Journal of Biomaterials and Nanobiotechnology, 2010, 1, 61-77
doi:10.4236/jbnb.2010.11008 Published Online October 2010 (http://www.SciRP.org/journal/jbnb)
61
Review Article: Immobilized Molecules Using
Biomaterials and Nanobiotechnology
Magdy M. M. Elnashar
Centre of Scientific Excellence, Polymers Department, Advanced Materials & Nanotechnology Laboratory, National Research Cen-
ter, Cairo, Egypt.
Received August 24th, 2010; revised October 15th, 2010; accepted October 21st, 2010.
ABSTRACT
Immobilized molecules using biomaterials and nanobiotechnology is a very interesting topic that touching almost all
aspects of our life. It uses the sciences of biology, chemistry, physics, materials engineering and computer science to
develop instruments and products that are at the cutting edge of some of today’s most promising scientific frontiers. In
this review article, the author based on his experience in this arena has tried to focus on some of the supports for im-
mobilization; the most important molecules to be immobilized such as DNA, cells, enzymes, metals, polysaccharides, etc
and their applications in medicine, food, drug, water treatment, energy and even in aerospace. He specified a special
section on what is new in the arena of supports and technologies used in enzyme immobilization and finally a recom-
mendation by the author for fu ture work with a special attention to up-to-date references.
Keywords: Immobilized molecules, Biotechnology, Enzymes, Biomaterials, Nanobiotechnology
1. Introduction
1.1. Some Important Definitions
1.1.1. Defi niti on of Biotechnology
The European Federation of Biotechnology defined bio-
technology as “the integration of natural sciences and
engineering in order to achieve the application of organ-
isms, cells, parts thereof and molecular analogues for
products and services” [1]. In other words, Biotech ap-
plications can be divided into 5 key sectors: biomedicine,
bioagriculture, industrial biotechnology, bioenergy, and
bioenvironment.
1.1.2. Defi niti on of Immobiliz ation
An immobilized molecule is one whose movement in
space has been restricted either completely or to a small
limited region by attachment to a solid structure. In gen-
eral the term immobilization refers to the act of the lim-
iting movement or making incapable of movement i.e.,
retard the movement [2].
1.2. History of Immobilization
Immobilization is a natural phenomenon existing in the
universe. Microorganisms in nature are irregularly dis-
tributed and often exist in Biofilms. Biofilms are sur-
face-attached microbial communities consisting of mul-
tiple layers of cells embedded in hydrated matrices [3].
Biofilms were first extensively studied during the 1940s
but it was not until the 1970s that it was appreciated that
their formation occurs in almost all natural environments.
A rock immersed in a stream, an implant in the human
body, a tooth, a water pipe or conduit, etc. are all sites
where Biofilms develop [4]. This natural phenomenon
encouraged humans to utilize it for his services.
1.3. What Can We Immobilize?
Many molecules have been immobilized and the majority
of them are biomolecules due to their biological and
biomedical applications. The following are examples of
some of these molecules:
 Proteins:
− Enzymes, antibodies, antigens, cell adhesion mole-
cules and “Blocking” proteins
 Peptides:
− Substances composed of amino acids
 Drugs:
− Anticancer agents, antithrombogenic agents, antibi-
otics, contraceptives, drug antagonists and peptide/pro-
tein drugs
 Saccharides:
− Sugars, oligosaccharides and polysaccharides

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology
62
 Lipids:
− Fatty acids, phospholipids, glycolipids and any fat-
like substances.
 Ligands:
− Hormone receptors, cell surface receptors, avidin
and biotin
− In immunology, small molecules that are bound to
another chemical group or molecule
 Nucleic acids and nucleotides:
− DNA, RNA
− High MW substances formed of sugars, phosphoric
acid, and nitrogen bases (purines and pyrimidines).
 Others:
− Conjugates or mixtures of any of the above
1.4. Methods of Immobilization
The methods of immobilization of the different mole-
cules are almost the same. However, according to Cao, L.
2005 [5] there is no general universally applicable
method of certain molecule immobilization. As enzyme
molecules alone or in combination with drugs, antibodies
and antigens, are the most used in industries, we will be
focusing on the immobilization techniques used for
enzymes as a model of other immobilized molecules. The
enzyme market in 2005 was around 2.65 billion dollars,
with an expected annual growth of more than 9% [6]. On
the industrial level, 75% of the enzymes were used,
which is around 2 billion dollars.
However, expensive enzymes are not favored to be
used in industries in the Free State as they are difficult to
be separated from the products (Figure 1(a)) and conse-
quently are lost after the first use. They were alterna-
tively immobilized on solid supports (Figure 1(b)) so
that they can be easily separated from the products by
simple filtration or using a fluidized magnetized bed re-
actor system [7-14].
The main advantage for enzyme immobiliza tion is the
easy separation of the enzyme from the reaction mixture
(substrates and products) and its reusability for tens of
time, which reduces the enzyme and the enzymatic
products cost tremendously. Beside this splendid advan-
tage, the immobilization process imparts many other ad-
vantages to the enzyme such as:
 The ability to stop the reaction rapidly by removing
the enzyme from the reaction solution (or vice
versa)
 Product is not contaminated with the enzyme
 Easy separation of enzyme from the product (espe-
cially useful in food and pharmaceutical industries)
 Enhancement of enzyme stability against pH, tem-
perature, solvents, contaminants, and impurities.
Immobilization provides a physical support for enzymes,
cells and other molecules. Immobilization of enzymes is
one of the main methods used to stabilize free enzymes
[7,8]. The support material and the main methods of im-
mobilization are key parameters in enzyme immobiliza-
tion. There are five principal methods for immobiliza-
tion of enzymes and cells (adsorption, covalent, entrap-
ment, encapsulation and crosslinking) and no one method
is perfect for all molecules or purposes. However, Katz-
bauer and Moser, 1995 [15] represented a classification
of combination between these methods.
Dialysis
Free
Enzyme
SubstrateProduct + Free EnzymeProduct Free
Enzyme
(a)
A = a1
Filtration
SubstrateProduct + Immobilised EnzymeProductImmob.
Enzyme
Immobilised
Enzyme
(b)
Figure 1. Schematic diagram of free and immobilized enzyme reactions. (a) Reaction of free enzyme with substrate and for-
mation of product, which has to be separated via dialysis; (b) Reaction of immobilized enzyme with substrate and formation
of product, which can be se parated via filtration or using a fluidized magnetized be d reactor system.

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology63
1.4.1. Adsorp t i on
Immobilization by adsorption is the simplest method and
involves reversible surface interactions between enzyme
and support material as shown in Figure 2. The proce-
dure of adsorption consists of mixing together the bio-
logical component(s) and a support with adsorption
properties, under suitable conditions of pH and ionic
strength for a period of incubation, followed by collec-
tion of the immobilized material and extensive washing
to remove the unbound biological components. The first
industrially used immobilized enzyme was prepared by
adsorption of amino acid acylase on DEAE-cellulose
[16]. Menaa et al. (2008a) [17] reported the role of hy-
drophobic surfaces of nanoporous silica glasses on pro-
tein folding enhancement.
Advantages of enzymes immobilized using the ad-
sorption technique:
 Reversibility, which enables not only the purifica-
tion of proteins but also the reuse of the carriers;
 Simplicity, which enables enzyme immobilization
under mild conditions;
 Possible high retention of activity because there is
no chemical modification [18];
 Cheap and qui c k method;
 No chemical changes to the support or enzyme oc-
curs.
Disadvantages of enzymes immobilized using the ad-
sorption technique:
 The immobilized enzymes prepared by adsorption
tend to leak from the carriers, owing to the rela-
tively weak interaction between the enzyme and the
carrier, which can be destroyed by desorption forces
such as high ionic strength, pH, etc,
 Contamination of product,
 Non-specific binding,
 Overloading on the support and
 Steric hindrance by the support.
Consequently, a number of variations have been de-
veloped in recent decades to solve this intrinsic drawback.
Examples are adsorption–cross-linking; modification–
adsorption; selective adsorption–covalent attachment;
and adsorption–coating, etc. For more details, the reader
is recommended to read the book of Cao L, 2005 [5].
1.4.2. Coval e n t Bi ndi n g
This method of immobilization involves formation of a
covalent bond between the enzyme and support material
as shown in Figure 3. Covalent bonds usually provide
the strongest linkages between enzyme and carrier, com-
pared with other types of enzyme immobilization meth-
ods. Thus, leakage of enzyme from the matrix used is
often minimized with covalently bound immobilized
enzymes [5]. The bond is normally formed between func-
tional groups present on the surface of the support and
functional groups belonging to amino acid residues on
the surface of the enzyme.
Multi-step immobilization is one of the technologies to
enhance enzyme covalent immobilization [19]. There are
many reaction procedures for coupling an enzyme to a
support via covalent bond however, most reactions fall
into the following categories: formation of an isourea
linkage; formation of a diazo linkage; formation of a pep-
tide bond or an alkylation reaction as shown in Table 1.
_
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_
+
Support
Enzyme
Figure 2. Immobilization of enzymes using the adsorption
technique.
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
Support
Enzyme
Figure 3. Immobilization of enzymes using the covalent
technique.
Table 1. Different methods for covalent binding of enzymes to supports.
Reaction Support – Enzyme Linkage
Diazotization SUPPORT--N=N---ENZYME
SUPPORT--CH2-NH---ENZYME
Alkylation and arylation SUPPORT--CH2-S---ENZYME
Schiff's base formation SUPPORT---CH=N---ENZYME
Amide bond formation SUPPORT---CO-NH---ENZYME
Amidation reaction SUPPORT---CNH-NH---ENZYME
Thiol-Disulfide interchange SUPPORT---S-S---ENZYME
Carrier binding with bifunctional reagents SUPPORT---O(CH2)2 N=CH(CH2)3 CH=N---ENZYME

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology
64
Advantages of enzymes immobilized using the cova-
lent technique:
 No leakage of enzyme.
 The enzyme can be easily in contact to substrate due
to the localization of enzyme on support materials.
 Increase of the thermal stability.
Disadvantages of enzymes immobilized using the co-
valent technique:
 The cost is quite high as the good supports are very
expensive (e.g. Eupergit C and Agaroses).
 Loss of enzyme activity (e.g. mismatched orienta-
tion of enzyme on the carriers such as involvement
of active centre in the binding).
1.4.3. Entrapment
Immobilization by entrapment differs from adsorption
and covalent binding as shown in Figure 4 in that en-
zyme molecules are free in solution, but restricted in
movement by the lattice structure of a gel [20]. The po-
rosity of the gel lattice is controlled to ensure that the
structure is tight enough to prevent leakage of enzyme or
cells, yet at the same time allows free movement of sub-
strate and product. The support also acts as a barrier and
can be advantageous as it protects the immobilized en-
zyme from microbial contamination by harmful cells,
proteins, and enzymes in the microenvironment [21].
Entrapment can be achieved by mixing an enzyme
with a polyionic polymer material, such as carrageenan,
and by crosslinking the polymer with multivalent cations,
e.g. hexamethylene diamine, in an ion-exchange reaction
to form a lattice structure that traps the enzymes, this is
termed ionotropic gelations.
Advantages of enzymes immobilized using the en-
trapment technique:
 Enzyme loading is very high
Disdvantages of enzymes immobilized using the en-
trapment technique:
 Enzyme leakage from the support.
 Diffusion of the substrate to the enzyme and of the
product away from the enzyme (diffusion limita-
tion).
1.4.4. Encapsulation
Encapsulation of enzymes as shown in Figure 5 can be
achieved by enveloping the biological components
within various forms of semipermeable membranes [22].
It is similar to entrapment in that the enzyme is free in
solution, but restricted in space. Large proteins or en-
zymes can not pass out of, or into the capsule, but small
substrates and products can pass freely across the
semipermeable membrane. Many materials have been
used to construct microcapsules varying from 10-100 m
in diameter. For example, nylon and cellulose nitrate
have proven popular. Ionotropic gelation of alginates
have proven it efficacy as well for encapsulation of
drugs, enzymes and cells [23]. On the nano scale level,
Menaa et al., 2008b, 2009 & 2010 [24-26] used Silica-
based nanoporous sol-gel glasses for the study of encap-
sulation and stabilization of some proteins.
Advantages of enzymes immobilized using the en-
capsulation technique:
 The enzymes could be encapsulated inside the cell.
 Possibility of coimmobilization. Where cells and/or
enzymes may be immobilized in any desired com-
bination to suit particular applications.
Disdvantages of enzymes immobilized using the en-
trapment technique:
 The problems associated with diffusion are acute and
may result in rupture of the membrane if products
from a reaction accumulate rapidly.
1.4.5. Crossl i n ki ng
This type of immobilization is support-free as shown in
Figure 6 and involves joining enzyme molecules to each
other to form a large, three-dimensional complex struc-
ture, and can be achieved by chemical or physical meth-
ods [19]. Chemical methods of crosslinking normally
Support
Enzyme
Figure 4. Immobilization of enzyme using the entrapment
technique.
Support
Enzyme
Figure 5. Immobilization of enzymes using the encapsula-
tion technique.

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology65
Enzyme
Figure 6. Crosslinking technique .
involve covalent bond formation between the enzymes
by means of a bi- or multifunctional reagent, such as
glutaraldehyde, dicarboxylic acid or toluene diisocyanate.
Flocculating agents, such as polyamines, polyethyle-
neimine, polystyrene sulfonates, and various phosphates,
have been used extensively to cross-link cells using
physical bonds. Crosslinking is rarely used as the only
means of immobilization, because poor mechanical
properties of the aggregates are severe limitations.
Crosslinking is most often used to enhance the other
methods of immobilization described.
Advantages of enzymes immobilized using the
crosslinking technique:
 The immobilization is support-free.
 Cross-linking between the same enzyme molecules
stabilises the enzymes by increasing the rigidity of
the structure
Disdvantages of enzymes immobilized using the
crosslinking technique:
 Harshness of reagents of crooslinking is a limiting
factor in applying this method to many enzymes.
 The enzyme may partially lose activity or become
totally inactivated in case the cross-linking reagent
reacted across the active site.
1.5. Examples of Matrices and Shapes for
Immobilization
Matrices for immobilization can be classified according
to their chemical composition as organic and inorganic
supports. The former can be further classified into natural
and synthetic matrices as in Table 2 [27].
The shape of the carrier can be classified into two
types, i.e. irregular and regular shapes such as (A): beads;
(B): fibres; (C): hollow spheres; (D): thin films; (E):
discs and (F): membranes. Selection of the geometric
properties for an immobilized molecule is largely de-
pendent on the peculiarity of certain applications.
Table 2. Chemical classification of enzyme matrices.
Organic Inorganic
Natural polymers
Polysaccharides
Cellulose
Dextran
Starch
Agar and agarose
Alginate
Carrageenans
Chitin and chitosan
Proteins
Collagen
Gelatin
Albumin
Ferritin
Minerals
Attapulgite clays
Bentonite
Kieselgur
Pumic stone
Hornblend
Diatomaceous earth
Sand
Synthetic polymers
Polystyrene
Polyacrylate and polymethacrylate
Polyacrylamides
Hydroxyalkyl methacrylate
Vinyl polymer
Maleic anhydride polymer
Polyethyleneglycol
Aldehyde-based polymer
Fabricated materials
Non-porous glass
Controlled pore glass
Controlled pore metal oxides
Alumina catalyst
Porous silica
Silochrome
Iron oxide
Stainless steel

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology
66
Gel disks are widely used in the literature. Researchers
usually use the casting method, e.g. a Petri dish, to make
a single film of gel and then cut it into disks using cork
borers. Elnashar et al., 2005 [28], invented a new
equipment to make many uniform films in one step and
with high accuracy using the equipment “Parallel Plates”
as shown in Figure 7.
Gel beads are mostly used in industries as they have
the largest surface area and can be formed by many tech-
niques such as the interphase technique, ionic gelation
methods, dripping method and the Innotech Encapsulator
[7,10]. The Innotech Encapsulator as shown in Figure 8
has the advantage of high bead production (50 – 3000
beads per second depending on bead size and encapsula-
tion-product mixture viscosity), which is suitable for the
scaling up production on the industrial scale.
Circular plates
Support
d(
Rod Spacing blocks Gel-Sheet
Beaker (1 L)
A B
Figure 7. Parallel plates equipment for making uniform
k-carrageenan gel disks.
Figure 8. Inotech Encapsulator IE-50 R.
1.6. Properties of Matrices for Immobilization
The supports on which molecules such as enzymes, an-
tibodies, antigens, etc will be immobilized are of great
interest. The term support or media is usually understood
to refer to a combination of a ligand that is firmly at-
tached often by covalent means, and a solid insoluble
matrix. These supports have to exhibit good chemical
and physical stability and contain available functional
groups to bind to the active molecule. To use a support
for immobilization of active molecules such as enzymes,
a range of fundamental properties are required, which are
summarised as follows [20].
a) Availability of matrix from a reliable commercial
source
b) Matrix has an abundance of easily derivatizable
functional groups
c) Matrix has good mechanical and chemical stability
d) Matrix has good capacity for the target molecule
e) Matrix material is “user friendly”
2. Applications of Immobilized Molecules
2.1. Drug Delivery Systems
Advanced drug delivery systems (ADDS) have found
applications in many biomedical fields [29,30]. Drug
delivery is a combination of material science, pharma-
ceutics and biology [31]. Adoption of different types of
membranes in ADDS has made it possible to release drug
in an optimal fashion according to the nature of a disease
[32]. Examples of drug delivery systems include glu-
cose-sensitive insulin and drug loaded magnetic nanopar-
ticles.
2.1.1. De ve l op m ent of Glucose-Sensi ti ve Insulin
The swelling or shrinking of smart hydrogel beads in
response to small changes in pH or temperature can be
used successfully to control drug release, because the
diffusion of the drug out of the beads depends on the gel
state [33].
Drug-delivery systems in which a drug is liberated in
response to a chemical signal (e.g. insulin release in
response to rising glucose concentration) can be
achieved using this system. The exposure of a glucose-
sensitive insulin releasing system to glucose resulted in
the oxidation of glucose to gluconic acid and thus a de-
crease in the pH, protonation and shrinking of the poly-
mer, leading to an increased release of insulin. The
polymer swells in size at normal body pH (pH = 7.4) and
closes the gates. It shrinks at low pH (pH = 4) when the
blood glucose level increases, thus opening the gates and
releasing the insulin from the nanoparticles [34].
2.1.2. Drug Loaded Magnetic Nanoparticles
Nanotechnology offers the means to send the drugs to

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology67
targeted sites, and has the drug released in a controlled
manner, which reduce side effects due to lower dosage
and minimize or prevent drug degradation by using
pathways other than gastrointestinal. Magnetic nanopar-
ticles are recently applied in various fields such as MRI
imaging, water treatment, hyperthermia and drug deliv-
ery systems. Drug loaded magnetic nanoparticles
(DLMNP) have several advantages such as: small parti-
cle size; large surface area; magnetic response; biocom-
patibility and non-toxicity. DLMNP is introduced
through injection and directed with external magnets to
the right organ, which requires smaller dosage because of
targeting, resulting in fewer side effects.
Recently, Yu et al., 2008 [35] reported a novel In Vivo
strategy for combined cancer imaging and therapy by
employing thermally cross-linked superparamagnetic
iron oxide nanoparticles as a drug-delivery carrier.
Whereas, Kettering et al., 2009 [36] used magnetic iron
nanoparticles with cisplatin adsorbed in them for drug
release in magnetic heating treatments for cancer.
2.2. Enzyme-Linked Immunosorbent Assays
(ELISA)
ELISA is a test used as a general screening tool for the
detection of antibodies or antigens in a sample [37].
ELISA technology links a measurable enzyme to either
an antigen or antibody. The procedure for detection of
Ab in patient’s sample as follows:
- Immobilize Ag on the solid support (well)
- Incubate with patient sample
- Add antibody-enzyme conjugate
- Amount of antibody-enzyme conjugate bound is
proportional to amount of Ab in the sample
- Add substrate of enzyme
- Amount of color is proportional to amount of Ab in
patient’s sample.
However, ELISA technique in some cases is regarded
as time consuming and it needs special equipment to run
the assay (not portable). Thus many techniques have
been developed to fasten the process such as that of Xin
et al., 2009 [38], where he developed a chemilumines-
cence enzyme immunoassay using magnetic particles to
monitor 17β-estradiol (E2) in environmental water sam-
ples. Another technique is using simple/rapid (S/R) test.
The development of simple/rapid S/R tests has been ex-
tended from pregnancy detection of HIV antibodies in
whole blood in addition to serum and plasma [39].
2.3. Antibiotics Production
Penicillins are the most widely used β-lactam antibiotics,
with a share of about 19 % of the estimated world-wide
antibiotic market (Table 3) [8,27].
Production of antibiotics is one of the key areas in the
field of applied microbiology. The conventional method
of production is in stirred tank batch reactors. Since it is
a no growth associated process, it is difficult to produce
the antibiotic in continuous fermentations with free-cells.
But it is a suitable case for cell immobilization, since
growth and metabolic production can be uncoupled
without affecting metabolite yields. Therefore, several
attempts have been made to immobilize various micro-
bial species on different supports matrices for antibiotic
production. The most widely studied system is the pro-
duction of penicillin G using immobilized cells of Peni-
cillium chrysogenum [4]. In a recent study by Elnashar et
al., they were successful to covalently immobilize pen-
cillin G acylase on carrageenan modified gels with reten-
tion of 100% activity after 20 reuses [9].
2.4. Medical Applications Particularly in
Therapy
Medical applications of immobilized enzymes include
diagnosis and treatment of diseases, among those enzyme
replacement therapies, as well as artificial cells and or-
gans, and coating of artificial materials for better bio-
compatibility [41]. Examples of potential medical uses of
immobilized enzyme systems are listed below. For more
applications, readers are encouraged to read the review
article of Soetan et al., 2010 [42], where he reviewed the
biochemical, biotechnological and other applications of
enzymes.
 Asparaginase (3.5.1.1) for leukemia
 Arginase (3.5.3.1) for cancer
 Urease (3.5.1.5) for artificial kidney, uraemic dis-
orders
 Glucose oxi da se (1.1.3.4) for artificial pancreas
 Carbonate dehydratase (4.2.1.1) and catalase
(1.11.1.6) for artificial lungs
 Glucoamylase (3.2.1.3) for glycogen storage dis-
ease
 Glucose-6-phosphate dehydrogenase (1.1.1.49) for
glucose-6-phosphate dehydrogenase deficiency
 Xanthine oxidase (1.1.3.22) for Lesch–Nyhan dis-
ease
 Phenylalanine ammonia lyase (4.3.1.5) for
phenylketonuria
 Urate oxidase (1.7.3.3) for hyperuricemia
 Heparinase (4.2.2.7) for extracorporeal therapy
procedures
In addition to the above applications, we will focus the
light on some important applications as solving the prob-
lem of lactose Intolerant people, production of fructose
for diabetics and for people on diet regimen, and treat-
ment of rheumatoid arthritis and joint diseases.

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology
68
2.4.1. Solving the Problem of Lactose Intolerant
People
β-galactosidase is widely used in milk industries for hy-
drolysis of lactose to glucose and galactose. Lactose is
the main carbohydrate contained in milk at a concentra-
tion between 5% and 10% (w/v) depending on the source
of milk [43]. Lactose could also be found in whey per-
meate at higher concentrations. The consumption of
foods with a high content of lactose is causing a medical
problem for almost 70% of the world population, espe-
cially in the developing countries, as the naturally present
enzyme (β-galactosidase) in the human intestine, loses its
activity during lifetime [44]. Undigested lactose in
chyme retains fluid, bacterial fermentation of lactose
results in production of gases, diarrhoea, bloating and
abdominal cramps after consumption of milk and other
dairy products.
Unfortunately, there is no cure to “lactose intoler-
ance”. This fact, together with the relatively low solubil-
ity and sweetness of lactose, has led to an increasing in-
terest in the development of industrial processes to hy-
drolyze the lactose contained in dairy products (milk and
whey) with both the free and immobilized conditions
[45]. The studies have shown that glucose and galactose,
the two monosaccharides hydrolosates of lactose (prod-
ucts hydrolyzed from lactose), are four times sweeter
than lactose, more soluble, more digestible [46], and can
be consumed by ‘lactose intolerant’ people. Interesting
results of immobilized β-galactosidase on thermostable
biopolymers of grafted carrageenan were obtained re-
cently by Elnashar and Yassin [10,13].
2.4.2. Fructose for Diabetics and for People on Diet
Regimen
People on diet regimen and patients suffering from dia-
betes are highly recommended to consume fructose
rather than any other sugar. Fructose can be produced
from starch by enzymatic methods involving α-amylase,
amyloglucosidase, and glucose isomerase, resulting in
the production of a mixture consisting of oligosaccha-
rides (8%), fructose (45%), and glucose (50%) [47].
However, separation of fructose from this high content
fructose syrup is costly and thus makes this method un-
economical. In industries, inulinases are used to produce
95% of pure fructose after one step of the enzymatic hy-
drolysis of inulin. Industrial inulin hydrolysis is carried
out at 60 °C to prevent microbial contamination and also
because it permits the use of higher inulin substrate con-
centration due to increased solubility. Elnashar et al.,
2009 and Danial et al., 2010, have succeeded recently to
produce a thermostable inulinolytic immobilized enzyme,
which would be expected to play an important role in
food and chemical industries, in which fructose syrup is
widely applied [7,10].
2.4.3. Treatment of Rheumatoid Arthritis and Joint
Diseases
Superoxide dismutase (SOD) and catalase (CAT) have
been encapsulated in biodegradable microspheres (MS)
to obtain suitable sustained protein delivery [48]. A
modified water/oil/water double emulsion method was
used for poly (D, L-lactide-co-glycolide) (PLGA) and
poly (D, L-lactide) PLA MS preparation co-encapsulat-
ing mannitol, trehalose, and PEG400 for protein stabili-
zation. SOD release from PLGA MS may be potentially
useful for long-term sustained release of the enzyme for
the treatment of rheumatoid arthritis or other intra-ar-
ticular and joint diseases (inflammatory manifestation).
2.5. Non Medical Applications of Immobilized
Enzymes
2.5.1. Treatment of Pesticide-Contaminated Waste
Application of pesticide in agriculture serves to lower the
cost of production, increase crop yields, provide better
quality produce and also reduce soil erosion. Although
pesticides are toxic and have adverse effect on human
health and the environment, their use is inevitable in
many cases as an effective means of controlling weeds,
insect, and fungus, parasitic and rodent pests. One of the
most important technologies to be applied for this ap-
proach is immobilized enzyme. The immobilized enzyme
is capable of breaking down a range of pesticide-con-
taminated waste as organophosphate insecticides [49,50].
2.5.2. Neutralizing Dangerous Chemical Gases or
Vapors
The use of immobilized enzymes in the national security
arena has shown to be promising. For example, they
could include infiltrating items such as air filters, masks,
clothing, or bandages with the concentrated immobilized
enzymes to neutralize dangerous chemical gases or va-
pors [51].
2.6. Purification of Proteins
Protein purification is an important objective in industrial
enzymes in order to increase the enzyme's specific activ-
ity and to obtain an enzyme in its pure form for a specific
goal. Affinity ligands is the most used technique for pu-
rification of target molecules as it can reduce the number
of chromatographic steps in purification procedures to
one or two steps. Immobilization of affinity ligands to an
insoluble support can be a powerful tool in isolation of
particular substances (e.g. protein) from a complex mix-
ture of proteins. Some examples of affinity ligands are
immobilized carbohydrate-binding proteins and immobi-
lized metal ions. Another technique for protein purifica-
tion is using Electric field gradient focusing (EFGF). For

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology69
more information on the principles and methods of pro-
tein purification, readers should refer to the “Handbook:
Purifying challenging proteins: principles and meth-
ods” in 2007 [52].
2.6.1. Immobilized Carbohydrates-Binding Proteins
Purification of proteins could be performed using immo-
bilized carbohydrates such as mannose, lactose and meli-
biose. For example, immobilized lactose on sepharose
4B™ will be selective for purification of lactase from a
mixture of other proteins. More information on this tech-
nique can be found in the book of Hermanson et al.,
1992 “Immobilized affinity ligand techniques” [53].
2.6.2. Electric Field Gradient Focusing (EFGF)
Electric field gradient focusing is a member of the family
of equilibrium gradient focusing techniques (e.g gel elec-
trophoresis). It depends on an electric field gradient and a
counter-flow to focus, concentrate and separate charged
analytes, such as peptides and proteins. Since analytes
with different electrophoretic mobilities have unique
equilibrium positions, EFGF separates analytes accord-
ing to their electrophoretic mobilities, similar to the way
isoelectric focusing (IEF: electrophoresis is a pH gradient
where the cathode is at a higher pH value than the anode)
separates analytes according to isoelectric points. The
constant counter flow is opposite to the electrophoretic
force that drives the analytes. When the electrophoretic
velocity of a particular analyte is equal and opposite to
the velocity of the counter flow, the analyte is focused in
a narrow band because at this position the net force on it
is zero.
However, EFGF avoids protein precipitation that often
occurs in IEF when proteins reach their isoelectric points
and, therefore, can be applied to a broad range of pro-
teins. Sun (2009) [54] in his Ph.D. thesis demonstrated
that protein concentration exceeding 10,000-fold could
be concentrated using such devices.
2.7. Extraction of Biomolecules Using Magnetic
Particles
The traditional methods for biomolecules purification
such as centrifugation, filtration, and chromatography
can today be replaced by the use of magnetic particles.
They are reactive supports for biomolecules capturing.
Their use is simple, fast, and efficient for the extraction
and purification of biomolecules. In the biomedical Weld,
numerous publications deal with the use of magnetic
particles for biomolecule extraction [55], cell sorting [56],
and drug delivery [57]. Magnetic beads are widely used
in molecular biology [58], medical diagnosis [59], and
medical therapy [55].
The major application concerns the extraction of bio-
molecules such as proteins [60], antibodies, and nucleic
acids [61]. Magnetic beads carrying antibodies are also
used for specific bacteria [55] and virus captures [58].
Krupey in 1994 [62] patented a method for virus capture
process. The method was based on interactions between
viruses and anionic polymers, leading to the precipitation
of complexes by charge neutralization. After the capture
step, viruses were extracted by centrifugation. At the
current time, to our knowledge, only one method using
magnetic beads has been published recently [63]. In these
studies, some DNA and RNA viruses were concentrated
more than 100 and 1000 times, respectively, using poly-
ethyleneimine (PEI)1-conjugated magnetic beads.
2.8. Heavy Metals Removal
Heavy metal pollution is an environmental problem of
worldwide concern. Several industrial wastewater
streams may contain heavy metals such as; Pb, Cr, Cd,
Ni, Zn, As, Hg, Cu, Ag. Traditionally, precipitation, sol-
vent extraction, ion-exchange separation and solid phase
extraction are the most widely used techniques to elimi-
nate the matrix interference and to concentrate the metal
ions. Many materials have been used to remove them
such as sorbents [64] (e.g. silica, chitosan, sponge, etc)
and biosorbents (e.g. immobilized algae) [65].
Biosorbents: can be defined as the selective seques-
tering of metal soluble species that result in the immobi-
lization of the metals by microbial cells such as cyano-
bacteria. It is the physicochemical mechanisms of inac-
tive (i.e. non-metabolic) metal uptake by microbial bio-
mass. Metal sequestering by different parts of the cell
can occur via various processes: complexation, chelation,
coordination, ion exchange, precipitation, reduction. Size
of immobilized bead for metals removal is a crucial fac-
tor for use of immobilized biomass in bio-sorption proc-
ess. It is recommended that beads should be in the size
range between 0.7 and 1.5 mm, corresponding to the size
of commercial resins meant for removing metal ions.
Abdel Hameed and Ebrahim, 2007 [63] in their review
article, has revealed some of the immobilized algae on
different matrices that have potential in heavy metals
removal due to its high uptake capacity and abundance.
2.9. Production of Biosensors
Biosensors are chemical sensors in which the recognition
system utilizes a biochemical mechanism [66]. A bio-
sensor is a sensing device made up of a combination of a
specific biological element and a transducer. The ”spe-
cific biological element” such as antibodies [67], en-
zymes [68], bacteria [69,70] and DNA [71] recognizes a
specific analyte such as pollutions (toxicity caused by
pesticides, phenols, mercury, arsenic, etc) and the
changes in the biomolecules are usually converted into
electrical signal (which is in turn calibrated to a certain

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology
70
scale) by a transducer.
2.10. Production of Biodiesel
The idea of using biodiesel as a source of energy is not
new [72], but it is now being taken seriously because of
the escalating price of petroleum and, more significantly,
the depletion of fossil fuels (oil and gas) within the next
35 years and the emerging concern about global warming
that is associated with burning fossil fuels [73]. Biodiesel
is much more environmentally friendly than burning fos-
sil fuels, to the extent that governments may be moving
towards making biofuels mandatory [74]. The global
market survey of biodiesel has shown a tremendous in-
crease in its production.
Biodiesel is made by chemical combination of any
natural oil or fat with an alcohol such as methanol and a
catalyst (e.g. lipases) for the transesterification process.
Transesterification is catalyzed by acids, alkalis [75] and
lipase enzymes [76]. Use of lipases offers important ad-
vantages as it is more efficient, highly selective, involves
less energy consumption (reactions can be carried out in
mild conditions), and produces less side products or
waste (environmentally favorable). However, it is not
currently feasible because of the relatively high cost of
the catalyst [77].
On the industrial level, a number of methods for the
immobilization of lipases on solid supports have been
reported [78]. Commercially available lipases are sup-
plied both as lyophilised powders, which contain other
components in addition to the lipase [79]. The immobi-
lized lipases most frequently used for biodiesel produc-
tion are lipase B from Candida Antarctica [80]. This is
supplied by Novozymes under the commercial name
Novozym 435® (previously called SP435) and is immo-
bilized on an acrylic resin. The Mucor miehei commer-
cial lipase (Lipozyme IM60 – Novozym) immobilized on
a macroporous anionic exchange resin has also been ex-
tensively used for the same purpose [81].
2.11. Life Detection and Planetary Exploration
Analytical techniques based on mass spectrometry have
been traditionally used in space science. Planetary ex-
ploration requires the development of miniaturized ap-
paratus for in situ life detection. Recently, a new ap-
proach is gaining acceptance in the space science com-
munity: the application of the well-known, highly spe-
cific, antibody–antigen affinity interaction for the detec-
tion and identification of organics and biochemical
compounds. Antibody microarray technology allows
scientists to look for the presence of thousands of differ-
ent compounds in a single assay and in just one square
centimeter. The detection of organic molecules of unam-
biguous biological origin is fundamental for the confir-
mation of present or past life.
Preservation of biomarkers on the antibody stability
under space environments, smaller biomolecules, such as
amino acids, purines, and fatty acids, are excellent bio-
markers in the search for life on Mars, but they may be
much less resistant to oxidative degradation. Recent
work by Kminek and Bada, 2006 [82] showed that
amino acids can be protected from radiolysis decomposi-
tion as long as they are shielded adequately from space
radiation. They estimated that it is necessary to drill to a
depth of 1.5 to 2 m to detect the amino acid signature of
life that became extinct about three billion years ago. A
microfabricated capillary [83] electrophoresis device
(kind of new immobilization technology) for amino acid
chirality determination was developed for extraterrestrial
exploration [84]. Recently, antibody microarray, a new
immobilization technology that kept the stability of anti-
body under space environment allowed it to be applied
for planetary exploration Exomars mission [85].
3. Recent Advances in Supports and
Technologies used in Enzyme
Immobilization
In the search for suitable supports for enzyme immobili-
zation, it was found that physical and chemical properties
(e.g. pore size, hydrophilic/hydrophobic balance, aq-
uaphilicity and surface chemistry) of support could exert
effect on enzyme immobilization and its catalytic proper-
ties [86]. Thus there was a need for new immobilization
techniques/supports to avoid such shortcomings [19].
The following are some examples of the recent carriers
and technologies used for enzyme immobilization.
3.1. New Carriers Used in Immobilization
3.1.1. New Carriers Used in Immobilization
Over the last few years, mesoporous support such as sil-
ica and silicates having pore size of 2–50 nm has been
developed and being considered as one of the most pro-
mising carriers for enzyme immobilization [87-91]. The
exploitation of novel carriers that enable high enzyme
loading and activity retention has become the focus of
recent attention [92]. The large surface areas and greater
pore volumes of these materials could enhance the load-
ing capacity of an enzyme and the large pores in the
support facilitate transport of substrate and product [93].
Functional mesoporous material resulted in exception-
ally high immobilization efficiency with enhanced stabil-
ity, while conventional approaches yielded far lower
immobilization efficiency [94]. Additionally, the increase
in the thermal stability of immobilized enzyme indicated
that protein inside a confined space could be stabilized
by some folding forces which did not exist in proteins in
bulk solutions [95]. Confinement of the support nanopore

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology71
could be similar to the macromolecular crowding [96],
and could also stabilize the enzyme at high temperature.
Nanoporous gold [97] and nanotube [98,99] have also
been used to immobilize enzymes. Most of the obtained
immobilized enzymes were used in the electrode prepa-
ration and biosensor applications. The modified porous
gold electrode shows an overall increased signal, and
therefore a better detection limit and higher sensitivity
when used as sensors.
3.1.2. Magnetic Hybrid Support
The use of magnetic supports for enzyme immobilization
enables a rapid separation in an easily stabilized fluidized
bed reactor for continuous operation of enzyme. It can
also reduce the capital and operation costs [100]. Due to
the functionalization [101] of enzyme and its suitable
microenvironment, magnetic materials were often em-
bedded in organic polymer or inorganic silica to form
hybrid support [102]. Recently, because of the low en-
zyme loading on the conventional magnetic beads, fur-
ther attention was paid to the magnetic mesoporous sup-
port [103]. Magnetite mesoporous silica hybrid support
was fabricated by the incorporation of magnetite to the
hollow mesoporous silica shells, which resulted in the
perfect combination of mesoporous materials properties
with magnetic property. The produced hybrid support has
shown to improve the enzyme immobilization [104].
3.2. New Technologies for Enzyme
Immobilization
3.2.1. Single Enzyme Nanoparticles
In the field of industrial enzymes, there is a great re-
search for improving the enzyme stability under harsh
conditions. As an innovative way of enzyme stabilization,
“single-enzyme nanoparticles (SENs)” technology was
rather attractive because enzymes in the nanoparticle
exhibited very good stability under harsh conditions
[107] have developed armored SENs that surround each
enzyme molecule with a porous composite organic/ in-
organic network of less than a few nanometers thick.
They significantly stabilized chymotrypsin and trypsin
and the protective covering around chymotrypsin is so
thin and porous that a large mass transfer limitation on
the substrate could not take place.
Yan et al. (2006) [106] provided a simple method that
yields a single enzyme capsule with enhanced stability,
high activity and uniformed size. The 2-step procedure
including surface acryloylation and in situ aqueous po-
lymerization to encapsulate a single enzyme in nanogel
to provide robust enzymes for industrial biocatalysis. The
immobilized horseradish peroxidase (HRP) exhibited
similar biocatalytic behavior (Km and kcat) to the free
enzyme. However, the immobilization process signifi-
cantly improved the enzyme\s stability at high tempera-
ture in the presence of polar organic solvent.
3.2.2. Enzymatic Immobilizat ion of Enzyme
The use of green chemistry rather than using harsh
chemicals is one of the main goals in enzyme industries
to avoid the partial denaturation of enzyme protein. An
emerging and novel technology is to fabricate solid pro-
tein formulations [108,109]. As model proteins, en-
hanced green fluorescent protein (EGFP) and glutathione
S-transferase (GST) were tagged with a neutral Gln-do-
nor substrate peptide for MTG (Leu-Leu-Gln-Gly,
LLQG-tag) at their C-terminus and immobilized onto the
casein-coated polystyrene surface [108].
Luciferase (Luc) and glutathione-S-transferase (GST)
ybbR-fusion proteins were immobilized onto PEGA resin
retaining high levels of enzyme activity using phospho-
pantetheinyl transferase (Sfp) mediating site-specific
covalent immobilization [109]. In general, the Sfp-cata-
lyzed surface ligation is mild, quantitative and rapid,
occurring in a single step without prior chemical modifi-
cation of the target protein.
3.2.3. Microwave Irradiation
The use of porous supports for immobilization of en-
zymes is difficult to distribute because of diffusion limi-
tations [110] and they often remain only on external
channel [111]. For enzymes having large dimensions,
such as penicillin acylase (PA), the mass transfer is even
slower. The immobilization of such enzyme to porous
materials can prove tedious using conventional tech-
niques [112].
Wang et al., 2008b & 2009a [95,113] have recently
succeeded to immobilize papain and PA using the ad-
sorption technique into the mesocellular siliceous foams
(MCFs) using microwave irradiation technology. Reac-
tion time of 80 and 140 s were enough for papain and PA
to attach on the wall of MCFs, respectively. The activi-
ties of papain and penicillin acylase immobilized with
microwave-assisted method were 779.6 and 141.8 U/mg,
respectively. In another experiment, macromolecules
crowding was combined with small molecular quenching
to perfect microwave-assisted covalent immobilization
[113].
3.2.4. Photoimmobilization Technology
In the field of immobilization of biomolecules, potential
applications of photoimmobilization using nitrene groups
could take place. Nitrene groups have a property of in-
sertion into C-H bond. When photoreactive polymer and
horseradish peroxidase or glucose oxidase are exposed to
ultraviolet (UV) light at 365 nm, the reactive nitrene
immobilizes the protein molecules in 10 to 20 min
through covalent bonding [114]. Horseradish peroxidase

Review Article: Immobilized Molecules using Biomaterials and Nanobiotechnology
72
(HRP) and glucose oxidase (GOD) have been immobi-
lized onto the photoreactive cellulose membrane by the
ultraviolet and sunlight [115]. They found that sunlight
intensity required for optimum immobilization was
21,625 lux beyond which no appreciable increase in im-
mobilization was observed. Moreover, sunlight exposure
gave better immobilization compared to 365 nm UV
light.
3.2.5. Ionic Liquids
Ionic liquids, the green solvents for the future, are com-
posed entirely of ions and they are salts in the liquid state.
In the patent and academic literature, the term “ionic
liquid” now refers to liquids composed entirely of ions
that are fluid around or below 100°C (e.g. ethanolamine
nitrate, m.p. 52-55oC). The date of discovery of the
“first” ionic liquid is disputed, along with the identity of
the discoverer. Room-temperature ionic liquids are fre-
quently colorless, fluid and easy to handle [116].
Versatile biphasic systems could be formed by con-
trolling the aqueous miscibility of ionic liquid [117].
Based on a biphasic catalytic system where the enzyme is
immobilized into an ionic liquid (IL), Mecerreyes and
co-workers [118] have reported a new method which
allows recycling and re-using of the HRP enzyme in the
biocatalytic synthesis of PANI. The HRP enzyme was
dissolved into the IL 1-butyl-3-methylimidazolium
hexafluorophosphate and the IL/HRP phase acts as an
efficient biocatalyst and can be easily recycled and re-
used several times. Due to the immiscibility between the
IL and water, the immobilized HRP could be simply re-
covered by liquid/ liquid phase separation after the bio-
catalytic reaction [119,120]. Although this new method is
faster and easier than the classical immobilization of
HRP into solid supports, it would not be widely applied
to the industrial production in the coming future because
of the ionic liquids' expenses.
4. Recommendation for the Future of
Immobilization Technology
At present, a vast number of methods of immobilization
are currently available. Unfortunately, there is no a uni-
versal enzyme support, i.e. the best method of immobili-
zation might differ from enzyme to enzyme, from appli-
cation to application and from carrier to carrier. Accord-
ingly, the approaches currently used to design robust
industrial immobilized enzymes are, without exception,
labeled as “irrational”, because they often result from
screening of several immobilized enzymes and are not
designed. As a consequence, some of the industrial en-
zymes are working below their optimum conditions.
Recently, Cao L. (2005) [5] in his book “Carrier
bound immobilized enzymes” tackled this problem as he
surmised that the major problem in enzyme immobiliza-
tion is not only the selection of the right carrier for the
enzyme immobilization but it is how to design the per-
formance of the immobilized enzyme.
The author of this review article is suggesting from
his point of view as he is working in that field for the last
ten years to follow these steps in order to get to this goal
in the shortest time:
1- build a data base containing all information on the
available biomolecules (enzymes, antibodies, etc) and
carriers (organic, inorganic, magnetic hybrid, ionic liq-
uids, etc) then
2- use the dry lab (bioinformatics) to validate the
probability of success and the efficiency of the immobi-
lization process then
3- starting the experiment in the wet lab.
The author believes that if this strategy could be per-
formed, we should expect immobilized molecules work-
ing at their optimum conditions, with higher stability and
efficiency, which will save money, time and effort for the
prosperity of human being.
5. Acknowledgements
The author would like to thank the Centre of Excellence
for Advanced Sciences, NRC, Egypt, the Research and
Development Innovation (RDI) program and the Science
and Technology Development Fund STDF/IMC for sup-
porting this work, and highly appreciates the efforts of
Mrs Joanne Yachou for her contribution towards editing.
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