Chemical Engineering Science 135 (2015) 21–32

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Chemical Engineering Science

journal homepage: www.elsevier.com/locate/ces

Advances in carrier-bound and carrier-free

immobilized nanobiocatalysts
Mengfan Wang a,c, Wei Qi b,c,d,n, Rongxin Su b,c,d, Zhimin He b
a
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
b
State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
c
Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin 300072, PR China
d
The Co-Innovation Center of Chemistry and Chemical Engineering of Tianjin, PR China

H I G H L I G H T S

Nanobiocatalysts have been developed as an attractive strategy for enzyme immobilization in biocatalysis and biosensor fields.

The carrier-bound nanobiocatalysts exhibit specific performances by combining enzymes with nanomaterials.

Cross-linked enzyme aggregates shows higher activity and stability than native enzymes as carrier-free nanobiocatalysts.

art ic l e i nf o a b s t r a c t

Article history: Although the immobilization of enzymes with improved activity and stability has been the subject of
Received 21 December 2014 growing interest for many years, new opportunities arose by implementing nanomaterials as immobiliz-
Received in revised form ing carriers. The nano-carriers not only offer larger surface area and lower mass-transfer limitation but
22 March 2015
also endow the native enzyme with specific performances due to their various physical or chemical
Accepted 29 March 2015
Available online 3 April 2015
properties. A carrier-free nanobiocatalyst, cross-linked enzyme aggregates (CLEAs), has been developed
for applications in industrial bioprocesses. This review illustrates the recent achievements in nanobio-
Keywords: catalysts, including the carrier-bound and carrier-free enzymes. These nanobiocatalysts provide enzymes
Immobilized enzyme with broad properties to serve in the fields of biocatalysis, biomedicine and biosensors.
Nanobiocatalyst
& 2015 Elsevier Ltd. All rights reserved.
Nanomaterial
Cross-linked enzyme aggregates
Biocatalysis
Biosensor

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2. Inorganic nanomaterial as carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1. Gold nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2. Magnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3. Carbon-based scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.4. Ordered mesoporous nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.5. Enzyme–inorganic hybrid nanoflowers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3. Organic nanomaterial as carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.1. Peptide-based scaffold. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2. Stimuli-responsive polymer nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.3. Single enzyme nanoparticles/nanogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. Carrier-free immobilized enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

n
Corresponding author at: State Key Laboratory of Chemical Engineering, School
of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
PR China. Tel.: þ 86 22 27407799; fax: þ 86 22 27407599.
E-mail address: qiwei@tju.edu.cn (W. Qi).

http://dx.doi.org/10.1016/j.ces.2015.03.051
0009-2509/& 2015 Elsevier Ltd. All rights reserved.
22
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1. Introduction
Enzymes, the biocatalysts in chemical reactions, have numerous
advantages, including high efficiency, specificity, regioselectivity, enan-
tioselectivity, mild reaction conditions, and environmentally friendly
processes (de Carvalho, 2011; Illanes et al., 2012). In the biosensor
field, enzymes play important roles, including in enzyme electrodes
and enzyme-linked immunosorbent assay (ELISA) (Sassolas et al.,
2012). However, native enzymes that dissolve in aqueous solution
are sensitive and fragile to temperature, pH and organic media, which
limit their applications in biocatalysis and biosensors. Therefore, the
strategies developed for stabilizing enzymes have attracted increasing
attention. Soluble enzymes increase the cost because they cannot be
recycled such as most chemical catalysts (DiCosimo et al., 2013).
Immobilization of enzymes onto solid carriers is an effective method
to stabilize enzymes because the carrier provides superior physical
stability to the soluble counterparts, and the carrier is easier to recover
from the medium at the end of a reaction (Cao et al., 2003). In a strict
sense, the carrier can be considered as a modifier that improves the
performance of enzymes. Accordingly, a significant number of syn-
thetic or natural carriers with different shapes, sizes, porous/nonpor-
ous structures, hydrophobic/hydrophilic properties and functionalized
surfaces have been designed for various enzyme immobilizations
(Garcia-Galan et al., 2011; Tran and Balkus, 2011).
In the past decades, nanomaterials have become the most flourish-
ing subject in the fields of optics, electronics, catalyst engineering and
biomedicine (Choi et al., 2014; Hubbell and Chilkoti, 2012; Lee et al.,
2014a; Ma et al., 2009; Zhang and Dai, 2012). The small unit size and
high surface-to-volume ratio make them an extraordinary option for
enzyme immobilization with higher protein loading and less mass-
transfer limitation. Furthermore, the unique optical, electronic, mag-
netic or mechanical properties of nanomaterials endow enzymes with
special properties and broaden the application fields of enzymes.
Cross-linked enzyme aggregates (CLEAs), which are well-known as
carrier-free immobilized enzymes, are another newly developed
nanobiocatalyst with size ranging from tens to hundreds of nan-
ometers (Sheldon, 2011b; Sheldon et al., 2013). The preparation of
CLEAs consists of two steps: precipitation and cross-linking. Due to the
Fig. 1. Schematic of thermophilic enzyme photothermal gold nanoparticle (TE–PGNs) synthesis and laser-induced activation (adapted from Blankschien et al. (2013)).
M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
simple preparation process, low cost, high unit activity and intrinsic
stability, CLEAs have been used in many unconventional biocatalysis
processes, such as non-aqueous media (Kartal et al., 2011; Yang et al.,
2012; Yu et al., 2014) and one-pot cascade reactions (Chmura et al.,
2013; Jung et al., 2013; Touahar et al., 2014).
In this review, we focus on the recent developments of nanobio-
catalysts including nanocarrier-bound and carrier-free enzymes. Inor-
ganic nanomaterial immobilization with gold nanoparticles, magnetic
nanoparticles, carbon-based scaffolds, ordered mesoporous nanoma-
terials and enzyme–inorganic hybrid nanoflowers, and organic nano-
material immobilization, including peptide-based scaffolds, stimuli-
responsive polymers and single-enzyme nanoparticles/nanogels, are
presented. Typical examples of enzymes benefiting from nanomater-
ials are illuminated. In addition, several improved preparation strate-
gies for CLEAs are presented to provide a new avenue for the
application of enzymes in chemical engineering, biotechnology, bio-
sensors and materials.
2. Inorganic nanomaterial as carrier
2.1. Gold nanoparticles
The high surface-to-volume ratio and easily functionalized surface
of gold nanoparticles (AuNPs) make them excellent carriers for
protein/enzyme immobilization. Multipoint Au–S bond formation is
a straight-forward way to fix functional molecules in the desired
geometry (Dreaden et al., 2012; Keighron et al., 2014). In some cases,
the tight chemisorption of proteins on AuNPs originates from the
binding of thiol and amino groups that exist in proteins (Zhao et al.,
2008). Anchoring groups are usually utilized for attachment of an
enzyme to the AuNPs surface. These anchoring groups include
thiolate, dithiolate, dithiocarbamate, amine, carboxylate, selenide,
isothiocyanate and phosphine (Brown et al., 2010; Hou et al., 2009;
Knight et al., 2009; Zhao et al., 2005). Additionally, some of the
thiolate ligands in alkanethiolate-stabilized AuNPs can be substituted
by other thiols through a thiol–thiol exchange reaction. The reaction
rate depends on the chain length and steric bulk of the leaving thiolate
 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
and incoming thiols as well as on the charge of the AuNPs (Hostetler
et al., 1999; Saha et al., 2012; Templeton et al., 1998). Lv et al. (2014)
described a reusable bioreactor that consists of porous polymer
monolithic supports with the pore surface coated with AuNPs. The
AuNPs serve as an intermediate ligand to immobilize lipase via the
non-covalent bond between the thiol and amine functionalities with
gold. After several cycles of operation, inactive lipase can be stripped
from the nanoparticles using 2-mercaptoethanol and subsequently
replaced by fresh enzyme.
Metal nanoparticles with localized surface plasmon resonance
(LSPR) properties at the specific incident wavelength generate
strong collective oscillations of electron plasma in metals, which
can be scattered in the form of light or heat (Daniel and Astruc,
2004; Giljohann et al., 2010). Based on this, Blankschien et al.
(2013) developed light-responsive, thermophilic enzyme photo-
thermal gold nanoparticle complexes (TE–PGNs) using gold nanor-
ods with a longitudinal resonance wavelength of 750–850 nm and
Aeropyrum pernix glucokinase (Glk) as the modal enzyme. By
illuminating TE–PGNs with a continuous laser (λ ¼ 800 nm),
photothermal heat was generated at the surface of the gold
nanorods. A calcium alginate matrix was used to encapsulate the
TE–PGNs to retain the heat around the TE–PGNs. As a result,
thermophilic Glk was activated, and the reaction rate increased
60% compared with free enzyme (Fig. 1).
Moreover, the excellent conductivity and biocompatibility make
AuNPs an attractive material for biosensors. AuNPs-based enzyme
electrodes can be prepared by immobilizing redox enzymes on AuNPs
and directly depositing them on the bulk electrode surface (Saxena et
al., 2011). Horseradish peroxidase (Li et al., 2010), hydrogen peroxidase
(Li et al., 2012) and cholesterol oxidase (Saxena et al., 2011) have been
immobilized onto AuNPs to provide an environment to improve the
electrocatalytic activity and enhance electron transfer from enzymatic
redox reactions to the electrode surfaces.
Fig. 2. The microfluidic chip for online tryptic digestion and ESI-MS (adapted from Le Nel et al. (2008)). The experimental setup (A), the microdevice (B) and the diagram (C).
23
2.2. Magnetic nanoparticles
Magnetic metal nanoparticles (MNPs), which usually have sizes
ranging from 10 to 100 nm, have been used in enzyme immobilization
due to their unique superparamagnetism, high surface area, large
surface-to-volume ratio and easy separation under external magnetic
fields (Garcia et al., 2011; Reddy et al., 2012). The ferrite colloids
magnetite (Fe3O4) and maghemite (γ-Fe2O3) are the main representa-
tives of MNPs. Immobilized enzymes based on iron magnetic nano-
particles have been applied in certain fields, such as immunoassays
(Yang et al., 2013b; Zhang et al., 2013), biosensors (Baratella et al.,
2013; Garcia et al., 2011), bioseparations (Kannan et al., 2013; Ma et al.,
2012) and drug delivery (Koppolu et al., 2012; Wang et al., 2014).
Wang et al. (2012) developed a simple and cost-effective method to
directly immobilize Candida rugosa lipase on alkyl silane modified
Fe3O4 magnetite nanoparticles through hydrophobic interaction. By
tuning the chain length of alkyl silane, the highest catalytic activity
was obtained with trimethoxyl octadecyl silane (C18) modified
nanoparticles, and 65% of the activity was retained after 7 recycles
due to the easy separation under a magnetic field. Covalent bonding
using bi-functional agents as cross-linkers is common in MNPs-based
enzyme immobilization (Alex et al., 2014; Kuo et al., 2012; Li et al.,
2007). Alex et al. (2014) modified MNPs with 3-aminopropyl triethoxy
silane (APTES) to change hydroxyl surface into amino groups. Then,
enzymes were bound to MNPs through double Schiff base reactions
between MNPs and glutaraldehyde and between glutaraldehyde and
enzymes. Kuo et al. (2012) covalently immobilized lipase on Fe3O4–
chitosan nanoparticles using N-(3-dimethylaminopropyl)-N'-ethylcar-
bodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linkers.
After 20 cycles, the immobilized lipase retained over 83% of its original
activity.
Based on the easy separation of magnetic nanoparticles, Le Nel
et al. (2008) developed a microfluidic chip to realize online tryptic
24 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
Fig. 3. The hybrid graphite film–carbon nanotube platform (A). SEM images of the top (B) and side (C) view of an as-prepared CNT array. Schematic of the enzyme protection
against microbiological attack (D). SEM images of the B. subtilis on the surface of the nanotube pattern (E) (adapted from Kondyurin et al. (2013)).
digestion and ESI-MS determination. Trypsin was covalently
immobilized onto magnetic nanoparticles and introduced into a
microchannel. Two permanent magnets were placed to trap the
trypsin-magnetic particles remaining around the microchannel.
When protein solution was pumped through the microchannel, it
was digested by the magnetic nanoparticles-bound trypsin in a
short time, and the resulting peptide fragments were separated
from the enzymes and delivered by a stream flowing directly to a
mass spectrometer (Fig. 2).
2.3. Carbon-based scaffold
Carbon-based nanomaterials, such as carbon nanotubes (CNTs),
graphene and graphene oxide (GO), with extraordinary mechan-
ical, electrical, thermal and biocompatible properties are excellent
materials in biosensors, gene delivery vectors and drug delivery
(Singh et al., 2005; Sun et al., 2014a; Vashist et al., 2011; Zhou et
al., 2014). Enzymes immobilized on carbon-based nanomaterials
allow researchers to take advantage of the mechanical strength,
conductivity or functionalization properties of carbon-based nano-
materials to improve enzyme behavior (Feng and Ji, 2011).
Pavlidis et al. (2012) reported the immobilization of several
recombinant hydrolytic enzymes of significant industrial interest on
amine-functionalized multi-wall CNTs and GO. Biochemical and
structural studies indicated that the immobilization approach and
the curvature of the nanomaterials significantly affect the immobiliza-
tion efficiency, activity, secondary structure and the operational
stability of immobilized enzymes. Kondyurin et al. (2013) developed
a novel platform consisting of a multilayered silicon/silicon oxide/Fe
substrate, an activated graphite-like carbon film and a dense forest of
vertically aligned multiwall carbon nanotubes through chemical vapor
deposition. Horseradish peroxidase and catalase were covalently
immobilized on the activated carbon film underneath the nanotubes.
Because bacteria cannot penetrate into the tall densely packed CNTs
forest, the enzymes were defended from microbial attack and shear
forces without impeding the flow of reactants and products (Fig. 3).
CNTs-immobilized enzymes play an important role in electroche-
mical sensors due to the enhanced electronic properties, large edge
plane/basal plane ratio and rapid electrode kinetics (Vashist et al.,
2011; Yang et al., 2015). Redox enzymes, such as glucose oxidase
(Palanisamy et al., 2014; Zhu et al., 2013b), horseradish peroxidase
(Gao et al., 2011; Periasarny et al., 2011), cellobiose dehydrogenases
(Tasca et al., 2013), superoxide dismutase (Tian et al., 2002; Zhu et al.,
2015) and laccase (Bourourou et al., 2013) are often deposited on
electrode surfaces with modified CNTs through adsorption or covalent
attachment. This combination has contributed to the rapid develop-
ment of enzyme-based sensors with higher sensitivity and selectivity.
2.4. Ordered mesoporous nanomaterials
Ordered mesoporous silicas, such as SBA-15, MCM-41 and MCF,
have attracted particular interest in enzyme immobilization
because of their large specific surface area, controlled porosity,
high mechanical strength and ease of modification (Lin et al.,
2014a; Zhou and Hartmann, 2013).
Jun et al. (2012) immobilized lipase into a meso-structured
onion-like silica (Meso-Onion-S) and used it as a nanoscale enzyme
reactor (NER). Meso-Onion-S has a 200–300 nm sized primary
meso-structured onion building unit, and each onion unit has
highly curved mesopores with a 10 nm diameter in a multishell
structure. Cross-linking was performed after absorbing enzymes
into pores, which resulted in improvement of enzyme loading
(3373.4%) and stability due to effective protection of the enzyme
from leaking under rigorous shaking (Fig. 4). The specific activity of
NER-lipase was 23 and 10 times higher than free lipase and
adsorbed lipase in transesterification reactions.
The catalytic properties of enzymes, such as the conversion
rate, selectivity, stability and recoverability, can be enhanced by
ionic liquids (ILs). Huang's group (Yang et al., 2013a; Zou et al.,
 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32 25

Fig. 4. Preparation of nanoscale enzyme reactor (NER) in Meso-Onion-S and the enzyme leaching of adsorbed enzymes (ADS) under rigorous shaking (200 rpm) but not with
NER (adapted from Lee et al. (2014b)).

Fig. 5. Enzyme–inorganic hybrid nanoflowers (adapted from Ge et al. (2012)). The proposed hybrid mechanism where yellow spheres indicate the protein molecules (A). The
SEM images of the α-lactalbumin, laccase, carbonic anhydrase and lipase nanoflowers (column 1–4) under the protein concentrations of 0.5, 0.1 and 0.02 mg/mL (row 1–3)
(B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
26 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
2013) developed a strategy to graft functionalized ILs onto a SBA-
15 surface to immobilize enzymes through ion interactions or
covalent bonding. Compared with plain SBA-15, ILs modified SBA-
15 had significantly promoted enzymatic properties, including
activity, thermal stability, reusability and storage stability. Lee
et al. (2014b) developed enzyme-/chemo-catalysts based on
mesoporous silica nanoparticles (MSN) for the cascading conver-
sion of glucose into 5-hydroxymethylfurfural (5-HMF). Fe3O4
nanoparticles (20 nm) were added to the precursor to form
Fe3O4-loaded MSN (Fe3O4@MSN) with a final particle size of
500–600 nm and a pore size of approximately 20 nm. Then, they
used Fe3O4@MSN as the host for adsorbing cellulase and glucose
isomerase to form cellulose–Fe3O4@MSN and isomerase–
Fe3O4@MSN. A maximum fructose yield of 51% was achieved
under the cascading catalysis (cellulose-to-glucose-to-fructose)
in aqueous media by cellulose–Fe3O4@MSN and isomerase–
Fe3O4@MSN. Due to the high stability and easy separation of
Fe3O4@MSN, the biocatalysts can be withdrawn at the end of each
reaction step, followed by chemical conversion of fructose into 5-
HMF with HSO3-MSN in organic media.
2.5. Enzyme–inorganic hybrid nanoflowers
Hyperactive enzyme–inorganic hybrid nanoflowers is a novel
and simple enzyme stabilization method created by Ge et al.
(2012). In phosphate buffered saline (PBS) containing enzyme,
primary crystals of copper phosphate (Cu3(PO4)2  3H2O) were
formed as nucleation sites after the addition of Cu2 þ . As a result,
Fig. 6. The self-assembly of BAmoc-peptides hydrogel and H2O2-triggered hydrogel degradation (A). Schematic representation of hybrid hydrogel responsive to various
analytes (B) (adapted from Ikeda et al. (2014)).
flower-like copper phosphate nanocrystals gradually grew from
these nucleation sites with enzyme proteins serving as “glue” to
bind the petals together (Fig. 5). Compared with free enzyme,
enzyme–inorganic hybrid nanoflowers showed 650% increased
activity for laccase in the oxidization reaction of syringaldazine
and  260% increased activity for carbonic anhydrase in the
hydration of CO2. To reveal the hyperactive mechanism of
enzyme–inorganic hybrid nanoflowers, Wang et al. (2013a)
designed three α-amylase–CaHPO4 hybrid nanocrystals with
nanoflower, nanoplate and parallel hexahedron structures. Both
the nanostructure morphology and the allosteric effect between
Ca2 þ and the amine group of the enzyme notably influence the
catalytic efficiency. Enzymes immobilized on nanoflowers are
more likely to contact substrate molecules due to the lower
mass-transfer limitation and larger enzyme loading based on the
high surface area of nanoflowers.
Moreover, enzyme–inorganic hybrid nanoflowers have been
applied to bioanalysis (Li et al., 2014; Lin et al., 2014b; Sun et al.,
2014b; Zhu et al., 2013c). Zhu et al. (2013c) used laccase–inorganic
hybrid nanoflowers in the rapid detection of phenol through
catalyzing the generation of visible products that are convenient
for either quantitative measurement or direct observation. Sun
et al. (2014b) synthesized multi-enzyme–inorganic hybrid nano-
flowers that included both glucose oxidase and peroxidase and
used them for the detection of glucose. Due to the close orienta-
tion of enzymes in the nanostructure, the two-enzyme cascade
reaction was conducted in one step to effectively convert glucose
into a chromogenic product.
 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32 27

Fig. 7. The enzymogel nanoparticle (A) and the cryo-TEM images of the particle with silica core and swollen/shrunken poly(acrylic acid) brush at pH 7 and pH 4.5 (B–D).
Degradation of a 500 μm thick cotton floss with free cellulase solution for 14 h (E, F), enzymogel for 2.5 h (G, H) and with magnetic field-directed deposition for 1 h (I, J)
(adapted from Kudina et al. (2014)).

3. Organic nanomaterial as carrier
3.1. Peptide-based scaffold
Peptide-based supramolecular hydrogels, formed by self-
assembly of amphiphilic peptides, have served as scaffolds for
tissue engineering, the matrix for biomineralization, and as
biomaterials for wound healing (He et al., 2014; Huang et al.,
2010; Khadka and Haynie, 2012). Due to the amphiphilicity,
adjustability and biocompatibility of peptides, peptide-based
hydrogels are an attractive matrix for enzyme immobilization
(Gao et al., 2010; Huang et al., 2014).
Wang et al. (2007) encapsulated methemoglobin into peptide
hydrogels via self-assembling of Fmoc-L-lysine and Fmoc-L-phenyla-
lanine. The encapsulated methemoglobin exhibited higher activity (up
to eight times) in the oxidation of pyrogallol in organic media
compared to unconfined methemoglobin in water. The improved
activity results from hydrophilic hydrogels promoting pyrogallol
across the microinterface and entering the hydrogel, similar to that
of reversed micelles. Moreover, the amphiphilic molecular structure
facilitates the approach of substrate to enzyme and the release of
product. To prevent enzymes from leaking, Hickling et al. (2014)
covalently attached octopeptides (VKVKVEVK) to pentaerythritol
tetranitrate reductase (PETNR). Two synthesis strategies were
explored for the preparation of peptide–enzyme conjugates: chemical
coupling (SPep-PETNR) of the peptide to the enzyme via click
chemistry and genetic expression (CPep-PETNR) of the full peptide–
enzyme conjugate. A small portion of peptide–enzyme conjugates
were doped with pure VKVKVEVK peptide, where the peptide
components contribute to the formation of β-sheet fibers through
28 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
self-assembly. As a result, PETNR was steadily immobilized on the
fiber surface.
Ikeda et al. (2014) developed a redox-responsive peptide-based
hydrogel by encapsulating redox enzyme into a H2O2-responsive
peptide matrix. The hydrogel was composed of di- and tri-peptides
that contained an H2O2-reactive boronoarylmethoxycarbonyl
(BAmoc) group at their N terminus. Once the enzymatic oxidation/
elimination occurred, the generated H2O2 triggered the removal of
BAmoc, which induced destabilization of the nanofibers and collapse
of the hydrogel. This responsive hydrogel can be used as a biomarker
for responding to disease-related molecules, such as glucose, lactose,
uric acid, choline, acetylcholine and sarcosine (Fig. 6). For example,
glucose molecules (input) can be recognized by the corresponding
glucose oxidase and converted into H2O2, which causes morpholo-
gical changes to the hydrogel (output). Thus, encapsulation of glucose
oxidase in this peptide hydrogel converts the H2O2 response into a
glucose response. They built Boolean logic gates (AND and OR) by
combining two types of enzymes, for example, nitroreductase and
choline oxidase, into hydrogel-enzyme hybrid materials, which were
able to sense multiple specific biochemicals.
3.2. Stimuli-responsive polymer nanomaterials
Stimuli-responsive polymer nanomaterials, which respond to
external stimulus changes, have attracted substantial attention in
drug delivery (Gupta et al., 2002; Roy et al., 2010; Schmaljohann,
2006; Stuart et al., 2010). Many polymer materials have been designed
to respond to physical or chemical/biochemical stimuli. Physical
stimuli include temperature, solvent composition, light, pressure,
electric fields and magnetic fields (Baumann et al., 2013; Katsuno
et al., 2013; Rauch et al., 2012), whereas chemical/biochemical stimuli
Fig. 8. The synthesis and cellular uptake of cationic single-protein nanocapsules with degradable and non-degradable polymeric shell (A). TEM (B) and AFM (C) images of
HRP nanocapsules. The formation of single-core nanoscale architecture (D) (adapted from Yan et al. (2010)).
include pH, ions and specific molecular recognition (Kang et al., 2014;
Zhou et al., 2013). With the help of stimuli-responsive materials,
enzymes can be absorbed or released at a certain time during a
reaction, which improves catalytic performance, stability and reusa-
bility of the enzyme.
Gan et al. (2012) developed a temperature-sensitive cross-linked
gelatin nanoparticles (CLGNs) immobilized enzyme. CLGNs were
prepared by coacervation, and glucoamylase was physically immobi-
lized onto CLGNs. Because the phase transition temperature of CLGNs
is 40 1C, the release of glucoamylase can be controlled when the
system temperature is above 40 1C. As most enzymes that offer high
catalytic performances in vivo display limited activities in organic
solvents, Zhu et al. (2013a) developed temperature-responsive poly-
mer nanoconjugates by covalently grafting Pluronic onto protein
molecules. This allows solubilization of nanoconjugates in toluene
with enhanced apparent activity for homogenous catalysis at 40 1C as
well as precipitation of nanoconjugates at 4 1C for recovery. Kudina
et al. (2014) designed core–shell structural enzymogel nanoparticles
with a magnetic core and negatively charged poly(acrylic acid) brush
scaffolds at pH 4–8. Cellulase, with a typical isoelectric of pH 4.9, can
be loaded onto the brush via electrostatic interaction at pH 4.5. By
adjusting the cellulose solution pH to 7, cellulase was released from
the polymer brush and maintained its activity as a free enzyme. The
free enzyme can be subsequently reloaded onto the enzymogel
nanoparticles by adjusting the pH to 4 and can be recovered under
a magnetic field. Furthermore, the enzyme in the brush exhibited
high activity in the hydrolysis of cellulose fibers, which makes it a
new type of phase boundary catalysis. The enzymogel nanoparticles
that adsorbed on the surface of cellulose fibers can cleave cellulose
chains with free cellulase shuttling between the polymer brush
interior and the brush–cellulose interface (Fig. 7).
 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
3.3. Single enzyme nanoparticles/nanogels
Single enzyme nanoparticles (Hegedus and Nagy, 2009; Kim and
Grate, 2003; Kim et al., 2006) and single enzyme nanogels were deve-
loped to stabilize enzymes by encapsulating single enzyme mole-
cules within a polymer network. The sizes of single enzyme nano-
particles/nanogels are usually several to several tens of nanometers
with very thin polymer layers, which provide higher surface area and
less mass transfer limitation to the substrate.
Single enzyme nanoparticles were first presented by Kim (Kim and
Grate, 2003; Kim et al., 2006). The preparation involves enzyme
surface modification, vinyl polymer growth from the enzyme surface,
and a silanol condensation reaction to form a polymer network
around each enzyme molecule. Later, Yan et al. (2010, 2006) devel-
oped a versatile procedure to synthesize single enzyme/protein
nanogels. After a mild modification with N-acryloxysuccinimide to
generate vinyl groups on the enzyme surface, in-situ polymerization
at room temperature was conducted in aqueous solution with
monomers and cross-linkers. By using glycerol dimethacrylate as the
degradable cross-linker, a delivery platform was obtained with
nanocapsules consisting of a protein core and a thin permeable
polymeric shell. This structure not only resisted protein against
protease but also enabled the collapse of the shell once inside and
the release of protein from the nanocapsule (Yan et al., 2010) (Fig. 8).
Additionally, Wang et al. (2013c) reported that the hydrophilic envi-
ronment contributed by the polyacrylamide gel network can faci-
litate enzyme catalysis in organic media. They presented a sub-
strate-imprinted lipase nanogel by first encapsulating lipase into
polyacrylamide nanogel, then lyophilizing the nanogel with palmitic
acid, a substrate of lipase, and finally extracting palmitic acid from the
nanogel by petroleum ether. Compared with native lipase, substrate-
imprinted lipase nanogel showed 200% activity in the transesterifica-
tion reaction between paranitrophenyl palmitate and ethanol in n-
heptane. Novel piezoelectric biosensor was also fabricated based on
single urease nanoparticles by immobilizing them onto nanoporous
alumina membranes for quantitative urea determination (Yang and
Zhang, 2013).
4. Carrier-free immobilized enzyme
Unlike carrier-bound enzyme immobilization, the carrier-free
immobilized enzyme is composed of pure proteins without extra
inactive mass as carrier, resulting in higher specific activity (Sheldon,
2011a, 2011b). The carrier-free immobilized enzymes are prepared by
directly cross-linking different enzyme preparations, such as dissolved
Fig. 9. Two strategies to prepare pouous CLEAs. Using starch as pore-making agent (A) and the structure of resulting p-CLEAs (B). Using multi-aldehyde macromolecule
(C) as cross-linker. The structure of p-CLEAs cross-linked by dextran polyaldehyde (D) and dialdehyde starch (E). (Adapted from Wang et al. (2011) and Zhen et al. (2013).)
29
enzyme, crystalline enzyme and physically aggregated enzyme, result-
ing in formation of cross-linked enzymes (CLEs), cross-linked enzyme
crystals (CLECs), and cross-linked enzyme aggregates (CLEAs) (Cao
et al., 2003; Roessl et al., 2010). CLEAs, as a nano-scaled biocatalyst,
have attracted attention in recent years due to their comparable
activities to native enzymes, easier preparation and lower cost. The
preparation of CLEAs consists of two steps: precipitation and cross-
linking. Firstly, soluble enzymes are precipitated into enzyme aggre-
gates by adding water miscible organic solvents, salts or nonionic
polymers. Secondly, the formed aggregates are cross-linked by a
bifunctional agent to produce CLEAs (Talekar et al., 2013).
Because most CLEAs are prepared in batch processes, the particle
size of CLEAs and the cross-linking degree may vary widely, which
will affect the enzyme activity and reaction kinetics (Nguyen and
Yang, 2014). Chen et al. (2006) synthesized CLEAs in CO2-expanded
micellar solutions. By changing the water-to-surfactant ratio (w0) and
the concentration of enzyme in the reverse micellar solution, dendritic
CLEAs can be tuned to 7–38 nm in diameter and exhibit 200% activity
compared with conventional CLEAs. Nguyen and Yang (2014) applied
a Y-shape millifluidic reactors to prepare uniform-sized cross-linked
cellulase aggregate by precisely controlling the mixing pattern and
spatial distribution of reactants. By tuning the parameters of the
precipitant concentration, glutaraldehyde concentration and flow
rates, the particle size of CLEAs can be controlled between 200 and
400 nm.
Another problem of CLEAs in practical application is internal mass-
transfer limitation, which result from its intrinsic supramolecular
structure. This results in an accessibility limit when the substrates
are macromolecules, such as proteins and polysaccharides. To over-
come this problem, Wang et al. developed two strategies to construct
porous CLEAs (Fig. 9) (Wang et al., 2011; Zhen et al., 2013). One
involves co-precipitating enzyme and starch, cross-linking and
removal of starch by amylase (Wang et al., 2011). Starch acts as the
pore-making agent, which cannot be cross-linked, and can be easily
removed via the hydrolysis of amylase. The other strategy uses
multi-aldehyde macromolecules (dextran polyaldehyde or dialdehyde
starch) as cross-linkers instead of glutaraldehyde. CLEAs prepared
using linear dextran polyaldehyde (MW¼ 2000 kDa) presented a
porous structure with low steric hindrance and thus exhibited
excellent activity toward macromolecular substrates (Zhen et al.,
2013). Both strategies exhibited similar porous structures in CLEAs,
which provided enhanced catalytic efficiencies on proteins (BSA and
ovalbumin) and polysaccharides (carob bean gum).
Due to the high stability of CLEAs, CLEAs of trypsin (CLEAs-T) were
successfully applied in rapid protein digestion for proteomics analysis
(Wang et al., 2013b). Unlike other immobilized enzymes, it is
30 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
unnecessary to separate CLEAs-T from the digestion solution before
MS measurement. Compared with conventional in-solution digestion
with free trypsin, CLEAs-T digestion allows a high dosage of enzyme
without autolysis. Furthermore, high temperature and ultrasound can
be combined during digestion to significantly decrease the digestion
time from more than 12 h to several minutes, which results in a large
increase in analytical precision and throughput.
Enantioselectivity is an important feature of enzymes as biocata-
lysts because the preparation of pure enantiomers of chiral inter-
mediates is a key step for many industrially relevant processes. Immo-
bilization has been approved as a useful option to improve enzyme
selectivity besides protein engineering and directed evolution meth-
ods. The effect of diffusion limitation, micro-environments, active site
structures and the freedom of the enzymes explain the improvement
of selectivity via immobilization (Mateo et al., 2007; Rodrigues et al.,
2013). Weiser et al. (2014) found that lipase from Pseudomonas
fluorescens (AK), lipase from Burkholderia cepacia (PS) and lipase B
from Candida antarctica (CaL B) showed increased enantiomer selec-
tivity in the kinetic resolution of racemic 1-phenylethanol when
immobilized as CLEAs. Moreover, when phenylalanine ammonialyase
(PAL) and bovine serum albumin (BSA) were co-immobilized as CLEAs
(PAL–BSA-co-CLEAs), they exhibited higher selectivity in the resolu-
tion of racemic phenylalanine derivatives both via the ammonia
elimination route and the ammonia addition route. PAL-CLEAs with-
out BSA only catalyzed the elimination reaction and became invalid in
the reverse route. This results from the looser packing of PAL in PAL–
BSA-co-CLEAs than PAL-CLEAs, which lessens the diffusion limitation.
5. Conclusions
Recent prosperity in nanomaterials and nanotechnologies enabled
the development of enzyme immobilization as nanobiocatalysts with
enhanced activity and stability. The unique properties of nanomater-
ials endow enzymes with special performance and broaden the
application fields of enzyme. Additionally, CLEAs, as a type of
carrier-free immobilized enzyme, have been developed to simplify
enzyme preparations but with higher unit activity. This review
illustrates the latest achievements in carrier-bound and carrier-free
nanobiocatalysts. The development of nanobiocatalyst will continue
and even speed up with the increasing demand for enzymes in the
fields of biocatalysis, biomedicine and biosensors.
Acknowledgment
This work was supported by the Ministry of Science and Technol-
ogy of the People's Republic of China (2012YQ090194), the 863
Program of China (2012AA06A303, 2013AA102204), the National Nat-
ural Science Foundation of China (21206113, 51173128), the Natural
Science Foundation of Tianjin (13JCQNJC09300) and the Beiyang
Young Scholar Project of Tianjin University (2013).
References
Alex, D., Mathew, A., Sukumaran, R.K., 2014. Esterases immobilized on aminosilane
modified magnetic nanoparticles as a catalyst for biotransformation reactions.
Bioresour. Technol. 167, 547–550.
Baratella, D., Magro, M., Sinigaglia, G., Zboril, R., Salviulo, G., Vianello, F., 2013. A
glucose biosensor based on surface active maghemite nanoparticles. Biosens.
Bioelectron. 45, 13–18.
Baumann, P., Balasubramanian, V., Onaca-Fischer, O., Sienkiewicz, A., Palivan, C.G.,
2013. Light-responsive polymer nanoreactors: a source of reactive oxygen
species on demand. Nanoscale 5, 217–224.
Blankschien, M.D., Pretzer, L.A., Huschka, R., Halas, N.J., Gonzalez, R., Wong, M.S.,
2013. Light-triggered biocatalysis using thermophilic enzyme–gold nanoparti-
cle complexes. ACS Nano 7, 654–663.
Bourourou, M., Elouarzaki, K., Lalaoui, N., Agnes, C., Le Goff, A., Holzinger, M.,
Maaref, A., Cosnier, S., 2013. Supramolecular immobilization of laccase on
carbon nanotube electrodes functionalized with (methylpyrenylaminomethyl)
anthraquinone for direct electron reduction of oxygen. Chem.—Eur. J. 19,
9371–9375.
Brown, S.D., Nativo, P., Smith, J.A., Stirling, D., Edwards, P.R., Venugopal, B., Flint, D.J.,
Plumb, J.A., Graham, D., Wheate, N.J., 2010. Gold nanoparticles for the improved
anticancer drug delivery of the active component of oxaliplatin. J. Am. Chem.
Soc. 132, 4678–4684.
Cao, L.Q., van Langen, L., Sheldon, R.A., 2003. Immobilised enzymes: carrier-bound
or carrier-free? Curr. Opin. Biotechnol. 14, 387–394.
Chen, J., Zhang, J.L., Han, B.X., Li, Z.H., Li, J.C., Feng, X.Y., 2006. Synthesis of cross-
linked enzyme aggregates (CLEAs) in CO2-expanded micellar solutions. Colloids
Surf. B: Biointerfaces 48, 72–76.
Chmura, A., Rustler, S., Paravidino, M., van Rantwijk, F., Stolz, A., Sheldon, R.A., 2013.
The combi-CLEA approach: enzymatic cascade synthesis of enantiomerically
pure (S)-mandelic acid. Tetrahedron: Asymmetry 24, 1225–1232.
Choi, S., Tripathi, A., Singh, D., 2014. Smart nanomaterials for biomedics. J. Biomed.
Nanotechnol. 10, 3162–3188.
Daniel, M.C., Astruc, D., 2004. Gold nanoparticles: assembly, supramolecular
chemistry, quantum-size-related properties, and applications toward biology,
catalysis, and nanotechnology. Chem. Rev. 104, 293–346.
de Carvalho, C., 2011. Enzymatic and whole cell catalysis: finding new strategies for
old processes. Biotechnol. Adv. 29, 75–83.
DiCosimo, R., McAuliffe, J., Poulose, A.J., Bohlmann, G., 2013. Industrial use of
immobilized enzymes. Chem. Soc. Rev. 42, 6437–6474.
Dreaden, E.C., Alkilany, A.M., Huang, X.H., Murphy, C.J., El-Sayed, M.A., 2012. The
golden age: gold nanoparticles for biomedicine. Chem. Soc. Rev. 41, 2740–2779.
Feng, W., Ji, P., 2011. Enzymes immobilized on carbon nanotubes. Biotechnol. Adv.
29, 889–895.
Gan, Z.H., Zhang, T., Liu, Y.C., Wu, D.C., 2012. Temperature-triggered enzyme
immobilization and release based on cross-linked gelatin nanoparticles. PLoS
One, 7.
Gao, W., Dong, H., Lei, J., Ji, H., Ju, H., 2011. Signal amplification of streptavidin–
horseradish peroxidase functionalized carbon nanotubes for amperometric
detection of attomolar DNA. Chem. Commun. 47, 5220–5222.
Gao, Y., Zhao, F., Wang, Q., Zhang, Y., Xu, B., 2010. Small peptide nanofibers as the
matrices of molecular hydrogels for mimicking enzymes and enhancing the
activity of enzymes. Chem. Soc. Rev. 39, 3425–3433.
Garcia-Galan, C., Berenguer-Murcia, A., Fernandez-Lafuente, R., Rodrigues, R.C.,
2011. Potential of different enzyme immobilization strategies to improve
enzyme performance. Adv. Synth. Catal. 353, 2885–2904.
Garcia, J., Zhang, Y., Taylor, H., Cespedes, O., Webb, M.E., Zhou, D.J., 2011. Multilayer
enzyme-coupled magnetic nanoparticles as efficient, reusable biocatalysts and
biosensors. Nanoscale 3, 3721–3730.
Ge, J., Lei, J.D., Zare, R.N., 2012. Protein–inorganic hybrid nanoflowers. Nat.
Nanotechnol. 7, 428–432.
Giljohann, D.A., Seferos, D.S., Daniel, W.L., Massich, M.D., Patel, P.C., Mirkin, C.A.,
2010. Gold nanoparticles for biology and medicine. Angew. Chem.—Int. Ed. 49,
3280–3294.
Gupta, P., Vermani, K., Garg, S., 2002. Hydrogels: from controlled release to pH-
responsive drug delivery. Drug Discov. Today 7, 569–579.
He, B., Yuan, X., Jiang, D.M., 2014. Molecular self-assembly guides the fabrication of
peptide nanofiber scaffolds for nerve repair. RSC Adv. 4, 23610–23621.
Hegedus, I., Nagy, E., 2009. Improvement of chymotrypsin enzyme stability as
single enzyme nanoparticles. Chem. Eng. Sci. 64, 1053–1060.
Hickling, C., Toogood, H.S., Saiani, A., Scrutton, N.S., Miller, A.F., 2014. Nanofibrillar
peptide hydrogels for the immobilization of biocatalysts for chemical transfor-
mations. Macromol. Rapid Commun. 35, 868–874.
Hostetler, M.J., Templeton, A.C., Murray, R.W., 1999. Dynamics of place-exchange
reactions on monolayer-protected gold cluster molecules. Langmuir 15,
3782–3789.
Hou, W.B., Dasog, M., Scott, R.W.J., 2009. Probing the relative stability of thiolate-
and dithiolate-protected Au monolayer-protected clusters. Langmuir 25,
12954–12961.
Huang, R.L., Qi, W., Jiang, N., Su, R.X., He, Z.M., 2010. Peptide based nanomaterials
and their technological applications. Prog. Chem. 22, 2328–2337.
Huang, R.L., Wu, S.K., Li, A.T., Li, Z., 2014. Integrating interfacial self-assembly and
electrostatic complexation at an aqueous interface for capsule synthesis and
enzyme immobilization. J. Mater. Chem. A 2, 1672–1676.
Hubbell, J.A., Chilkoti, A., 2012. Nanomaterials for drug delivery. Science 337,
303–305.
Ikeda, M., Tanida, T., Yoshii, T., Kurotani, K., Onogi, S., Urayama, K., Hamachi, I., 2014.
Installing logic-gate responses to a variety of biological substances in supra-
molecular hydrogel–enzyme hybrids. Nat. Chem. 6, 511–518.
Illanes, A., Cauerhff, A., Wilson, L., Castro, G.R., 2012. Recent trends in biocatalysis
engineering. Bioresour. Technol. 115, 48–57.
Jun, S.H., Lee, J., Kim, B.C., Lee, J.E., Joo, J., Park, H., Lee, J.H., Lee, S.M., Lee, D., Kim, S.,
Koo, Y.M., Shin, C.H., Kim, S.W., Hyeon, T., Kim, J., 2012. Highly efficient enzyme
immobilization and stabilization within meso-structured onion-like silica for
biodiesel production. Chem. Mater. 24, 924–929.
Jung, D.H., Jung, J.H., Seo, D.H., Ha, S.J., Kweon, D.K., Park, C.S., 2013. One-pot
bioconversion of sucrose to trehalose using enzymatic sequential reactions in
combined cross-linked enzyme aggregates. Bioresour. Technol. 130, 801–804.
Kang, Y., Ha, W., Liu, Y.Q., Ma, Y., Fan, M.M., Ding, L.S., Zhang, S., Li, B.J., 2014. pH-
responsive polymer–drug conjugates as multifunctional micelles for cancer-
drug delivery. Nanotechnology, 25.
 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
Kannan, K., Mukherjee, J., Gupta, M.N., 2013. Use of polyethyleneimine coated Fe3O4
nanoparticles as an ion-exchanger for protein separation. Sci. Adv. Mater. 5,
1477–1484.
Kartal, F., Janssen, M.H.A., Hollmann, F., Sheldon, R.A., Kilinc, A., 2011. Improved
esterification activity of Candida rugosa lipase in organic solvent by immobi-
lization as cross-linked enzyme aggregates (CLEAs). J. Mol. Catal. B: Enzym. 71,
85–89.
Katsuno, C., Konda, A., Urayama, K., Takigawa, T., Kidowaki, M., Ito, K., 2013.
Pressure-responsive polymer membranes of slide-ring gels with movable
cross-links. Adv. Mater. 25, 4636–4640.
Keighron, J.D., Akesson, S., Cans, A.-S., 2014. Coimmobilization of acetylcholinester-
ase and choline oxidase on gold nanoparticles: stoichiometry, activity, and
reaction efficiency. Langmuir: ACS J. Surf. Colloids 30, 11348–11355.
Khadka, D.B., Haynie, D.T., 2012. Protein- and peptide-based electrospun nanofibers
in medical biomaterials. Nanomed.: Nanotechnol. Biol. Med. 8, 1242–1262.
Kim, J., Grate, J.W., 2003. Single-enzyme nanoparticles armored by a nanometer-
scale organic/inorganic network. Nano Lett. 3, 1219–1222.
Kim, J., Jia, H.F., Lee, C.W., Chung, S.W., Kwak, J.H., Shin, Y., Dohnalkova, A., Kim, B.G.,
Wang, P., Grate, J.W., 2006. Single enzyme nanoparticles in nanoporous silica: a
hierarchical approach to enzyme stabilization and immobilization. Enzyme
Microb. Technol. 39, 474–480.
Knight, E.R., Leung, N.H., Thompson, A.L., Hogarth, G., Wilton-Ely, J., 2009. Multi-
metallic arrays: bi-, tri-, tetra-, and hexametallic complexes based on Gold
(I) and Gold(III) and the surface functionalization of gold nanoparticles with
transition metals. Inorg. Chem. 48, 3866–3874.
Kondyurin, A., Levchenko, I., Han, Z.J., Yick, S., Mai-Prochnow, A., Fang, J., Ostrikov,
K., Bilek, M.M.M., 2013. Hybrid graphite film–carbon nanotube platform for
enzyme immobilization and protection. Carbon 65, 287–295.
Koppolu, B., Bhavsar, Z., Wadajkar, A.S., Nattama, S., Rahimi, M., Nwariaku, F.,
Nguyen, K.T., 2012. Temperature-sensitive polymer-coated magnetic nanopar-
ticles as a potential drug delivery system for targeted therapy of thyroid cancer.
J. Biomed. Nanotechnol. 8, 983–990.
Kudina, O., Zakharchenko, A., Trotsenko, O., Tokarev, A., Ionov, L., Stoychev, G.,
Puretskiy, N., Pryor, S.W., Voronov, A., Minko, S., 2014. Highly efficient phase
boundary biocatalysis with enzymogel nanoparticles. Angew. Chem.—Int. Ed.
53, 483–487.
Kuo, C.H., Liu, Y.C., Chang, C.M.J., Chen, J.H., Chang, C., Shieh, C.J., 2012. Optimum
conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles.
Carbohydr. Polym. 87, 2538–2545.
Le Nel, A., Krenkova, J., Kleparnik, K., Smadja, C., Taverna, M., Viovy, J.-L., Foret, F.,
2008. On-chip tryptic digest with direct coupling to ESI-MS using magnetic
particles. Electrophoresis 29, 4944–4947.
Lee, G.J., Attri, P., Choi, E.H., Kwon, Y.W., Krasnikov, I., Seteikin, A., 2014a. Optical and
structural properties of nanobiomaterials. J. Nanosci. Nanotechnol. 14, 221–249.
Lee, Y.C., Dutta, S., Wu, K.C.W., 2014b. Integrated, cascading enzyme-/chemocata-
lytic cellulose conversion using catalysts based on mesoporous silica nanopar-
ticles. ChemSusChem 7, 3181.
Li, F., Feng, Y., Wang, Z., Yang, L.M., Zhuo, L.H., Tang, B., 2010. Direct electrochemistry
of horseradish peroxidase immobilized on the layered calcium carbonate–gold
nanoparticles inorganic hybrid composite. Biosens. Bioelectron. 25, 2244–2248.
Li, S.F., Zhu, X.Y., Zhang, W., Xie, G.M., Feng, W.L., 2012. Hydrogen peroxide
biosensor based on gold nanoparticles/thionine/gold nanoparticles/multi-
walled carbon nanotubes–chitosans composite film-modified electrode. Appl.
Surf. Sci. 258, 2802–2807.
Li, Y., Xu, X., Deng, C., Yang, P., Zhang, X., 2007. Immobilization of trypsin on
superparamagnetic nanoparticles for rapid and effective proteolysis. J. Pro-
teome Res. 6, 3849–3855.
Li, Z., Zhang, Y., Su, Y., Ouyang, P., Ge, J., Liu, Z., 2014. Spatial co-localization of multi-
enzymes by inorganic nanocrystal–protein complexes. Chem. Commun. 50,
12465–12468.
Lin, J.F., Zhao, B.H., Cao, Y., Xu, H., Ma, S.H., Guo, M.Y., Qiao, D.R., 2014a. Rationally
designed Fe-MCM-41 by protein size to enhance lipase immobilization,
catalytic efficiency and performance. Appl. Catal. A: Gen. 478, 175–185.
Lin, Z., Xiao, Y., Yin, Y., Hu, W., Liu, W., Yang, H., 2014b. Facile synthesis of enzyme–
inorganic hybrid nanoflowers and its application as a colorimetric platform for
visual detection of hydrogen peroxide and phenol. ACS Appl. Mater. Interfaces
6, 10775–10782.
Lv, Y., Lin, Z., Tan, T., Svec, F., 2014. Preparation of reusable bioreactors using
reversible immobilization of enzyme on monolithic porous polymer support
with attached gold nanoparticles. Biotechnol. Bioeng. 111, 50–58.
Ma, C., Li, C.Y., He, N.Y., Wang, F., Ma, N.N., Zhang, L.M., Lu, Z.X., Ali, Z., Xi, Z.J., Li, X.L.,
Liang, G.F., Liu, H.N., Deng, Y., Xu, L.J., Wang, Z.F., 2012. Preparation and
characterization of monodisperse core–shell Fe3O4 @ SiO2 microspheres and
its application for magnetic separation of nucleic acids from E. coli BL21.
J. Biomed. Nanotechnol. 8, 1000–1005.
Ma, H., Liu, M.S., Jen, A.K.Y., 2009. Interface-tailored and nanoengineered polymeric
materials for (opto)electronic devices. Polym. Int. 58, 594–619.
Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., Fernandez-Lafuente, R.,
2007. Improvement of enzyme activity, stability and selectivity via immobiliza-
tion techniques. Enzyme Microb. Technol. 40, 1451–1463.
Nguyen, L.T., Yang, K.L., 2014. Uniform cross-linked cellulase aggregates prepared in
millifluidic reactors. J. Colloid Interface Sci. 428, 146–151.
Palanisamy, S., Cheemalapati, S., Chen, S.-M., 2014. Amperometric glucose biosen-
sor based on glucose oxidase dispersed in multiwalled carbon nanotubes/
graphene oxide hybrid biocomposite. Mater. Sci. Eng. C—Mater. Biol. Appl. 34,
207–213.
31
Pavlidis, I.V., Vorhaben, T., Tsoufis, T., Rudolf, P., Bornscheuer, U.T., Gournis, D.,
Stamatis, H., 2012. Development of effective nanobiocatalytic systems through
the immobilization of hydrolases on functionalized carbon-based nanomater-
ials. Bioresour. Technol. 115, 164–171.
Periasarny, A.P., Yang, S., Chen, S.-M., 2011. Preparation and characterization of
bismuth oxide nanoparticles-multiwalled carbon nanotube composite for the
development of horseradish peroxidase based H2O2 biosensor. Talanta 87,
15–23.
Rauch, S., Eichhorn, K.J., Oertel, U., Stamm, M., Kuckling, D., Uhlmann, P., 2012.
Temperature responsive polymer brushes with clicked rhodamine B: synthesis,
characterization and swelling dynamics studied by spectroscopic ellipsometry.
Soft Matter 8, 10260–10270.
Reddy, L.H., Arias, J.L., Nicolas, J., Couvreur, P., 2012. Magnetic nanoparticles: design
and characterization, toxicity and biocompatibility, pharmaceutical and biome-
dical applications. Chem. Rev. 112, 5818–5878.
Rodrigues, R.C., Ortiz, C., Berenguer-Murcia, A., Torres, R., Fernandez-Lafuente, R.,
2013. Modifying enzyme activity and selectivity by immobilization. Chem. Soc.
Rev. 42, 6290–6307.
Roessl, U., Nahalka, J., Nidetzky, B., 2010. Carrier-free immobilized enzymes for
biocatalysis. Biotechnol. Lett. 32, 341–350.
Roy, D., Cambre, J.N., Sumerlin, B.S., 2010. Future perspectives and recent advances
in stimuli-responsive materials. Prog. Polym. Sci. 35, 278–301.
Saha, K., Agasti, S.S., Kim, C., Li, X.N., Rotello, V.M., 2012. Gold nanoparticles in
chemical and biological sensing. Chem. Rev. 112, 2739–2779.
Sassolas, A., Blum, L.J., Leca-Bouvier, B.D., 2012. Immobilization strategies to
develop enzymatic biosensors. Biotechnol. Adv. 30, 489–511.
Saxena, U., Chakraborty, M., Goswami, P., 2011. Covalent immobilization of
cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive
amperometric detection of cholesterol in real samples. Biosens. Bioelectron. 26,
3037–3043.
Schmaljohann, D., 2006. Thermo- and pH-responsive polymers in drug delivery.
Adv. Drug Deliv. Rev. 58, 1655–1670.
Sheldon, R.A., 2011a. Characteristic features and biotechnological applications of
cross-linked enzyme aggregates (CLEAs). Appl. Microbiol. Biotechnol. 92,
467–477.
Sheldon, R.A., 2011b. Cross-linked enzyme aggregates as industrial biocatalysts.
Org. Process Res. Dev. 15, 213–223.
Sheldon, R.A., van Pelt, S., Kanbak-Aksu, S., Rasmussen, J.A., Janssen, M.H.A., 2013.
Cross-linked enzyme aggregates (CLEAs) in organic synthesis. Aldrichimica
Acta 46, 81–93.
Singh, R., Pantarotto, D., McCarthy, D., Chaloin, O., Hoebeke, J., Partidos, C.D., Briand,
J.P., Prato, M., Bianco, A., Kostarelos, K., 2005. Binding and condensation of
plasmid DNA onto functionalized carbon nanotubes: toward the construction of
nanotube-based gene delivery vectors. J. Am. Chem. Soc. 127, 4388–4396.
Stuart, M.A.C., Huck, W.T.S., Genzer, J., Muller, M., Ober, C., Stamm, M., Sukhorukov,
G.B., Szleifer, I., Tsukruk, V.V., Urban, M., Winnik, F., Zauscher, S., Luzinov, I.,
Minko, S., 2010. Emerging applications of stimuli-responsive polymer materials.
Nat. Mater. 9, 101–113.
Sun, H., She, P., Lu, G.L., Xu, K.L., Zhang, W., Liu, Z.N., 2014a. Recent advances in the
development of functionalized carbon nanotubes: a versatile vector for drug
delivery. J. Mater. Sci. 49, 6845–6854.
Sun, J.Y., Ge, J.C., Liu, W.M., Lan, M.H., Zhang, H.Y., Wang, P.F., Wang, Y.M., Niu, Z.W.,
2014b. Multi-enzyme co-embedded organic–inorganic hybrid nanoflowers:
synthesis and application as a colorimetric sensor. Nanoscale 6, 255–262.
Talekar, S., Joshi, A., Joshi, G., Kamat, P., Haripurkar, R., Kambale, S., 2013.
Parameters in preparation and characterization of cross linked enzyme aggre-
gates (CLEAs). RSC Adv. 3, 12485–12511.
Tasca, F., Ludwig, R., Gorton, L., Antiochia, R., 2013. Determination of lactose by a
novel third generation biosensor based on a cellobiose dehydrogenase and aryl
diazonium modified single wall carbon nanotubes electrode. Sens. Actuators
B: Chem. 177, 64–69.
Templeton, A.C., Hostetler, M.J., Kraft, C.T., Murray, R.W., 1998. Reactivity of
monolayer-protected gold cluster molecules: steric effects. J. Am. Chem. Soc.
120, 1906–1911.
Tian, Y., Mao, L., Okajima, T., Ohsaka, T., 2002. Superoxide dismutase-based third-
generation biosensor for superoxide anion. Anal. Chem. 74, 2428–2434.
Touahar, I.E., Haroune, I., Ba, S., Bellenger, J.P., Cabana, H., 2014. Characterization of
combined cross-linked enzyme aggregates from laccase, versatile peroxidase
and glucose oxidase, and their utilization for the elimination of pharmaceu-
ticals. Sci. Total Environ. 481, 90–99.
Tran, D.N., Balkus, K.J., 2011. Perspective of recent progress in immobilization of
enzymes. ACS Catal. 1, 956–968.
Vashist, S.K., Zheng, D., Al-Rubeaan, K., Luong, J.H.T., Sheu, F.S., 2011. Advances in
carbon nanotube based electrochemical sensors for bioanalytical applications.
Biotechnol. Adv. 29, 169–188.
Wang, J.J., Gong, C., Wang, Y.N., Wu, G.L., 2014. Magnetic nanoparticles with a pH-
sheddable layer for antitumor drug delivery. Colloids Surf. B—Biointerfaces 118,
218–225.
Wang, J.Q., Meng, G., Tao, K., Feng, M., Zhao, X.B., Li, Z., Xu, H., Xia, D.H., Lu, J.R.,
2012. Immobilization of lipases on alkyl silane modified magnetic nanoparti-
cles: effect of alkyl chain length on enzyme activity. PLoS One, 7.
Wang, L.-B., Wang, Y.-C., He, R., Zhuang, A., Wang, X., Zeng, J., Hou, J.G., 2013a. A
new nanobiocatalytic system based on allosteric effect with dramatically
enhanced enzymatic performance. J. Am. Chem. Soc. 135, 1272–1275.
32 M. Wang et al. / Chemical Engineering Science 135 (2015) 21–32
Wang, M.F., Jia, C.X., Qi, W., Yu, Q.X., Peng, X., Su, R.X., He, Z.M., 2011. Porous-CLEAs
of papain: application to enzymatic hydrolysis of macromolecules. Bioresour.
Technol. 102, 3541–3545.
Wang, M.F., Mu, Y.Y., Qi, W., Su, R.X., He, Z.M., 2013b. Intensive protein digestion
using cross-linked trypsin aggregates in proteomics analysis. ChemPlusChem
78, 407–412.
Wang, Q., Yang, Z., Wang, L., Ma, M., Xu, B., 2007. Molecular hydrogel-immobilized
enzymes exhibit superactivity and high stability in organic solvents. Chem.
Commun., 1032–1034.
Wang, R., Zhang, Y.F., Huang, J.H., Lu, D.N., Ge, J., Liu, Z., 2013c. Substrate imprinted
lipase nanogel for one-step synthesis of chloramphenicol palmitate. Green
Chem. 15, 1155–1158.
Weiser, D., Varga, A., Kovacs, K., Nagy, F., Szilagyi, A., Vertessy, B.G., Paizs, C., Poppe,
L., 2014. Bisepoxide cross-linked enzyme aggregates-new immobilized bioca-
talysts for selective biotransformations. ChemCatChem 6, 1463–1469.
Yan, M., Du, J.J., Gu, Z., Liang, M., Hu, Y.F., Zhang, W.J., Priceman, S., Wu, L.L., Zhou, Z.
H., Liu, Z., Segura, T., Tang, Y., Lu, Y.F., 2010. A novel intracellular protein delivery
platform based on single-protein nanocapsules. Nat. Nanotechnol. 5, 48–53.
Yan, M., Ge, J., Liu, Z., Ouyang, P.K., 2006. Encapsulation of single enzyme in nanogel
with enhanced biocatalytic activity and stability. J. Am. Chem. Soc. 128,
11008–11009.
Yang, J., Hu, Y., Jiang, L., Zou, B., Jia, R., Huang, H., 2013a. Enhancing the catalytic
properties of porcine pancreatic lipase by immobilization on SBA-15 modified
by functionalized ionic liquid. Biochem. Eng. J. 70, 46–54.
Yang, M.Z., Guan, Y.P., Yang, Y., Xia, T.T., Xiong, W.B., Wang, N., Guo, C., 2013b.
Peroxidase-like activity of amino-functionalized magnetic nanoparticles and
their applications in immunoassay. J. Colloid Interface Sci. 405, 291–295.
Yang, N., Chen, X., Ren, T., Zhang, P., Yang, D., 2015. Carbon nanotube based
biosensors. Sens. Actuators B: Chem. 207, 690–715.
Yang, X.E., Zheng, P., Ni, Y., Sun, Z.H., 2012. Highly efficient biosynthesis of sucrose-
6-acetate with cross-linked aggregates of Lipozyme TL 100 L. J. Biotechnol. 161,
27–33.
Yang, Z.P., Zhang, C.J., 2013. Single-enzyme nanoparticles based urea biosensor.
Sens. Actuators B: Chem. 188, 313–317.
Yu, C.Y., Wei, P., Li, X.F., Zong, M.H., Lou, W.Y., 2014. Using ionic liquid in a biphasic
system to improve asymmetric hydrolysis of styrene oxide catalyzed by cross-
linked enzyme aggregates (CLEAs) of mung bean epoxide hydrolases. Ind. Eng.
Chem. Res. 53, 7923–7930.
Zhang, M., Dai, L.M., 2012. Carbon nanomaterials as metal-free catalysts in next
generation fuel cells. Nano Energy 1, 514–517.
Zhang, X., Wang, H.B., Yang, C.M., Du, D., Lin, Y.H., 2013. Preparation, characteriza-
tion of Fe3O4 at TiO2 magnetic nanoparticles and their application for
immunoassay of biomarker of exposure to organophosphorus pesticides.
Biosens. Bioelectron. 41, 669–674.
Zhao, W.A., Chiuman, W., Lam, J.C.F., McManus, S.A., Chen, W., Cui, Y.G., Pelton, R.,
Brook, M.A., Li, Y.F., 2008. DNA aptamer folding on gold nanoparticles: from
colloid chemistry to biosensors. J. Am. Chem. Soc. 130, 3610–3618.
Zhao, Y., Perez-Segarra, W., Shi, Q.C., Wei, A., 2005. Dithiocarbamate assembly on
gold. J. Am. Chem. Soc. 127, 7328–7329.
Zhen, Q.N., Wang, M.F., Qi, W., Su, R.X., He, Z.M., 2013. Preparation of beta-
mannanase CLEAs using macromolecular cross-linkers. Catal. Sci. Technol. 3,
1937–1941.
Zhou, M., Peng, Z., Liao, S.Q., Li, P.W., Li, S.D., 2014. Design of microencapsulated
carbon nanotube-based microspheres and its application in colon targeted drug
delivery. Drug Deliv. 21, 101–109.
Zhou, S.Z., Bismarck, A., Steinke, J.H.G., 2013. Ion-responsive alginate based
macroporous injectable hydrogel scaffolds prepared by emulsion templating.
J. Mater. Chem. B 1, 4736–4745.
Zhou, Z., Hartmann, M., 2013. Progress in enzyme immobilization in ordered
mesoporous materials and related applications. Chem. Soc. Rev. 42, 3894–3912.
Zhu, J.Y., Zhang, Y.F., Lu, D.N., Zare, R.N., Ge, J., Liu, Z., 2013a. Temperature-
responsive enzyme–polymer nanoconjugates with enhanced catalytic activities
in organic media. Chem. Commun. 49, 6090–6092.
Zhu, L., Deng, C., Chen, P., You, X.D., Su, H.B., Yuan, Y.H., Zhu, M.F., 2013b. Glucose
oxidase biosensors based on carbon nanotube non-woven fabrics. New Carbon
Mater. 28, 342–348.
Zhu, L., Gong, L., Zhang, Y.F., Wang, R., Ge, J., Liu, Z., Zare, R.N., 2013c. Rapid
detection of phenol using a membrane containing laccase nanoflowers. Chem.
—Asian J. 8, 2358–2360.
Zhu, X., Niu, X., Zhao, H., Tang, J., Lan, M., 2015. Immobilization of superoxide
dismutase on Pt–Pd/MWCNTs hybrid modified electrode surface for superoxide
anion detection. Biosens. Bioelectron. 67, 79–85.
Zou, B., Hu, Y., Jiang, L., Jia, R., Huang, H., 2013. Mesoporous material SBA-15
modified by amino acid ionic liquid to immobilize lipase via ionic bonding and
cross-linking method. Ind. Eng.Chem. Res. 52, 2844–2851.