Vis/UV/NIR spectroscopy by monitoring the absorbance at a wavelength of 665 nm, which was attributed to the appearance of the oxidized form of redox dye DA67. Results in Figure 18 clearly show that the immobilized enzymes were still capable of carrying the mentioned sequential reaction within the LbL film.

Figure 18. Time course of the conversion of the sequential enzymatic reaction catalyzed by the multienzyme film of GOD and POD. The conversion was monitored by the absorption changes at 665 nm due to formation of oxidized DA67. The film was immersed in the substrate solution [56 pM D-glucose, 200 pM DA67] at the point indicated by the arrow.84 Source: Biotechnol Bioeng, 51, (2), 1996, 163−7. Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Copyright 1996 John Wiley & Sons, Inc. This material is reproduced with permission of John Wiley & Sons, Inc.

In 2007, Kreft et al.86 reported a novel “shell-in-shell” LbL microcapsule having two distinct and separated compartments, where POD and GOD were entrapped in the inner and outer compartments, respectively. Following a 6-step procedure (Figure 19) and using the CaCO3 core dissolution technique, the authors were able to create two spaces separated by a semipermeable PE multilayer membrane. Step one comprises M


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Figure 19. General route for the synthesis of shell-in-shell microcapsules. (A) initial core; (B) core−shell particle; (C) ball-in-ball particle; (D) ballin-ball with an external PE bilayer; E = shell-in-shell microcapsule; PEM: polyelectrolyte multilayer.86 Source: Angew. Chem., Int. Ed. Engl. 46, (29), 2007, 5605−8. Kreft, O.; Prevot, M.; Mohwald, H.; Sukhorukov, G. B. Copyright 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This material is reproduced with permission of John Wiley & Sons, Inc.

Figure 20. Sequential enzymatic process based on GA and GOD and its experimental setup.85 Reprinted from reference 85, Copyright 1996, with permission from Elsevier.

product) molecules to diffuse in and out, whereas large enzyme molecules are retained in separated compartments.86 10.2. Glucoamylase and Glucose Oxidase. Separate molecular layers of glucoamylase (GA) and GOD (pI’s of GA and GOD are 4.2 and 4.3, respectively) were assembled on an ultrafilter (Molcut II LC, cutoff molecular weight 5 kDa, Millipore) by LbL adsorption using PEI as a polycation.85 The Molcut II system is composed of an upper cup, an ultrafilter, and a lower cup (Figure 20). Protein alternate films were assembled on the filter, which was fixed on the bottom of the upper cup. Aqueous PEI solution was placed in the upper cup for 15 min at room temperature, and then the inside of the upper cup was washed carefully with water for 2 min. Aqueous PSS solution was then placed in the upper cup for 15 min at room temperature, and the cup was washed again. These procedures were repeated three times to produce precursor layers of (PEI/PSS)4, then continued to build the following film structures using aqueous GA and GOD solutions:

coprecipitation of calcium carbonate from its precursors CaCl2 and Na2CO3 in the presence of POD and magnetite nanoparticles. In the second step, five bilayers of PSS/PAH were deposited. The resulting core−shell particles were subjected in the third step to a second coprecipitation process, this time in the presence of GOD leading to “ball-in-ball particles” formation. These particles are characterized by a polyelectrolyte multilayer that is “sandwiched” between two calcium carbonate compartments. During the second coprecipitation step, the formation of single core CaCO3 particles filled with GOD only could take place. Owing to the magnetic properties of the inner compartment, it was possible to collect the “ball-in-ball” particles by applying a magnetic field while all the nonmagnetic coproducts were removed in the washing step (step 4). In step 5, the building process is finalized by depositing a terminal PSS/PAH bilayer. In the last step, EDTA treatment led to the dissolution of all calcium carbonate constituents and, thus, the formation of a shell-in-shell capsule. The sequential action of GOD and POD encapsulated in the shell-in-shell microcapsules was tested using glucose and amplex red. Oxidation of glucose by GOD leads to the formation of H2O2, which in the presence of POD is used to convert amplex red, into the highly fluorescent resorufin. The coupling of both enzymatic reactions was proven successful by CLSM images, due to the semipermeable character of the polyelectrolyte shell, which allows small substrate (and

(1) Film 1: filter + (PEI/PSS)4 + (PEI/GOD)2 + (PEI/ PSS)2 + (PEI/GA)2 + PEI (2) Film 2: filter + (PEI/PSS)4 + (PEI/GOD)2 + (PEI/ PSS)10 + (PEI/GA)2 + PEI (3) Film 2: filter + (PEI/PSS)4 + (PEI/GA)2 + (PEI/PSS)2 + (PEI/GOD)2 + PEI (4) Film 2: filter + (PEI/PSS)4 + (PEI/GA)2 + (PEI/PSS)10 + (PEI/GOD)2 + PEI N


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Figure 21. (a) Schematic illustration of the structure of the bienzyme multilayer film. E1 refers to glucose oxidase and E2 refers to glucoamylase. (b) The bipolar quaternary ammonium salt NC6BPC6N.88 Source: Macromol Chem Phys, 197, 1996, 147−153. Sun, Y.; Zhang, X.; Sun, C.; Wang, B.; Shen, J. Copyright 1996 Hüthig & Wepf Verlag, Zug. This material is reproduced with permission of John Wiley & Sons, Inc.

Film formation on the filter was confirmed by X-ray photoelectron spectroscopy (XPS) elemental analysis. Only carbon and oxygen were observed from a bare filter membrane (cellulose). After polyions and proteins had been assembled, nitrogen and sulfur were also detected. The enzymatic activities of these films were examined using the experimental setup depicted in Figure 20. An aqueous solution of “water-soluble starch” was placed on the enzymeimmobilized ultrafilter in the upper cup. Filtration was started by applying pressure to the upper cup with a syringe to achieve a constant flow rate. Then, 1 mL of PIPES buffer was added to the upper cup and the same pressure was applied. The latter procedure was repeated several times in order to wash out the filterable components. As per the reaction scheme shown in Figure 20, the glycosidic bonds in starch are hydrolyzed by GA producing glucose, which is converted to gluconolactone by GOD with H2O2 as a coproduct. Then unreacted starch, H2O2, and glucose were quantified. The highest reaction yield was obtained with film 2, and the second highest yield was with film 1. In these two films, the arrangement of protein layers against the direction of flow agrees with the order of the sequential enzymatic reactions. By contrast, when the order of the two protein layers was reversed, as in films 3 and 4, lower yields were obtained. These results indicated that appropriate arrangement of the two enzymes is crucial for obtaining high yields in the sequential reactions, since starch is hydrolyzed to glucose in the outer GA layers, and the glucose produced is subsequently converted to gluconolactone and H2O2 in the inner GOD layers. By contrast, in the case of films 3 and 4, starch had to pass through GOD layers and separator layers, (PEI/PSS)n, before it reached GA layers, and the glucose produced had to diffuse back to GOD layers against the flow of the solution. This, of course, slows down the whole process. In particular, diffusion of high molecular-weight starch through multilayers would be rate limiting, and facile contact of starch with GA layers is important. The role of the spacer layer that separates the GA layers from the GOD layers becomes evident from a comparison of the activities of films 1 and 2. It is curious that higher yields of glucose and H2O2 were obtained using film 2 rather than using film 1, regardless of the initial starch concentration. This cannot be explained by the ease of diffusion. Reports have shown that for multicomponent assemblies, film growth is somewhat smaller in the first few steps when the assembled pair is

changed. The GA-PEI layers might not be ideally prepared in film 1 because film 1 has thinner spacer layers of PEI−PSS than film 2, so a smaller amount of GA will be immobilized on film 1, reducing the total activity. Another plausible mechanism is related to the inhibition of GA activity by gluconolactone. Small molecules such as gluconolactone and H2O2 should be able to diffuse backward to the GA layers relatively easily. They are inhibitors of GA and reduce its enzymatic activity. A mixed type inhibition of the GA by gluconolactone was reported.87 The extent of this inhibition must be larger in film 1 than in film 2, because the spacer layer of the latter is thicker. This novel approach of combining ultrafiltration with the use of a layered protein film allowed the successful separation of substrate and products without any further procedure.85 The concept of sequential action of GA and GOD was implemented by Sun et al., to develop a maltose sensor.88 In principal, GA breaks maltose into two molecules of D-glucose, which are subsequently oxidized by GOD to gluconic acid with the production of hydrogen peroxide: glucoamylase

Maltose + H 2O ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ 2 D‐glucose glucoseoxidase

B‐D‐glucose + O2 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ gluconic acid + H 2O2

H 2O2 → O2 + 2e− + 2H+

The oxidation current of H2O2 produced in the enzymatic reaction has a linear relationship with the concentration of maltose within a certain range. The electrode structure is illustrated in Figure 21a: the enzyme multilayer films are fabricated on a gold electrode, so that GOD and GA are alternatingly sandwiched between the bipolar quaternary ammonium salt NC6BPC6N (Figure 21b). In detail, a gold electrode was polished with aluminum powder, sonicated in fresh water and allowed to air-dry. The clean gold electrode was immersed for 24 h at room temperature in an ethanolic solution of 3-mercaptopropionic acid to give one monolayer of self-assembled film. Afterward, it was transferred into the NC6BPC6N solution (pH = 8), thus a positively charged surface was obtained. The modified cationic electrode was deposited with two layers of GOD followed by two layers of GA alternating with NC6BPC6N. The resulting modified electrode was applied as a maltose sensor. So when it was immersed into the maltose solution, maltose first reacted with outside GA layers to produce glucose. The resulting b-DO


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glucose then diffused into the assembly and reacted with interior GOD layers to produce H2O2, which oxidized on the surface of the gold electrode. It was shown that the time required to reach 95% of the steady-state current is less than 60 s after the addition of the maltose sample. The calibration curve for the determination of maltose was measured. A linear relationship up to 6 mmol L−1 was obtained between the current response and maltose concentration. In the experiment, the authors also found that no current is observed when the bare gold electrode, the electrode with 3-mercaptopropionic acid and NC6BPC6N layers, and finally the electrode with a protein bilayer of GA alone were immersed in the experimental solutions that contained maltose. The above experimental results showed that only when GA and GOD were fabricated rationally on the surface of the gold electrode can the sensor be used to determine maltose.88 10.3. Glucose Sensitive Multilayers Based on Sequential Actions of Multiple Enzymes. Insulin is the mainstay of drug therapy for patients with insulin-dependent diabetes mellitus (IDDM or Type I diabetes). However, there are several limitations to the delivery of insulin, such as poor permeability because of its high molecular weight, lack of lipophilicity, and short half-life of 5−20 min as a result of the inactivation and digestion by various proteolytic enzymes. Most patients need to administer at least three or four injections of insulin per day to reach near normal glycemia control, resulting in inconvenience and side-effects, and thus low patient compliance.89 Different approaches of LbL coatings were investigated to meet this challenge. Attempts ranged from using insulin as a model protein90 up to the design of multilayer films that are glucose sensitive showing on/off release pattern according to the presence/absence of glucose molecules in the surrounding medium.91Sodium chloride was typically used to salt out insulin aggregates from mild HCl solutions (pH 1−3).19,89 Insulin aggregates are positively charged at this low pH (insulin PI=5.5), which makes it possible to coat them with a layer of a negative polyelectrolyte. After washing 2−3 times, another layer of a positive polyelectrolyte is applied. Qi et al. fabricated glucose-sensitive multilayer shells as insulin carriers via LbL assembly.92 The design depends on an assembly of alternating layers of GOD and CAT on insulin crystals stabilized by glutaraldehyde cross-linking through the formation of CN bonds between each protein and glutaraldehyde through a Schiff’s base reaction. In principal, GOD converts glucose into gluconic acid releasing molecular oxygen and producing H2O2. The production of gluconic acid lowers the pH value at the surface of the shells and enhances the permeability of shells and the dissolution of insulin. This enhancement of permeability was attributed to the fact that acidic pH weakens or partially breaks the Schiff’s base complex rendering the shells more lose and thus more permeable. In addition, the decrease in pH favors a higher insulin solubility in water, thus facilitating the release from the system.92 Nevertheless, GOD activity may suffer decay with time due to peroxide-introduced degradation, leading to low sensitivity to glucose. In this scenario, it is the role of CAT to convert the aggressive H2O2into H2O and O2, with most of the oxygen produced consumed by GOD. For this reason, the two enzymatic reactions were coupled together by codeposition of alternating layers of GOD and CAT on insulin particles, and cross-linked by glutaraldehyde.92

Figure 22 shows a proof of this concept, where about 40% of insulin was seen to be released from the CAT/GOD multilayer

Figure 22. Release profiles of coated insulin particles in PBS (black) and in glucose solution (red), respectively. Each error bar represents the mean of three measurements at least (±SD).92 Reprinted from reference 92, Copyright 2009, with permission from Elsevier.

shells after 3 h of incubation in a glucose solution. Then the release rate gradually decreased until 6 h and finally the rate went back to zero. While in PBS buffer, little insulin was released from the CAT/GOD multilayer shells. Therefore, the CAT/GOD multilayer shells coated onto insulin particles can tune the release of insulin with response to external glucose. Unfortunately, the main drawback of the described approach was the loss of activity of a high percentage (57%) of GOD upon cross-linking by glutaraldehyde. Thus, a nonbonding attachment may be better for the preservation of enzyme activity. This was the motive for Chen et al.91 to introduce a novel multilayer design of glucose-sensitive LBL multilayer film based on a 21-arm star polymer (globular shape with multiple arms connected at a central core), showing an on−off controlled release of insulin in response to glucose concentration changes (Figure 23). The LBL multilayer film consisted of positively charged poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) star polymer and negatively charged insulin and GOD. Considering that the pI values of insulin and GOD are 5.5 and 4.2, respectively, the pH conditions of adsorption solutions (pH 6) make them both negatively charged to be absorbed on a layer of positively charged 21-arm star PDMAEMA. Thus, positively charged star PDMAEMA was applied as the first layer on negatively charged quartz slides, followed by a layer of negatively charged insulin, and so on until four (star PDMAEMA/insulin) bilayers were fabricated continuously. Those were covered by another four (star PDMAEMA/GOD) bilayers. A top layer of star PDMAEMA was intentionally added as the surface layer to prevent the leakage of GOD and control the release of built-in insulin after being immersed in glucose solution. The use of electrostatic attraction as the dominant driving force for film construction is less harsh on the enzymatic activity of GOD compared to cross-linking with glutaraldehyde in the previous example.92 The mechanism of insulin release from the multilayer film (Figure 23) depends on the ability of GOD to convert glucose into gluconic acid, and thus lower the pH microenvironment within the LBL multilayer film. The decrease in pH will induce star PDMAEMA to rearrange the film morphology due to the changes in ionization of the polyelectrolyte to accomplish the distinctive geometry of “star structure”, since the arms of star P


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Figure 23. Schematic representation of the on−off regulation of insulin release from the glucose-sensitive self-assembled multilayer film based on 21arm star polymer.91 Reprinted from reference 91, Copyright 2010, with permission from Elsevier.

11. CHALLENGES AND FUTURE PERSPECTIVES LbL techniques have acquired enormous interest in the last 10 years, and this is clearly manifested in the increase in publications in literature investigating different aspects related to this technology, starting from a material science point of view and ending with applications in many fields such as surface functionalization, biosensing and drug delivery. The versatility of the technique, the freedom to use a wide pool of polymers and/or building units, the different end products and geometries (films, spheres, capsules, etc.), and the fine control on the building process are powerful tools that enable scientists to achieve new applications that could not be done before. In the field of biomacromolecules, one of LbL’s unique advantages is the sole use of aqueous solutions and the avoidance of relatively harsh conditions related to organic solvents and heat treatment that significantly affect the activity of these molecules. However, a wider look on the current standing point can raise the question of why such a promising technology was not turned into products so far? A deeper look shows that most research efforts are still locked in the lab phase, and production techniques are only efficient at small scale. To move this technology further, some problems have to be overcome. A major challenge is developing a better production technique that enables large scale production of LbL structures, as the current used processes depend mainly on successive cycles of suspension−centrifugation−resuspension, which makes the production time too lengthy. In addition, the large volumes needed and wasted during production is quite a challenge. To elaborate on this, most of published works use 10 mL of PE solution (1 mg/mL) to coat 1 g of particles, and a significant amount of added PE is lost and not adsorbed. This means that for producing 1 kg of coated particles, 100 L of PE solution would be needed for one layer, and stable structures are composed of 10−20 layers on average (i.e., 1000−2000 L), which means great amounts of PEs are lost. One approach that was recently described is using automated filtration techniques for LbL deposition, where particles are soaked in the PE solution, which is then removed by filtration. However, the problem of waste of time and ingredients is still present. Another significant challenge that prevents the move forward from in vitro to in vivo studies is the use of non-biocompatible/ biodegradable PEs, which are considered toxic to living organisms. PSS, PAH, PEI, and PDMAEMA are widely used in many LbL designs. That would be accepted with regards to lab uses as chemical reactors and electrodes, but will not find its way to marketed products intended for use in humans. Luckily, many research groups have shifted toward the use of biopolymers such as chitosan and alginate, which are safe and accepted for internal use. However, biopolymers are much weaker polyelectrolytes, and thus different LbL design strategies should be adopted.

PDMAEMA would become highly ionized and more stretched resulting in an “open” status of the film to release insulin. Moreover, it is also reasonable that part of the insulin has changed to become positively charged while the pH of microenvironment decreases to below its isoelectric point (5.5). Electrostatic repulsion between star PDMAEMA and positively charged insulin can also increase its release. Since GOD has a lower isoelectric point (4.2) and higher molecular weight (∼154 kDa) than insulin (MW 5.8 kDa), its leakage from the LBL multilayer film under the same conditions should be much less than that of insulin. On the other hand, when the film is in glucose-free medium, GOD could no longer exert its functions to produce gluconic acid. The pH of the microenvironment within the LBL multilayer film will gradually increase back to neutral pH, which enables the star PDMAEMA arms to relax and become less ionized and stretched, restoring the compact nature of the multilayer film and decreasing insulin release. In addition, insulin within the film will restore its negative charge, which enhances the electrostatic attraction and thus leads to more anchoring to star PDMAEMA layers.91 To evaluate and challenge the glucose-sensitivity of the LbL multilayer film, repeated “on−off” release of insulin was performed by immersing the film alternately in PBS with or without glucose. As shown in Figure 24, it is obvious that more

Figure 24. Repeated on−off release of insulin from the [(Star PDMAEMA/Insulin)4 + (Star PDMAEMA/GOD)4 + Star PDMAEMA] multilayer films in response to stepwise glucose challenge at 37 °C.91 Reprinted from reference 91, Copyright 2010, with permission from Elsevier.

insulin was released from the film with star PDMAEMA when glucose was present in the medium. Stepwise glucose treatment challenge reveals quick “on” and “off” responses for insulin release from the LBL multilayer film.91 Q


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(12) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117−6123. (13) Borden, M. A.; Caskey, C. F.; Little, E.; Gillies, R. J.; Ferrara, K. W. Langmuir 2007, 23 (18), 9401−8. (14) Lvov, Y.; Ariga, K.; Onda, K.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195−6203. (15) Tedeschi, C.; Caruso, F.; Mohwald, H.; Kirstein, S. J. Am. Chem. Soc. 2000, 122, 5841−5848. (16) Watanabe, S.; Regen, S. L. J. Am. Chem. Soc. 1994, 116, 8855− 8856. (17) Cheung, J. H.; Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2712. (18) Macdonald, M.; Rodriguez, N. M.; Smith, R.; Hammond, P. T. J. Controlled Release 2008, 131 (3), 228−34. (19) Fan, Y. F.; Wang, Y. N.; Fan, Y. G.; Ma, J. B. Int. J. Pharm. 2006, 324 (2), 158−67. (20) Moraes, M. L.; de Souza, N. C.; Hayasaka, C. O.; Ferreira, M.; Rodrigues Filho, U. P.; Riul, A., Jr.; Zucolotto, V.; Oliveira, O. N., Jr. Mater. Sci. Eng. C 2009, 29, 442−447. (21) Volodkin, D. V.; Balabushevitch, N. G.; Sukhorukov, G. B.; Larionova, N. I. Biochemistry (Moscow) 2003, 68 (2), 236−41. (22) Zhu, H.; McShane, M. J. Langmuir 2005, 21 (1), 424−30. (23) Tiourina, O. P.; Antipov, A. A.; Sukhorukov, G. B.; Larionova, N. I.; Lvov, Y.; Möhwald, H. Macromol. Biosci. 2001, 1, 209−214. (24) Lvov, Y.; Mohwald, H. Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Marcel Dekker: New York, 2000. (25) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films. 1992, 210/211, 710−712. (26) Cassier, T.; Lowack, K.; Decher, G. Supramol. Sci. 1998, 5, 309− 315. (27) Caruso, F.; Schüler, C. Langmuir 2000, 16, 9595−9603. (28) Mansouri, S.; Winnik, F. M.; Tabrizian, M. Expert Opin. Drug Delivery 2009, 6 (6), 585−97. (29) Pavlukhina, S.; Sukhishvili, S. Adv. Drug Delivery Rev. 2011, 63 (9), 822−36. (30) Antipov, A. A.; Sukhorukov, G. B. Adv. Colloid Interface Sci. 2004, 111 (1−2), 49−61. (31) De Koker, S.; De Cock, L. J.; Rivera-Gil, P.; Parak, W. J.; Auzely Velty, R.; Vervaet, C.; Remon, J. P.; Grooten, J.; De Geest, B. G. Adv. Drug Delivery Rev. 2011, 63 (9), 748−61. (32) Delcea, M.; Mohwald, H.; Skirtach, A. G. Adv. Drug Delivery Rev. 2011, 63 (9), 730−47. (33) Wohl, B. M.; Engbersen, J. F. J. Controlled Release 2011, 158 (1), 2−14. (34) Chelikani, P.; Fita, I.; Loewen, P. C. Cell. Mol. Life Sci. 2004, 61 (2), 192−208. (35) Balabushevich, N. G.; Zimina, E. P.; Larionova, N. I. Biochemistry (Moscow) 2004, 69 (7), 763−9. (36) Balabushevich, N. G.; Tiourina, O. P.; Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2003, 4 (5), 1191−7. (37) Caruso, F.; Trau, D.; Mohwald, H.; Renneberg, R. Langmuir 2000, 16, 1485−1488. (38) Caruso, F.; Niikura, K.; Furlong, N.; Okahata, Y. Langmuir 1997, 13, 3427−3433. (39) Jin, W.; Shi, X.; Caruso, F. J. Am. Chem. Soc. 2001, 123 (33), 8121−2. (40) Yu, A.; Caruso, F. Anal. Chem. 2003, 75 (13), 3031−7. (41) Kim, S.; Park, J.; Cho, J. Nanotechnology 2010, 21 (37), 375702. (42) Balabushevitch, N. G.; Sukhorukov, G. B.; Moroz, N. A.; Volodkin, D. V.; Larionova, N. I.; Donath, E.; Mohwald, H. Biotechnol. Bioeng. 2001, 76 (3), 207−13. (43) Fritz, H.; Wunderer, G. Arzneim.-Forsch. 1983, 33, 479−494. (44) Tiourina, O. P.; Sukhorukov, G. B. Int. J. Pharm. 2002, 242 (1− 2), 155−61. (45) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16 (9−12), 849−56. (46) Shin, Y. J.; Kameoka, J. J. Ind. Eng. Chem. 2012, 18, 193−197.

12. CONCLUSIONS LbL technology offers a versatile tool to implement a wide variety of architectures and designs. In this review we provide an overview of the recent developments in the use of LbL to encapsulate enzymes in films, spheres, capsules, and so on (Table 1). Special importance was given to the encapsulation of multiple enzymes and their sequential action in LbL films. The flexibility of the technique together with the large selection pool of polyelectrolytes made it possible to obtain many LbL structures that were able, in many cases, to show superiority over other encapsulation techniques in terms of stability and activity of the encapsulated enzyme. Yet, techniques introduced here represent only a few of the potential possibilities for LbL assembly, and further applications will require compositional variations and alterations in architecture for more optimization. We think that the advantages offered by this technology would result in various important opportunities and applications in the fields related to enzymatic processes.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +41223796945. E-mail address: Gerrit.Borchard@unige. ch. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS (IN ALPHABETICAL ORDER): β-GLS, β-glucosidase; CAT, catalase; CE, cholesterol esterase; COX, cholesterol oxidase; GA, glucoamylase; GI, glucose isomerase; GOD, glucose oxidase; LbL, layer by layer; MF, melamine formaldehyde; PAH, poly(allylamine hydrochloride); PDADMAC, poly(diallyldimethylammonium) chloride; PDDA, polydiallyldimethylammonium chloride; PEI, polyethylenimine; PEs, polyelectrolytes; pI, isoelectric point; POD, peroxidase; PS particles, polystyrene particles; PSS, polystyrene sulfonate; QCM, quartz crystal microbalance; UV, ultraviolet; Vis, visible light

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REFERENCES

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Biomacromolecules

Review

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