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Cryogel Poly(acrylamide): Synthesis, Structure and Applications

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DOI: 10.1080/15422119.2013.795902

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Cryogel Poly(acrylamide): Synthesis, Structure and

Applications
a a b b
B. M.A. Carvalho , S. L. Da Silva , L. H.M. Da Silva , V. P.R. Minim , M. C.H. Da Silva
b c b
, L. M. Carvalho & L. A. Minim
a
Department of Chemistry, Biotechnology and Bioprocess , Federal University of São
João Del Rei , Ouro Branco , MG , 36420-000 , Brazil
b
Department of Food Technology , Federal University of Viçosa , Viçosa , MG ,
36570-000 , Brazil
c
Department of Veterinary , Federal University of Viçosa , Viçosa , MG , 36570-000 ,
Brazil
Accepted author version posted online: 09 Jun 2013.

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Applications, Separation & Purification Reviews, DOI: 10.1080/15422119.2013.795902

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 ACCEPTED MANUSCRIPT

Cryogel Poly(acrylamide): Synthesis, Structure and

Applications

B.M.A. Carvalho1*, S.L Da Silva1, L.H.M Da Silva2, V.P.R. Minim2,

M.C.H. Da Silva2, L.M. Carvalho3, L.A. Minim2.

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1
Department of Chemistry, Biotechnology and Bioprocess, Federal University of São João Del

Rei, Ouro Branco, MG 36420-000, Brazil.

2
Department of Food Technology, Federal University of Viçosa, Viçosa, MG 36570-000,

Brazil.

3
Department of Veterinary, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil.

*Corresponding Author: E-mail: brunamara.carvalho@gmail.com

Received on September 8, 2012; revised on February 3, 2013; accepted on April 9, 2013.

Abstract

This review focuses on the description of macromolecular gels formed as a result of

polymerization, under freezing conditions, from monomer solutions. Materials resulting from

this polymerization are known as cryogels and have particular properties, such as porosity of a

specific nature. They are attractive in terms of biotechnological applications. The cryogels have

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interconnected macropores and their preparation method from aqueous solutions in freezing

conditions is a process that combines the crystallization of a solvent with the copolymerization of

monomers. Cryogels have recently been used as adsorbents for the efficient separation and

purification of biomacromolecules such as proteins, DNA, cell organelles, viruses and enzymes

that are present in solutions. Despite the wide application of cryogels, improvements in their

preparation are necessary to minimize problems, such as the formation of hydrogels, instead of

cryogels. Hydrogels form due to immediate polymerization after addition of the redox initiator
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before solvent freezing. The synthesis and use of cryogels as chromatographic supports are also

discussed.

Keywords:

Cryogel, Biomacromolecules, Purification, Isolation, Supermacroporosity.

1. Introduction

1.1. History

Polymer gels have been studied over 6 decades as separation materials for

biomolecules. During this period, many researchers have developed new ways of producing such

gels in order to minimize problems and expand alternatives. Hydrogels are polymeric gels with

large water contents in the native materials. If hydrogels are synthesized at subzero temperatures,

they may be named “cryogels”. Studies on cryogels started in the 1970s, being of continuous

interest since then. To a large extent, the applications of cryogels are related to their

macroporous structure, allowing the effective mass transport of macromolecular solutes and the

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migration of cells, high biocompatibility and good mechanical properties in these highly

hydrated systems that contain strongly associated water (SAW). Problems such as the diffusion

of occluded flow homogeneity for coupling cells and ligands, common in polymeric gels, have

been minimized with the development of these polymeric gels made under cryogenic conditions.

Significant variations in the structural characteristics of cryogels, such as distribution

of pore size and pore wall thickness, may be obtained through changes in polymerization
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conditions. These include; changes in the water/organic co-solvent ratios, ionic strength, pH,

cooling rate, gradients of temperature and solute concentrations, as well as alterations in

precursor composition (1-3).

During nearly 45 years of studies, cryogels (from the Greek κριοσ [Kryos] which

means ice) have been studied for the development of new and specific gels; such as cryogels

with open porous structures and unique double-continuous macroporous networks (4); analysis

and clinical significance of cryogel obtained from patients with Rheumatoid arthritis (5),

inorganic cryogels (6) and poly(acrylamide) cryogels designed for chromatography of

bioparticles (7), nucleation-controlled polymerization of human monoclonal immunoglobulin G

cryoglobulins (8), and cryo- structuration of polymer systems (9).

The use of microbiological cryogels was initiated in the 1980s for immobilizing cells via

mechanical concentration (10). However, the potential of cryogels for the isolation of cells in

cell culture and other applications has been perceived only very recently (11-13).

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1.2. Fundamental definitions

Gels are colloidal systems with characteristics of low flow-ability, demonstrating a dispersed

form of continuous and branched structures that are normally interpenetrated by a liquid phase

(14). Gels containing only a small amount of the dispersed phase (1-3%) have a certain degree

of rigidity and elasticity, where the rigidity results from the interaction between particles that are

chemically bound together. The organization of primary units can occur by joining chains by
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covalent bonds, hydrogen bonds, dipolar forces and van der Waals forces. The gels are usually

classified according to the continuous phase used, i.e., water, ethanol or air for hydrogel or

lyogel, alcoogel and airgel, respectively.

Polymer gels consist of a 'polymer-immobilized solvent' system, in which macromolecules

connected by chemical bonds form a 3D structure (where bonds, in most cases, remain

unchanged over time) (11). The conformation of the chain is determined by the nature of the

bonds and the gel production method. The role of the solvent (water, alcohol, air) contained

within the polymer network is crucial because the solvent does not allow for the formation of a

compact polymer mass, thereby preventing the collapse of the system.

According to Lozinsky et al. (15), there are two main methods of producing gels. The first uses

a limited swelling of a non-crosslinked polymer or via swelling of a xerogel (solid formed from a

gel by drying with unhindered shrinkage). The second and most commonly used method

employs the formation of a colloidal gel in a liquid system in which water is the dispersion

medium. When the initial solution or sol-gel forming precursor is frozen, "cryotropic

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gelatinization", cryo-gelling or cryo-structuration occurs. How this specific type of product

synthesis in polymeric gels is obtained by the so-called cryogels (15).

Crystallization of the solvent is the main differentiating factor between cryogelatinization and

cooling-induced gelling. The latter products are psychotropic gels, i.e. gels obtained at

conditions of lower temperature, for example, from the gelling of gelatin, without the occurrence

of the phase transition of the solvent. These gels are thermo-reversible, thereby differentiating
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them from cryogels formed under freezing conditions.

2. Polyacrylamide cryogels

Among the numerous publications describing a variety of cryogel systems, the

poly(acrylamide) (PAAm) cryogel is the most representative and widely used cryogel for a broad

range of applications (16-19) and commonly obtained by redox-initiating systems. The critical

problem in these systems is the formation of hydrogels instead of cryogels, because the

polymerization starts almost immediately after adding the redox initiator before the freezing of

the solvent. Poly(acrylamide) cryogels have a spongy morphology, which is primarily dictated

by the temperature used for gelling the cryotropic.

2.1. Fundamentals of the mechanism of synthesis of polyacrylamide cryogels

In the production of polymeric gels, the components used for constructing the matrices are

usually acrylamide, N, N'-methylenebis(acrylamide), tetramethylenediamine, also denominated

TEMED, and ammonium persulfate. When ammonium persulfate is dissolved in water, there is

formation of free radicals. If these free radicals are then placed in contact with acrylamide, a

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radical reaction occurs. "Activated" acrylamide can then react with successive molecules of

acrylamide with the production of long polymer chains. A solution of these polymers, although

viscous, does not form gel as these long chains can slide over each other. Gel formation requires

the linking of several chains together. This is achieved by means of polymerization in the

presence of N, N'-methylenebis(acrylamide), a compound containing two acrylamide units

coupled at the ends, in non-reactive terminals (20). Polymerization takes place to produce chains

of acrylamide (Figure 1). The pore size of the network is determined by two parameters: (1) the
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content of acrylamide used per unit volume of reaction medium and (2) the degree of cross-

linking.

Independently of the total content of acrylamide per unit volume, the mean pore size reaches a

minimum when N, N'-methylenebis(acrylamide) is used at 5% (Table 1). Therefore, in many

formulations, the content of bis(acrylamide) is set at 5% to control the pore size. Pore size can

then be controlled by varying the total content of acrylamide. Figure 2 shows the distribution of

pore size produced as a function of varying the concentrations (w/v) of acrylamide. The buffer

solution, TEMED, is usually added to catalyze the formation of the gel due to its presence in the

form of free radicals. The nature of the buffer is another factor to be considered in the

production of polyacrylamide gels. Buffers are required (1) to maintain a constant pH within the

acrylamide gel and/or (2) as an electrolyte to conduct current through an electric field in case of

electrophoretic use.

Cryotropic gelling in polymeric systems is considered as common polymerization method (as

explained above), but under freezing conditions, acrylamide (AAm) and bis (acrylamide)

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(BisAAM) react somewhat differently. In the case of freeze-gelation, the freezing temperature

may vary from about -10°C to -30°C (21). Thus, it is intriguing to observe a cryogel

morphology differing completely from that of traditional gels. To explore the differences, we

shall discuss the process that occurs in the micro and macroscopic freezing of solutions during

which cryo-gelling occurs. In pioneering studies, Butler and Bruice (22) and Pincock (23)

showed that, in moderately frozen solutions, part of the solvent maintains the liquid state,

although this solvent is unstructured (also called the unfrozen liquid microphase). Substances
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dissolved in these systems concentrate in the region of the non-frozen solvent (a process called

cryo-concentration), thereby allowing further chemical reactions to occur, although the entire

sample appears to be a solid block. Experiments to detail the kinetics and thermodynamics of

the reactions involved showed the existence of an unfrozen microphase (24). It is clear now that,

at moderately low temperatures, a macroscopic system consists of two solid phases, one phase

constituting a frozen polycrystalline pure solvent, and the other an unfrozen microphase liquid

containing almost all of the dissolved components present in the original solution. As the

volume of microphase is less than the volume of the initial solution, it presents high

concentrations of dissolved substances. Thus, high concentrations in the microphase speed up

chemical reactions and these can spread faster than reactions in a homogeneous solution above

the freezing point, despite the lower temperature.

When the initial solution contains molecules of low molecular weight, or macromolecular gel

precursor, the moderate cryotropic freezing promotes the formation of the gels (Figure 3).

According to Lozinsky et al. (15), for a system to be considered as moderately frozen, it should

retain a fraction of non-frozen solvent (usually, this refers to a region colder by less than few tens

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of degrees than the freezing point of the pure solvent). Thus, after the mixture containing the

gel-forming agent is frozen, the formation of ice crystals from the solvent occurs (Figure 3)

while a so-called unfrozen liquid microphase (UFLMP) is formed. Although being only a small

part of the total initial volume, the UFLMP is of great importance for the formation of the

cryogel. Cryo-concentration occurs where the gel forming reagents are concentrated and the

formation of polymer chains occurs more quickly when compared to polymerization in the liquid

medium. The ice crystals from the solvent act as pore formers and, during freezing, these
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crystals grow and merge to form interconnected porosity (15). The size and shape of the pores

and the volume of the UFLMP depend on several factors, such as concentration of precursors,

nature of the solvent, the type of cryogenic treatment system, and temperature, among other

factors.

2.2. The supercooled liquid microphase

The cryogel system is frozen heterogeneously and consists of a solid phase (frozen crystal

solvent) and unfrozen liquid microphase. The volume of the microphase depends on the nature

of the solvent, the initial concentration of dissolved substances, and the presence of mixtures of

soluble and insoluble molecules. The unfrozen microphase is shown in Figure 3 on a scale that

does not reflect the true relationship between the frozen and unfrozen regions. The unfrozen

region usually forms 0.1 - 10% of the total sample (25-29). At high concentrations, dissolved

substances begin to precipitate out of the liquid microphase due to limited solubility; however, if

the dissolved substances are consumed in a chemical reaction, for example, due to gel formation,

precipitated substances are redissolved in the microphase.

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Crystallization of the frozen solvent is a crucial step in the satisfactory formation of a

homogeneous supermacroporous phase with interconnected molecules within the cryogel. After

the thawing of the frozen samples, the gel formed presents a supermacroporous structure. The

frozen crystals of the solvent act as a pore-forming agent and the melting of the crystals forms

cavities of cryogels. Due to the lowered entropy caused by a decrease in the excess of the Gibbs-

free energy in the gel-solvent interface, the surface tension of the system increases as the

temperature lowers, this causes the wells (initially with sharp angles) to later become rounded.
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The temperature used during the cryogenic treatment has a major effect on the porosity of the

spongy cryogels (30). The arrangement of the particles and their maintenance in their place in

the lattice are determined by the existence of a minimum energy, corresponding to the

optimization of the bonds formed between the particles. The quantity of nuclei formed at

temperatures far from the freezing point of water is greater than that formed at temperatures of

nearly freezing point; therefore ice crystals formed by water molecules that are arranged around

these nuclei have a smaller diameter in environments where the temperature is farther from the

freezing point of water.

Lozinsky (30), demonstrated that for polyacrylamide (PAAm) cryogels formed at three negative

temperatures (-10°C, -20°C and -30°C), the largest pores are formed at the highest freezing point

while the smaller pores were obtained at lower temperatures (Figure 4). In this Figure, the shape

of the interconnected pores of capillary size can be observed, which may allow mass transfer by

diffusion of substances as well as processing of liquids and low viscosity biological suspensions

(viruses, organelles, cells) via these pores. The hydrodynamic characteristics of the cryogel

matrices can be controlled by varying the initial concentration of gel-forming agents and the

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conditions of the cryogenic treatment. Figure 5 shows the flow of water through a column in

which the PAAm cryogels were prepared with an initial ratio of co-monomers of acrylamide and

N, N'-methylenebis (acrylamide), and cryotropic gelling temperatures (30). Larger pores lead to

higher flows with the same driving pressure. The lower the degree of polymer cross-linking in

the macropore wall (low content of N, N'-methylenebisacrylamide), the stronger the expansion

during the gelling phase, thus decreasing the size of the capillaries and preventing flow. No

special equipment is required for the synthesis of the cryogel poly(acrylamide) matrix. The
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equipment used is simple and inexpensive. For the process of cryogelling, the solution must

remain at freezing temperature; thus, a simple ice bath can be used for the synthesis of the

matrix.

3. Characterization of cryogels

Cryogels have a continuous system of interconnected macropores, ranging in size from 10-100

μm, and differ in their low resistance to flow and diffusion of solutes of any size. These

characteristics of the chromatographic matrices provide advantages when compared to expanded

bed chromatography, where special equipment is required, for example, for processing non-

clarified broth (31-32). The cryogels also differ from fixed bed traditional chromatography,

which has a stationary phase packed in a column that is incapable of processing solutions

containing particulate materials, such as suspensions of cells or non-clarified cell homogenates,

despite its high capacity and resolution. Particulate material contained in these solutions

decreases the bed porosity, resulting in an increased flow resistance in the column, and

culminating in blockage. The macroporous cryogels (especially in the form of monolithic

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columns) are currently the only available form of chromatography on a macroporous bed for

processing microbial cells and non-clarified mammalian broth (33).

Some methods, described below, have been used to characterize these chromatographic matrices.

For example, pore structures can be viewed by scanning electron microscopy. One tracer pulse

may be applied for measuring the residence time distribution (RTD) of a liquid column at

different cryogel flow rates. More details about this method can be found in Persson (34). The
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behavior of the flow resistance of cryogel matrices can be evaluated by the ratio of the

hydrostatic pressure drop through the column flow rate versus the corresponding value of the

water and hydraulic permeability, which can be determined using the equation (35):

Qw μ w L
kw = (1)
Δp w A

where Qw is the flow of water through the column, μ w is the viscosity of water, L is the length of

the column, A is the cross-section and Δp w is the pressure drop.

Figure 6 presents experimental results of the hydraulic flow resistance in the Fe3O4 nanoparticle-

embedded cryogel column systems. The water inside the swollen cryogel can be encountered in

different forms: polymer-bound water, water in small pores and water in large pores; this latter,

responsible for the porosity of the matrix cryogel may be removed mechanically by compressing

the cryogel due to the elasticity of the material. The porosity thus can be estimated by measuring

the content of free water present and the bed volume, as previously described by Yao et al. (35).

The porosity is given by:

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mw − ms
ϕ= (2)
ρ wV0

where mw and ms are, respectively, the mass of the sample saturated with water and sample

weight after wet compression to remove the free water within the pores, as reported by (36, 7),

ρw is the density of water, and V0 is the difference between V1 and V2, where V2 is the final

volume after immersion of the sample cryogel in a graduated cylinder containing water, and V1

is the known volume of water in a graduated cylinder. Figure 7 shows a scanning electron
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microscope (SEM) photograph of a polyacrylamide cryogel. The pore size is noticeable when

comparing the structure of the cryogel.

The elasticity modules of cryogel may be calculated in the linear range by Young’s modulus (37

cited by 17) by testing compression and deformation by the following equation:

F/A
E= (3)
Δh / h

where E is the modulus of elasticity, F is the applied force, A is the area of the sample, Δh is

the variation in length after the compression of the sample, and h is the initial length of the

sample. Hajizadeh et al, 2013, studying the elasticity of gels containing MIP (molecularly

imprinted cryogel) or NIP (non-imprinted polymer) (up to 30% for both gels), confirmed that

both types of gel presented comparable mechanical strength, with E values of around 3.8 kPa.

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4. Applications

Cryogels have recently been introduced as a new matrix for applications in various bioseparation

processes (3). Cryogels have interconnected pores of different sizes, allowing the mass to flow

through the pores with a low and purely convective mass-transfer resistance. Thus, these

characteristics are attractive means for the direct capture of biomacromolecules.

4.1. Cells and viruses

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In the field of microbiology, recent studies have demonstrated the efficiency of cryogels in

processing suspensions of cells and viruses. Cells and purification applications for cell cultures

were made possible by the combination of properties such as macroporosity, biocompatibility,

good elasticity, chemical and physical stability and ease of preparation, making these cryogel

materials suitable for a wide range of applications in microbiology research. The demand for

high purity bioproducts, including viruses and plasmid DNA as gene therapy vectors, and virus

particles as components of vaccines, has had a great influence on downstream processing. Thus,

technologies involving the use of polymeric supermacroporous materials (or cryogels), which

allow for the processing of different cell populations are of great interest. One of the most

attractive features of the cryogels is their porosity, which is useful for processing fluids including

particulate suspensions of microbial cells (11, 12, 38). These macropores are obtained by the

synthesis of hydrogel cryogelling (or gelling at temperatures below 0°C), as demonstrated in

Figure 3.

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The peculiar structure of the pores of the cryogel, in combination with its elasticity and high

mechanical stability, has encouraged its use as a matrix for the immobilization of cells. Two

main ways to immobilize cells are commonly used, depending on the application potential of the

immobilized cell biocatalysts (ICBs) and the pore size desired for the cryogel matrix (39). These

methods rely on the immobilization of the cryogel due to mechanical concentration (Figure 8a)

and immobilization on the surfaces of the pores within the cryogel via specific interactions or by

adsorption (Figure 8b).

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According to Plieva et al. (39), normally, the immobilization of microbial cells via mechanical

cryogel concentration is simple. The cell suspension is mixed with a solution of monomer

(polymer) gel precursors until a stable cell suspension is achieved. After addition of

polymerization compounds (in this case for free radical polymerization), the cell suspension is

frozen and the temperature regimes vary with the geometry required. These cryogels may be

prepared in the form of beads, sheets, and monoliths, or can be synthesized in a protective open

plastic (Figure 8a). ICBs with pore size up to 1-2 μm are produced in most cases, in the form of

pellets or sheets (10, 40). ICBs with large interconnected pores of sizes up to 200 μm are often

prepared for high flow rates in monolithic columns or disks (41-42) or are produced inside an

open protective film (defined as a macroporous gel particles, MGPs) to keep the ICBs stable

under intense agitation (39, 43).

The long-term use of a single ICB is important for the development of an efficient industrial

process. An important question concerns the regeneration of the ICBs by simple incubation at an

average growth rate to restore the activity of bioconversion. Another important concern is the

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preservation of ICBs to prevent contamination after prolonged use. Thus, selected

microorganisms in extremely unfavorable conditions would be the best choice for the preparation

of IBCs (39).

Another strategy for immobilization includes the preparation of cryogel groups with specific

ligands used for connection with the cells (Figure 8b). The presence of appropriate ligands on

the pore surface within macroporous cryogels has made it possible to link them with specific
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microorganisms (7, 44-46) and mammalian cells (47-48).

High-density cell suspensions can be applied in monolithic cryogel columns without blocking

macropores (49). Thus, yeast cells can be immobilized on the surface of cryogel monolithic

columns packed with ion exchange groups for tertiary amine groups with a continuous

circulation of a dense suspension of cells through the column. The isolation and separation of

cells using cryogels (chromatographic separation of microbial cells) is generally regarded as

difficult and many factors must be taken into account for this application, including the large size

of cells reflected by low diffusivity, surface chemistry and other complexes (49). In fact, due to

the lack of sorbents with appropriate high porosity for processing cells, cell chromatography has

hitherto been used frequently (33). With their large pores (10-100 μm) and mechanically strong

interconnected pore walls, cryogels can withstand high flow rates with macropore

interconnectivity and with virtually no compression, thus allowing the convective mass transport

of solutes and particles of at least 10 μm in size (7, 34). When there are no specific ligands on

the matrix cryogel, cells pass freely through the column without blockage by the monolithic

pores. However, when specific ligands are present on the surface of the pores, the microbial

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cells can bind to them and be eluted from the column cryogel in high yield and with a viability

governed by conventional methods of elution (7, 44, 45, 50).

4.2. Proteins and cryogels with copper cations

Cryogels with chelated metals have been used for affinity chromatography. The technique of

Immobilized Metal Affinity Chromatography (IMAC), introduced by Porath et al. (51), is based

on the ability of proteins to be reversibly attached to metal ions through coordination bonds. The
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protein acts as a Lewis base, donating electrons to the metal cation (52). The metal, which

interacts with the protein, is immobilized by means of a chelating agent, which in turn is

covalently bound via a spacer to the matrix. The compounds used as chelating agents are: tris

(carboxymethyl) ethylenediamine (TED, a pentadentate), nitrilotriacetic acid (NTA, a

tetradentate), carboxymethylated aspartic acid (CM-Asp, a tetradentate) and iminodiacetic acid

(IDA, a tridentate). The number of bonds formed between the ion and immobilized chelating

determines the overall affinity of the adsorbent for the protein, and thus the stronger the

immobilization of metal, the weaker the interaction with the protein. IDA, a tridentate chelator, is

most commonly used. The interactions between immobilized metal cations and the protein are

complex and combine electrostatic, hydrophobic and coordination effects.

Cryogels based on polyacrylamide loaded with Cu2+ by IDA (iminodiacetic acid) ligands

(cryogel-IDA-Cu2+) have been investigated by several research groups (7, 33, 36, 44, 53-56).

Plieva et al. (7), used Cu2+-IDA cryogels for the adsorption of lysozyme and Teilum et al. (55),

used Cu2+-IDA cryogels to immobilize liver mitochondria and studied a specific protein released

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by these mitochondria. Recently, cryogels with chelated metal were also used for surface

analysis and quantification of cells (38).

4.3. Enzymes and polyacrylamide cryogels

In order to isolate and purify urokinase, Kumar et al. (57) developed a process for isolating the

enzyme in a cell-culture bioreactor. Polyacrylamide cryogels can be used for this purpose as a

matrix for affinity chromatography on immobilized metal (IMAC). A comparative study was
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made between the matrix cryogel and an affinity matrix based on conventional Sepharose for the

continuous capture of urokinase. The activation of the matrix (cryogel polyacrylamide and

Sepharose) was performed with Cu2+ (using iminodiacetic acid-IDA) as a good binder for IMAC

due to its affinity for proteins and the fact that it is a low cost metal to facilitate regeneration of

the stationary phase. According to the authors, urokinase showed significant affinity for copper

mainly due to the presence of 17 histidine residues (His) on its surface; these groups contain

residues that interact with Cu2+ imidazole. As for the comparative study of the efficiency of the

Cu2+-IDA polyacrylamide cryogel and the IDA-Cu2+ sepharose column for the capture of

urokinase, it was shown that the cryogel was selective and showed a better operational efficiency

compared with the Sepharose column. Thus, the authors concluded that IMAC, coupled with the

use of arrays of supermacroporous cryogel, are an ideal technique for the capture of urokinase

under physiological pH conditions in a continuous system. Arvidsson et al. (45) showed that

cryogel supermacroporous arrays are attractive for the handling of microbial cells in affinity

chromatography with recoveries of Escherichia coli ranging from 70-80%.

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4.4. Other chelating cations

Although the majority of studies focus on the application of Cu2+-IDA cryogels for the

separation and purification of biomolecules or microbial cells, other transition metal ions, such

as Zn+2, Ni+2, Fe+2 and Co+2 are also suitable for IMAC (58, 59). Studies with cryogels in

nanotechnology applications have recently been presented. Yao et al. (35) proposed a new

continuous supermacroporous cryogel embedded with nano-sized particles for use in the
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chromatography of proteins (BSA). The synthesis of cryogels was made by copolymerization of

cryogenic radicals of a solution of acrylamide (AAm), N, N'-methylene-bis-acrylamide

(MBAAm), allyl glycidyl ether (AGE) and Fe3O4 nanoparticles under freezing. The matrix,

besides showing high porosity (pore 10-50μm) possessed high liquid permeability and a low

coefficient of axial dispersion in a wide range of flow velocity. The adsorption capacity of the

protein (BSA) in the array of the cryogel compared better with the same matrix cryogel (reported

in previous studies) without embedded nanoparticles.

4.5. Non acrylamide cryogels in the food industry

Cryogels present alternative solutions to problems with processed foods. Lopez-Fouz et al. (60)

proposed the removal of the bitter taste of citrus juices using cryogels of polyvinyl alcohol

(PVA)-polyethylene glycol (PEG) immobilized with Rhadococcus fascians. This microorganism

produces enzymes that metabolize Limonin (composite family of limonoids that are primarily

responsible for the bitter taste of citrus juices). The effect of the PVA, PEG, loaded cells, and

pH on efficiency of R. fascians in Limonin degradation was studied. The stability of PVA-PEG-

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R. fascians-beds following the passage of orange juice was another focus of the study. The

concentrated cells could be used for three complete cycles (runs 24 to 20°C), retaining an

effectiveness of 35.6% hydrolysis of Limonin. Moreover, following the process, the authors did

not alter the main physical and chemical parameters of the citrus juice, except decrease the bitter

taste.

Naringin is another compound responsible for the bitter taste of citrus fruit juices. Busto et al.
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(61) studied the immobilization of naringinase (from cultures of Aspergillus niger CECT 2088)

in polymer matrices of PVA cryogel containing biocatalytic activity. The effects of

concentration of the matrix, enzyme load and pH on efficiency of immobilization were studied.

The naringinase concentrate can be reused for 6 cycles (24 hours runs at 20°C), retaining 36%

efficiency for the hydrolysis of naringin juice concentrate.

The unique combination of the properties of cryogels, such as their macroporous structure,

biocompatibility and chemical and mechanical robustness, provides new opportunities for the

design of macroporous materials. Moderate conditions for immobilization and high mechanical

stability of cryogels allow the preparation of effective biocatalysts to be employed in aqueous or

low aqueous solutions. The release of cells bound by affinity ensures high viability and recovery

of desorbed cells during chromatography. Furthermore, the large interconnected pores of the

monolithic cryogel allow rapid processing of large volumes of liquid phase. Thus, the

macroporous cryogel filter can be used for analytical detection and selective capture of specific

cells in water effluent.

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5. Cryogels compared to other permeation media

A critical point of modern biotechnological processes is the separation and purification of

biomolecules from complex media, such as liquid suspensions of fermented media with or

without cells, fragments of such suspensions, and industrial waste. The chromatographic process

at industrial preparative scale is generally used during the stages of separation and purification of

biological materials. Traditional fixed-bed chromatography, packed with a stationary phase in a

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column, however, suffers from a drawback, namely, it is incapable of processing solutions

containing particulate materials, for example, suspensions of cells or non-clarified cell

homogenates, despite its high capacity and resolution. Particulate material contained in these

solutions decreases porosity, resulting therefore in increased flow resistance in the blocked

column. To overcome this drawback expanded bed chromatography was proposed, where the

mobile phase flow maintains the particles in the adsorbent suspension, to increase the porosity of

the bed and allow the particulate material to pass through the column without increasing flow

resistance. The density and size allow adequately designed particles to have a restricted

movement relative to a stationary position and a flow regime is maintained in the column.

However, despite all the advantages of expanded bed chromatography, columns, and special

types of equipment are required. Chromatographic columns with the characteristics of a fixed

bed, but with pores sufficiently large to allow solutions containing cell debris and particulate

matter in general may be drained through the column without clogging are ideally suited for the

separation and purification of biomolecules. In this case, the bed could have a system of

continuous interconnected porous particles rather than convective pores, where the process is

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limited only in the interstitial inter-particle spaces. Such structures are inherent to so-called

"cryogels", i.e. polymeric gels formed in freezing conditions.

The significant reduction in the influence of mass transport phenomena of cryogels matrices

compared with matrices of other materials, has led to the increased use of cryogels as supports

for chromatography. Regarding the purification of bovine lactoferrin, Billakanti et al. (62),

found values of q (adsorption capacity at equilibrium) approximately 10 times higher than the
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values found by Iafisco et al. (63) to study the adsorption of bovine lactoferrin on hydroxyapatite

nanocrystals. Regarding the dynamic capacity of the adsorbent, the cryogels (64) have also

proved effective when compared to other types of adsorbents (66).

Thus, this unique combination of properties has become highly attractive and advantageous for

cryogelation applications in the bioseparation area. Additionally, cryogel synthesis is relatively

simple, which is an important advantage of this technique.

6. Conclusion

The supermacroporous monolithic AAm-cryogel columns are promising separation media for the

biotechnologist. The monolith structures have uniformly-distributed interconnected macropores

of different sizes (typical of freeze-thawed cryogels prepared via free radical polymerization).

The size and morphology of the pores of cryogels are easily adjustable and controlled by the

concentration of the monomers used and the freezing temperature.

The separation of particulate matter and capture of soluble material in particle containing media

as well as direct capture of proteins and other biomolecules from crude extracts, or even

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unprocessed fermentation media are areas of interest that have encouraged the development of

this type of material.

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Table 1. Effect of the concentration of bis(acrylamide) on the size of the porous medium (radius
Å or 0.1 nm).

Total acrylamide + % N,N'-methylenebis(acrylamide)

N,N'-methylenebis

(acrylamide) (% w/v) 1 5 15 25
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6.5 24 Å 19 Å 28 Å -

8.0 23 Å 16 Å 24 Å 36 Å

10.0 19 Å 14 Å 20 Å 30 Å

12.0 17 Å 9Å - -

15.0 14 Å 7Å - -

a
Data taken from Cooper (20).

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Figure 1 – Reactions forming cross-links in chains of acrylamide.
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Figure 2 - Influence of the concentration of acrylamide on gel pore size (reprinted with
permission of Wiley from Cooper (20)).
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Figure 3- Main Scheme for the formation of polymeric cryogels: 1, high molecular weight
precursor; 2, solvent; 3, precursor or low molecular weight soluble substances; 4, polycrystals of
frozen solvent; 5, unfrozen liquid microphase (ULMP); 6, cross-linked polymer gel; 7,
supermacroporus sponge.
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Figure 4 - Scanning electron microscopy images of PAAm cryogel samples formed from
aqueous 3% acrylamide and N, N'-methylene-bisacrylamide (30:1 molar ratio of monomers) at -
10°C (a), -20°C (b) and -30°C (c). (Adapted with permission of Spinger from Lozinsky (30)).
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Figure 5 - Hydrodynamic properties of columns made with the Figure 4 cryogels in
polyacrylamide (PAAm) at -10°C (1), -20°C (2) and -30°C (3) with a 3% aqueous solution of
AAm and N, N'-methylenebis-AAm using various ratios of monomers (hydrostatic pressure of
300 mm H2O or 3 kPa or 0.5 p.s.i.). (Adapted with of Springer from Lozinsky (30)).
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Figure 6 - Experimental and fitted flow rates as a function of pressure drop in the Fe3O4
nanoparticle-embedded cryogel column (μw =1×10−3 Pa s = 1 cP, L = 6.8×10−2 m = 68 mm, A =
2×10−4 m2). (●) Experimental and (─) fitted data. (Reprinted with permission of Elsevier from
Yao et al. (35)).
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Figure 7 - Scanning electron microscope photograph of the cryogel synthesized at -10oC by
cryoconcentration of monomers: AAm / MBAAm = 30:1.
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Figure 8 - Schematic representation of the two main approaches for cell immobilization. (a) via
mechanical concentration of cells within the cryogels during its preparation in different formats,
and (b) binding cells in the already prepared cryogel system, carrying specific ligands (Reprinted
with permission of Elsevier from Plieva, et al. (49)).
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