|
__________________________________________________________
Biotechnology and Bioengineering
Volume 37, Issue 7 , Pages 614 - 626
Published Online: 18 Feb 2004
Copyright © 1991 John Wiley & Sons, Inc.
Article
A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing
Victor Goetz, Magali Remaud, David J. Graves *
Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6393
*Correspondence to David J. Graves, Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania
19104-6393
__________________________________________________________
_________________________________________________________
Weapons-Grade Anthrax:
http://www.asph.org/document.cfm?page=847
Summary of 2004 Environmental Health Conference
Introduction
The fifth annual Environmental and Occupational Health
conference, "Environmental Health Risk: Assessment, Management
and Communications", was held from July 11-13, 2004 in
Minneapolis, MN. The generous support provided by the Health
Resources Services Administration, the Centers for Disease Control
and Prevention, and the National Institute for Environmental Health
Sciences made it possible for this year's effort to be our most
successful yet. We would also like to acknowledge NSF
International for their support of the conference. The efforts of the
Association of Schools of Public Health Environmental and
Occupational Health Council resulted in a valuable and memorable
experience for all the participants.
The presenters were:
John Cicmanec, DVM, MS, U.S. EPA
Overview of Environmental Health Issues related to agriculture
John L. Cicmanec, DVM, MS, U.S. Environmental Protection
Agency: Weapons-Grade Anthrax:
The Infectious Dose-50 in
Rhesus Monkeys Derived from a
Biologically-based Model for Use
in Human Risk Assessment�
One of the significant discoveries following the bioterrorist attacks
of October 2001 was that a modified form of Bacillus anthracis
(Ames strain) was the causative agent. Physical alteration of the
inoculum had occurred; the electrostatic charge had been altered
and the resulting spores were 1 to 3 microns in diameter. Eight
separate inhalation studies have been identified in which non-human
primates were used for inhalation exposure to B. anthracis to
determine the Infectious Dose50 ( Druett 1953, Henderson,1949,
Estep 2003,etc.) Depending on the spore particle size and strain
used, these values ranged from 4000 to 682,000 spores. Although
some studies used spores that were one micron in diameter,
conventional spore preparations will aggregate so that many of the
inhaled particles will often range from four to twelve microns. The
primary advantage that is gained through the use of spores with an
altered electrostatic charge is that particles do not clump and
essentially all of the inoculum can be deposited directly in the lungs.
In order to adjust for the amount of the conventional inoculum in the
non-human primate studies that was deposited in nasal passages,
pharynx, and tracheobronchial regions, a methodology that has been
developed for chemical particulate inhalation exposure (using the
MMAD and sigma g) has been used as a model. Conveniently,
cadmium chloride and radio-labeled polystyrene microspheres share
the same target cell, alveolar macrophages, as anthrax spores.
Through the use of ranking the dose response of these two
surrogates and zones of deposition in the respiratory tract, similar
dosage adjustments can be made for anthrax spores of various
particle sizes. Use of this model enables us to predict that the ID50
for the modified form of anthrax is at least 15 to 500 times lower
than for conventional spores. (This presentation does not represent
US EPA policy)
http://www3.interscience.wiley.com/cgi-bin/abstract/107622942/
ABSTRACT
Biotechnology and Bioengineering
Volume 37, Issue 7 , Pages 614 - 626
Published Online: 18 Feb 2004
Copyright © 1991 John Wiley & Sons, Inc.
Abstract | References | Full Text: PDF (1554k) |
A novel magnetic silica support for use in chromatographic and
enzymatic bioprocessing
Victor Goetz, Magali Remaud, David J. Graves *
Department of Chemical Engineering, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6393
*Correspondence to David J. Graves, Department of Chemical
Engineering, University of Pennsylvania, Philadelphia, Pennsylvania
19104-6393
Keywords
chromatography • immobilized enzyme • magnetically stabilized
fluidized bed • silica
Abstract
A limited number of support matrices have so far been developed for
use in magnetically stabilized fluidized bed (MSFB) applications. We
have developed a versatile magnetic silica support which can be
derivatized readily for both adsorption chromatography and enzyme
immobilization by well-known techniques. A magnetic pellicular bead
is prepared by electrostatically depositing alternating layers of
colloidal silica and cationic polymer onto macroscopic nickel core
particles. The polymer is then burned out and the silica partially
sintered to yield a porous shell with 5-80 m2/g of surface area. This
magnetic composite was tested as a support for immobilizing
invertase. Sucrose was continuously converted to its component
monosaccharides with nearly constant activity over the first 8 days
and retention of 50% of initial activity after 25 days.
Received: 24 July 1990; Accepted: 13 September 1990
Digital Object Identifier (DOI)
10.1002/bit.260370704 About DOI
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1: Biotechnol Prog. 1990 Nov-Dec;6(6):452-7.
Plant cell culture using a novel bioreactor: the magnetically stabilized fluidized bed.
Bramble JL, Graves DJ, Brodelius P.
Department of Chemical Engineering, University of Pennsylvania, Philadelphia 19104.
A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages
for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through
the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes
particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of
the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch
mode is compared to the results obtained in shake flask culture.
PMID: 1366835 [PubMed - indexed for MEDLINE]
1: Biotechnol Prog. 2003 Nov-Dec;19(6):1721-7. Related Articles,Links
Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption.
Tong XD, Sun Y.
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
China.
A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed
(MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic
Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive
triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for
protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS.
The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity
was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions
and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with
those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB,
higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is
promising for use in liquid MSFB for protein adsorption.
Publication Types:
1: Biotechnol Prog. 2003 Nov-Dec;19(6):1721-7. Related Articles,Links
Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption.
Tong XD, Sun Y.
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,
China.
A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed
(MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic
Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive
triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for
protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS.
The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity
was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions
and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with
those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB,
higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is
promising for use in liquid MSFB for protein adsorption.
Publication Types:
Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed
Handan Yavuz1 , Ridvan Say2 , Müge Andaç1 , Necmi Bayraktar3 and Adil Denizli1
1Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey
2Department of Chemistry, Anadolu University, Ankara, Turkey
3Faculty of Medicine, Urology Department, Hacettepe University, Ankara, Turkey
BioMagnetic Research and Technology 2004, 2:5 doi:10.1186/1477-044X-2-5
Published 26 August 2004
Abstract
Background
Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic
anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III)
in dialysis solutions may give rise to these disorders.
Methods
Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced
by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4).
Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g.
These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed.
Results
Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was
observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using
Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation
constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system),
indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed
and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity.
Conclusions
Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude
that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1366835&dopt=Abstract
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BioMagnetic Research and Technology
Volume 2
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Review
.
Magnetic techniques for the isolation and purification of proteins and peptides
Ivo Safarik1, 2 and Mirka Safarikova1
1Laboratory of Biochemistry and Microbiology, Institute of Landscape Ecology, Academy of Sciences, Na Sadkach 7, 370 05
Ceske Budejovice, Czech Republic
2Department of General Biology, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic
BioMagnetic Research and Technology 2004, 2:7 doi:10.1186/1477-044X-2-7
The electronic version of this article is the complete one and can be found online at: http://www.biomagres.com/content/2/1/7
Received 10 November 2004
Accepted 26 November 2004
Published 26 November 2004
© 2004 Safarik and Safarikova; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures
can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic
separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies,
strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help
of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques.
Outline Introduction
Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References
Isolation, separation and purification of various types of proteins and peptides, as well as of other specific molecules,
is used in almost all branches of biosciences and biotechnologies. Separation science and technology is thus very important
area necessary for further developments in bio-oriented research and technology. New separation techniques, capable of treating
dilute solutions or solutions containing only minute amounts of target molecules in the presence of vast amounts of accompanying
compounds in both small and large-scale processes, even in the presence of particulate matter, are necessary.
In the area of biosciences and biotechnology the isolation of proteins and peptides is usually performed using variety
of chromatography, electrophoretic, ultrafiltration, precipitation and other procedures, affinity chromatography being one
of the most important techniques. Affinity ligand techniques represent currently the most powerful tool available to the downstream
processing both in term of their selectivity and recovery. The strength of column affinity chromatography has been shown in
thousands of successful applications, especially in the laboratory scale. However, the disadvantage of all standard column
liquid chromatography procedures is the impossibility of the standard column systems to cope with the samples containing particulate
material so they are not suitable for work in early stages of the isolation/purification process where suspended solid and
fouling components are present in the sample. In this case magnetic affinity, ion-exchange, hydrophobic or adsorption batch
separation processes, applications of magnetically stabilized fluidized beds or magnetically modified two-phase systems have
shown their usefulness.
The basic principle of batch magnetic separation is very simple. Magnetic carriers bearing an immobilized affinity or
hydrophobic ligand or ion-exchange groups, or magnetic biopolymer particles having affinity to the isolated structure, are
mixed with a sample containing target compound(s). Samples may be crude cell lysates, whole blood, plasma, ascites fluid,
milk, whey, urine, cultivation media, wastes from food and fermentation industry and many others. Following an incubation
period when the target compound(s) bind to the magnetic particles the whole magnetic complex is easily and rapidly removed
from the sample using an appropriate magnetic separator. After washing out the contaminants, the isolated target compound(s)
can be eluted and used for further work.
Magnetic separation techniques have several advantages in comparison with standard separation procedures. This process
is usually very simple, with only a few handling steps. All the steps of the purification procedure can take place in one
single test tube or another vessel. There is no need for expensive liquid chromatography systems, centrifuges, filters or
other equipment. The separation process can be performed directly in crude samples containing suspended solid material. In
some cases (e.g., isolation of intracellular proteins) it is even possible to integrate the disintegration and separation
steps and thus shorten the total separation time [1]. Due to the magnetic properties of magnetic adsorbents (and diamagnetic
properties of majority of the contaminating molecules and particles present in the treated sample), they can be relatively
easily and selectively removed from the sample. In fact, magnetic separation is the only feasible method for recovery of small
magnetic particles (diameter ca 0.1 – 1 μm) in the presence of biological debris and other fouling material
of similar size. Moreover, the power and efficiency of magnetic separation procedures is especially useful at large-scale
operations. The magnetic separation techniques are also the basis of various automated procedures, especially magnetic-particle
based immunoassay systems for the determination of a variety of analytes, among them proteins and peptides. Several automated
systems for the separation of proteins or nucleic acids have become available recently.
Magnetic separation is usually very gentle to the target proteins or peptides. Even large protein complexes that tend
to be broken up by traditional column chromatography techniques may remain intact when using the very gentle magnetic separation
procedure [2]. Both the reduced shearing forces and the higher protein concentration throughout the isolation process positively
influence the separation process.
Separation of target proteins using standard chromatography techniques often leads to the large volume of diluted protein
solution. In this case appropriate magnetic particles can be used for their concentration instead of ultrafiltration, precipitation
etc. [3].
The purpose of this review is to summarize various methodologies and strategies which can be employed for the isolation
and purification of target proteins and peptides with the help of magnetic materials. An extensive list of realised purification
procedures documents the efficiency of magnetic separation techniques. All these information will help the scientists to select
the optimal magnetic material and the purification procedure.
Outline Necessary materials and equipment
Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References
Figures
Figure 1
Examples of batch magnetic separators applicable for magnetic separation of proteins and peptides
The basic equipment for laboratory experiments is very simple. Magnetic carriers with immobilized affinity or hydrophobic
ligands, magnetic particles prepared from a biopolymer exhibiting affinity for the target compound(s) or magnetic ion-exchangers
are usually used to perform the isolation procedure. Magnetic separators of different types can be used for magnetic separations,
but many times cheap strong permanent magnets are equally efficient, especially in preliminary experiments.
Magnetic carriers and adsorbents can be either prepared in the laboratory, or commercially available ones can be used.
Such carriers are usually available in the form of magnetic particles prepared from various synthetic polymers, biopolymers
or porous glass, or magnetic particles based on the inorganic magnetic materials such as surface modified magnetite can be
used. Many of the particles behave like superparamagnetic ones responding to an external magnetic field, but not interacting
themselves in the absence of magnetic field. This is important due to the fact that magnetic particles can be easily resuspended
and remain in suspension for a long time. In most cases, the diameter of the particles differs from ca 50 nm to approx. 10
μm. However, also larger magnetic affinity particles, with the diameters up to millimetre range, have been successfully
used [4]. Magnetic particles having the diameter larger than ca 1 μm can be easily separated using simple magnetic
separators, while separation of smaller particles (magnetic colloids with the particle size ranging between tens and hundreds
of nanometers) may require the usage of high gradient magnetic separators.
Commercially available magnetic particles can be obtained from a variety of companies. In most cases polystyrene is used
as a polymer matrix, but carriers based on cellulose, agarose, silica, porous glass or silanized magnetic particles are also
available. Examples of magnetic particles used (or usable) for proteins and peptides separation can be found elsewhere [5-7].
Particles with immobilised affinity ligands are available for magnetic affinity adsorption. Streptavidin, antibodies,
protein A and Protein G are used most often in the course of protein and peptides isolation. Magnetic particles with above
mentioned immobilised ligands can also serve as generic solid phases to which native or modified affinity ligands can be immobilised
(e.g., antibodies in the case of immobilised protein A, protein G or secondary antibodies, biotinylated molecules in the case
of immobilised streptavidin).
Also some other affinity ligands (e.g., nitrilotriacetic acid, glutathione, trypsin, trypsin inhibitor, gelatine etc.)
are already immobilised to commercially available carriers. To immobilise other ligands of interest to both commercial and
laboratory made magnetic particles standard procedures used in affinity chromatography can be employed. Usually functional
groups available on the surface of magnetic particles such as -COOH, -OH or -NH2 are used for immobilisation, in some cases
magnetic particles are available already in the activated form (e.g., tosylactivated, epoxyactivated etc).
In the laboratory magnetite (or similar magnetic materials such as maghemite or ferrites) particles can be surface modified
by silanization. This process modifies the surface of the inorganic particles so that appropriate functional groups become
available, which enable easy immobilisation of affinity ligands [8]. In exceptional cases enzyme activity can be decreased
as a result of usage of magnetic particles with exposed iron oxides. In this case encapsulated microspheres, having an outer
layer of pure polymer, will be safer.
Biopolymers such as agarose, chitosan, kappa carrageenan and alginate can be easily prepared in a magnetic form. In the
simplest way the biopolymer solution is mixed with magnetic particles and after bulk gel formation the magnetic gel formed
is mechanically broken into fine particles [9]. Alternatively biopolymer solution containing dispersed magnetite is dropped
into a mixed hardening solution [4] or water-in-oil suspension technique is used to prepare spherical particles [10].
Basically the same procedures can be used to prepare magnetic particles from synthetic polymers such as polyacrylamide,
poly(vinylalcohol) and many others [11].
In another approach used standard affinity or ion-exchange chromatography material was post-magnetised by interaction
of the sorbent with water-based ferrofluid. Magnetic particles accumulated within the pores of chromatography adsorbent thus
modifying this material into magnetic form [12,13]. Alternatively magnetic Sepharose or other agarose gels were prepared by
simple contact with freshly precipitated or finely powdered magnetite [12,14].
Magnetoliposomes (magnetic derivatives of standard liposomes), either in the original form or after immobilization of
specific proteins, have the potential for the separation of antiphospholipid antibodies [15], IgG antibodies [16] and other
proteins of interest [17].
Recently also non-spherical magnetic structures, such as magnetic nanorods have been tested as possible adsorbent material
for specific separation of target proteins [18].
Magnetic separators are necessary to separate the magnetic particles from the system. In the simplest approach, a small
permanent magnet can be used, but various magnetic separators employing strong rare-earth magnets can be obtained at reasonable
prices. Commercial laboratory scale batch magnetic separators are usually made from magnets embedded in disinfectant-proof
material. The racks are constructed for separations in Eppendorf micro-tubes, standard test tubes or centrifugation cuvettes,
some of them have a removable magnetic plate to facilitate easy washing of separated magnetic particles. Other types of separators
enable separations from the wells of microtitration plates and the flat magnetic separators are useful for separation from
larger volumes of suspensions (up to approx. 500 – 1000 ml). Examples of typical batch magnetic separators are shown
in Fig. 1.
Flow-through magnetic separators are usually more expensive, and high gradient magnetic separators (HGMS) are the typical
examples. Laboratory scale HGMS is composed from a column packed with fine magnetic grade stainless steel wool or small steel
balls which is placed between the poles of an appropriate magnet. The suspension is pumped through the column, and magnetic
particles are retained within the matrix. After removal the column from the magnetic field, the particles are retrieved by
flow and usually by gentle vibration of the column.
For work in dense suspensions, open gradient magnetic separators may be useful. A very simple experimental set-up for
the separation of magnetic affinity adsorbents from litre volumes of suspensions was described [19].
Currently many projects require the analysis of a high number of individual proteins or variants. Therefore, methods are
required that allows multiparallel processing of different proteins. There are several multiple systems for high throughput
nucleic acid and proteins preparation commercially available. The most often used approach for proteins isolation is based
on the isolation and assay of 6xHis-tagged recombinant proteins using magnetic beads with Ni-nitriloacetic acid ligand [20].
The commercially available platforms can be obtained from several companies such as Qiagen, USA (BioRobot and BioSprint series),
Tecan, Japan (Te-MagS) or Thermo Electron Corporation, USA (KingFisher).
Outline Basic principles of magnetic separations of proteins and peptides
Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References
Magnetic separations of proteins and peptides are usually convenient and rapid. Nevertheless, several hints may be helpful
to obtain good results.
Proteins and peptides in the free form can be directly isolated from different sources. Membrane bound proteins have to
be usually solubilized using appropriate detergents. When nuclei are broken during sample preparation, DNA released into the
lysate make the sample very viscous. This DNA may be sheared by repeated passage up and down through a 21 gauge hypodermic
syringe needle before isolation of a target protein. Alternatively, DNase can be added to enzymatically digest the DNA.
Magnetic beads in many cases exhibit low non-specific binding of non-target molecules present in different samples. Certain
samples may still require preclearing to remove molecules which have high non-specific binding activity. If preclearing is
needed, the sample can be mixed with magnetic beads not coated with the affinity ligand. In the case of immunomagnetic separation,
magnetic beads coated with secondary antibody or with irrelevant antibodies have been used. The non-specific binding can also
be minimised by adding a non-ionic detergent both in the sample and in the washing buffers after isolation of the target.
In general, magnetic affinity separations can be performed in two different modes. In the direct method, an appropriate
affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting the affinity towards target compound(s)
is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds
then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the
solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate
magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilised secondary antibodies,
protein A or protein G are used for capturing of the complex. Alternatively the free affinity ligands can be biotinylated
and magnetic particles with immobilised streptavidin or avidin are used to capture the complexes formed. In both methods,
magnetic particles with isolated target compound(s) are magnetically separated and then a series of washing steps is performed
to remove majority of contaminating compounds and particles. The target compounds are then usually eluted, but for specific
applications (especially in molecular biology, bioanalytical chemistry or environmental chemistry) they can be used still
attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA etc.
The two methods perform equally well, but, in general, the direct technique is more controllable. The indirect procedure
may perform better if affinity ligands have poor affinity for the target compound.
In most cases, magnetic batch adsorption is used to perform the separation step. This approach represents the simplest
procedure available, enabling to perform the whole separation in one test-tube or flask. If larger magnetic particles (with
diameters above ca 1 μm) are used, simple magnetic separators can be employed. In case magnetic colloids (diameters
ranging between tens and hundreds of nanometres) are used as affinity adsorbents, high-gradient magnetic separators have usually
to be used to remove the magnetic particles from the system.
Alternatively magnetically stabilised fluidised beds (MSFB), which enable a continuous separation process, can be used.
The use of MSFB is an alternative to conventional column operation, such as packed-bed or fluidised bed, especially for large-scale
purification of biological products. Magnetic stabilisation enables the expansion of a packed bed without mixing of solid
particles. High column efficiency, low pressure drop and elimination of clogging can be reached [21,22].
Also non-magnetic chromatographic adsorbents can be stabilized in magnetically stabilized fluidized beds if sufficient
amount of magnetically susceptible particles is also present. The minimum amount of magnetic particles necessary to stabilize
the bed is a function of various parameters including the size and density of both particles, the magnetic field strength,
and the fluidization velocity. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be
used in the bed for continuous separations [23].
Biocompatible two phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation
of biologically active compounds, subcellular organelles and cells. One of the disadvantages of this system is the slow separation
of the phases when large amounts of proteins and cellular components are present. The separation of the phases can be accelerated
by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. This
method seems to be useful when the two phases have very similar densities, the volumetric ratio between the phases is very
high or low, or the systems are viscous. Magnetically enhanced phase separation usually increases the speed of phase separation
by a factor of about 10 in well-behaved systems, but it may increase by a factor of many thousands in difficult systems. The
addition of ferrofluids and/or iron oxide particles was shown to have usually no influence on enzyme partioning or enzyme
activity [24,25].
Proteins and peptides isolated using magnetic techniques have to be usually eluted from the magnetic separation materials.
In most cases bound proteins and peptides can be submitted to standard elution methods such as the change of pH, change of
ionic strength, use of polarity reducing agents (e.g., dioxane or ethyleneglycol) or the use of deforming eluents containing
chaotropic salts. Affinity elution (e.g., elution of glycoproteins from lectin coated magnetic beads by the addition of free
sugar) may be both a very efficient and gentle procedure.
Outline Examples of magnetic separations of proteins and peptides
Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References
Tables
Table 1
Examples of proteinases and peptidases purified by magnetic techniques
Table 2
Purification of lysozyme by magnetic techniques
Table 3
Examples of polysaccharide and disaccharide hydrolases purified by magnetic techniques
Table 4
Examples of other enzymes purified by magnetic techniques
Table 5
Examples of antibodies purified by magnetic techniques
Table 6
Examples of DNA/RNA/oligonucleotide/aptamer binding proteins purified by magnetic techniques
Table 7
Purification of albumin and haemoglobin by magnetic techniques
Table 8
Examples of other proteins purified by magnetic techniques
Table 9
Examples of peptides purified by magnetic techniques
Magnetic affinity and ion-exchange separations have been successfully used in various areas, such as molecular biology,
biochemistry, immunochemistry, enzymology, analytical chemistry, environmental chemistry etc [26-29]. Tables 1, 2, 3, 4, 5,
6, 7, 8, 9 show some selected applications of these techniques for proteins and peptides isolation.
In the case of proteins and peptides purifications, no simple strategy for magnetic affinity separations exists. Various
affinity ligands have been immobilised on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting
the affinity for target enzymes or lectins. Immunomagnetic particles, i.e. magnetic particles with immobilised specific antibodies
against the target structures, have been used for the isolation of various antigens, both molecules and cells [5] and can
thus be used for the separation of specific proteins.
Magnetic separation procedures can be employed in several ways. Preparative isolation of the target protein or peptide
is usually necessary if further detailed study is intended. In other cases, however, the magnetic separation can be directly
followed (after elution with an appropriate buffer) with SDS electrophoresis. Magnetically separated proteins and peptides
can also be used for further mass spectroscopy characterization [30,31]. The basic principles of magnetic separations can
be used in the course of protein or peptide determination using various types of solid phase immunoassays. Usually immunomagnetic
particles directly capture the target analyte, or magnetic particles with immobilised streptavidin are used to capture the
complex of biotinylated primary antibody and the analyte. The separated analyte is then determined (usually without elution)
using an appropriate method. A combination of magnetic separation with affinity capillary electrophoresis is also possible
[32].
Enzyme isolation is usually performed using immobilised inhibitors, cofactors, dyes or other suitable ligands, or magnetic
beads prepared from affinity biopolymers can be used (see Tables 1, 2, 3, 4).
Genetic engineering enables the construction of gene fusions resulting in fusion proteins having the combined properties
of the original gene products. To date, a large number of different gene fusion systems, involving fusion partners that range
in size from one amino acid to whole proteins, capable of selective interaction with a ligand immobilized onto magnetic particles
or chromatography matrices, have been described. In such systems, different types of interactions, such as enzyme-substrate,
receptor-target protein, polyhistidines-metal ion, and antibody-antigen, have been utilized. The conditions for purification
differ from system to system and the environment tolerated by the target protein is an important factor for deciding which
affinity fusion partner to choose. In addition, other factors, including protein localization, costs for the affinity matrix
and buffers, and the possibilities of removing the fusion partner by site-specific cleavage, should also be considered [33,34].
As an example, isolation of recombinant oligohistidine-tagged proteins is based on the application of metal chelate magnetic
adsorbents [35,36]. This method has been used successfully for the purification of proteins expressed in bacterial, mammalian,
and insect systems.
Antibodies from ascites, serum and tissue culture supernatants can be efficiently isolated using magnetic particles with
immobilized Protein A, Protein G or anti-immunoglobulin antibodies. Protein A, isolated from Staphylococcus aureus, binds
the Fc region of IgG of most mammalian species with high affinity, leaving antigen specific sites free. Protein G, isolated
from Streptococcus sp., reacts with a larger number of IgG isotypes. It has a higher binding affinity to immunoglobulins than
Protein A, however, it also interacts with the Fab regions of IgG, although the affinity is ten times lower than for the Fc
region [37]. Antiphospholipid antibodies were successfully isolated using magnetoliposomes [15].
Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules.
Aptamers binding proteins can be immobilised to magnetic particles and used for isolation of target proteins.
DNA/RNA binding proteins (e.g., promoters, gene regulatory proteins and transcription factors) are often short-lived and
in low abundance. A rapid and sensitive method, based on the immobilization of biotinylated DNA/RNA fragments containing the
specific binding sequence to the magnetic streptavidin particles, can be used. The bound DNA/RNA binding proteins are usually
eluted with high salt buffer or change of pH [38].
Other types of proteins were isolated using specific affinity-based procedures. For example, plasminogen immobilized on
magnetic particles was used to separate scrapie and bovine spongiform encephalopathy associated prion protein PrPSc from its
conformer which is a cellular protein called PrPC. In fact, plasminogen represents the first endogenous factor discriminating
between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes [39,40].
Magnetic separation was also successfully used for the recovery of proteins expressed in the form of inclusion bodies,
involving at first chemical extraction from the host cells, then adsorptive capture of the target protein onto small magnetic
adsorbents, followed by rapid collection of the product-loaded supports with the aid of high gradient magnetic fields [41].
A new approach for analytical ion-exchange separation of native proteins and proteins enzymatic digest products has been
described recently [31]. Magnetite particles were covered with a gold layer and then stabilized with ionic agents. These charged
stabilizers present at the surface of the gold particles are capable of attracting oppositely charged species from a sample
solution through electrostatic interactions. Au@magnetic particles having negatively charged surfaces are suitable probes
for selectively trapping positively charged proteins and peptides from aqueous solutions. The species trapped by the isolated
particles were then characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) after a simple
washing.
Magnetic solid phase extraction (MSPE) enables to preconcentrate target analytes from larger volumes of solutions or suspensions
using relatively small amount of magnetic specific adsorbent. Up to now this procedure was used for preconcentration of low-molecular
weight xenobiotics [42,43] but using suitable magnetic adsorbents the MSPE could be used to preconcetrate target proteins
and peptides as well.
Sometimes the removal of certain proteins will reveal functions involving the depleted proteins or will help in the course
of subsequent protein isolation. As an example, Dynabeads have been used to remove involved proteins from Xenopus egg extracts
for analyses of the cell mitosis mechanisms [44,45]. Rapid removal of contaminating proteolytic enzymes from the crude samples
could increase yields of sensitive proteins due to the limitation of their proteolysis [46].
A combination of mechanical cell disintegration and magnetic batch affinity adsorption was used to simplify the isolation
of intracellular proteins. Magnetic glass beads were used because of their hardness and rigidity [1].
An example of quite different protein purification strategy can also be mentioned. Proteins associated with the endocytic
vesicles of Dictyostelium discoideum were separated after magnetic isolation of the vesicles that was accomplished by feeding
the amoebae with dextran-stabilized iron oxide particles. The cells were broken, the labelled vesicles were magnetically separated
and then disrupted to release proteins which were resolved by SDS-PAGE. After „in-gel“ digestion with
endoproteinase Lys-C or Asp-N the generated peptides were used for amino acid sequencing. This strategy allowed the identification
of the major protein constituents of the vesicles [47]. Analogous procedure was used for the separation and study of peroxisomes
proteins when at first peroxisomes were separated using magnetic beads with immobilized specific antibodies and then the protein
content of the separated peroxisomes was analysed [48].
Outline Conclusions
Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References
Standard liquid column chromatography is currently the most often used technique for the isolation and purification of
target proteins and peptides. Magnetic separation techniques are relatively new and still under development. Magnetic affinity
particles are currently used mainly in molecular biology (especially for nucleic acids separation), cell biology and microbiology
(separation of target cells) and as parts of the procedures for the determination of selected analytes using magnetic ELISA
and related techniques (especially determination of clinical markers and environmental contaminants). Up to now separations
in small scale prevail and thus the full potential of these techniques has not been fully exploited.
It can be expected that further development will be focused at least on two areas. The first one will be focused on the
laboratory scale application of magnetic affinity separation techniques in biochemistry and related areas (rapid isolation
of a variety of both low- and high-molecular weight substances of various origin directly from crude samples thus reducing
the number of purification steps) and in biochemical analysis (application of immunomagnetic particles for separation of target
proteins from the mixture followed by their detection using ELISA and related principles). Such a type of analysis will enable
to construct portable assay systems enabling e.g. near-patient analysis of various protein disease markers. New methodologies,
such as the application of chip and microfluidics technologies, may result in the development of magnetic separation processes
capable of magnetic separation and detection of extremely small amount of target biologically active compounds [49].
In the second area, larger-scale (industrial) systems are believed to be developed and used for the isolation of biologically
active compounds directly from crude culture media, wastes from food industry etc., integrating three classical steps (clarification,
concentration and initial purification) into a single unit operation [50]. It is not expected that extremely large amounts
of low cost products will be isolated using magnetic techniques, but the attention should be focused onto the isolation of
minor, but highly valuable components present in raw materials. Of course, prices of magnetic carriers have to be lowered
and special types of low-cost, biotechnology applicable magnetic carriers and adsorbents prepared by simple and cheap procedures
have to become available. The existence of inexpensive and effective magnetic separators enabling large-scale operations is
necessary, as well.
In the near future quite new separation strategies can appear. A novel magnetic separation method, which utilizes the
magneto-Archimedes levitation, has been described recently and applied to separation of biological materials. By using the
feature that the stable levitation position under a magnetic field depends on the density and magnetic susceptibility of materials,
it was possible to separate biological materials such as haemoglobin, fibrinogen, cholesterol, and so on. So far, the difference
of magnetic properties was not utilized for the separation of biological materials. Magneto-Archimedes separation may be another
way for biological materials separation [51].
It can be expected that magnetic separations will be used regularly both in biochemical laboratories and biotechnology
industry in the near future.
Acknowledgements
The research is a part of ILE Research Intention No. AV0Z6087904. The work was supported by the Ministry of Education
of the Czech Republic (Project No. ME 583) and Grant Agency of the Czech Academy of Sciences (Project No. IBS6087204).
Outline References
Abstract
Introduction
Necessary materials and equipment
Basic principles of magnetic separations of proteins and peptides
Examples of magnetic separations of proteins and peptides
Conclusions
Acknowledgements
References
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J Magn Magn Mater 2001, 225:101-108. [Publisher Full Text]
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16. Zollner TCA, Zollner RD, de Cuyper M, Santana MHA: Adsorption of isotype "E" antibodies on affinity magnetoliposomes.
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Originally Published IVD Technology March 2005
Assay Development
Paramagnetic microparticles for optimized biological separations
Polymer-based particles with highly irregular surfaces enhance the
existing advantages of the technology through their large surface
area and high density.
Debra A. Sesholtz, Mitchell T. Gore, Robert L. Frescatore, and Kim
M. Stever
Magnetically responsive particles have been utilized for a variety of
laboratory applications, including nucleic acid separation, protein
purification, cell isolation, therapeutics, and diagnostics. Their use
can greatly simplify biological separations and reduce assay times.
Often, they eliminate the need for column chromatography or
centrifugation.
In addition, magnetic separation procedures are usually scalable,
both up and down, and often allow the establishment of systems
coupling fully automated, high-throughput separation with molecule
detection. As with most biological separation procedures, the issues
of binding capacity and specificity are critical. Higher binding
capacities characteristic of magnetic microparticles are usually
associated with increased sensitivity and lower cost per assay. And
magnetic carriers have recently been finding employment in clinical
and biomedical applications in the area of targeted drug delivery.
This article reviews applications for magnetic-particle-based
technology, with a focus on the enhanced characteristics of
irregular, high-surface-area magnetic particles.
Magnetic Particle Anatomy
Magnetic microparticles are composed primarily of ferrimagnetic
materials. In cell separations, the magnetic labels most commonly
used are ferrimagnetic magnetite (Fe3O4) and maghemite
(g-Fe2O3). Although the particles under discussion are often
referred to as magnetic—and for ease they will be here—they are,
strictly speaking, superparamagnetic. This term designates particles
that will be responsive to a magnetic field but that will not
themselves become magnetized. Such particles disperse easily once
they are removed from the magnetic field.
Some magnetic particles are polymer based. In these, the
magnetically responsive material is added to a polymer matrix by
one of two processes: either it is entrapped during the
polymerization process, or it is attached to the particle after
polymerization. The particles are then usually overgrown in either
case, in order to encapsulate the magnetic material and provide
functional groups on the surface.
Figure 1. A BioMag superparamagnetic particle is a crystalline
aggregate of magnetite encapsulated by aminosilane and, as shown
by this transmission electron micrograph, has a highly irregular
shape. Its surface area relative to mass is more than 100 m2/g (click
to enlarge).
Magnetic particles can also be composed of crystalline aggregates
that are encapsulated by any of a variety of polymers such as
polystyrene, dextran, or silanes. With this type of particle, as with
polymer-based particles, it is important that the magnetic material
be completely encapsulated. This is to prevent leaching of the iron
complexes, which can cause problems in biological systems.
Practical Advantages
The introduction of magnetic particles in biological applications such
as immunology, cell separation, molecular biology, and diagnostics
has made processes for separating cells, DNA, and proteins faster
and easier.
In cell separation applications, a particular type of cell can be
isolated from a complex mixture by numerous means, among them
fluorescence-assisted cell sorting via flow cytometry;
density-gradient-based methods; and magnetic- particle-based
methods. Cell isolations based on magnetically responsive particle
technology offer many advantages over other procedures. The main
one is that they are relatively simple to perform. Target cells can be
isolated from fairly crude mixtures, including blood, tissue
homogenates, stool, soil, microbial culture media, water, and food,
among others. Also, the procedures are gentle, can be scaled up or
down as necessary, and can replace centrifugal, filtration, or
chromatographic separation. These attractive properties allow the
development of automated cell isolation systems.
Magnetic particles are used also in the study of biological processes
using whole cells, where the goal may be simply to detect the
presence of pathogens such as Escherichia coli, Salmonella, and
Staphylococcus in clinical, food, or environmental samples.1-3
Another objective may be to detect the expression of a eukaryotic
cell surface marker.4,5 Researchers may also find it useful to isolate
a particular cell type in order to study it in a controlled manner. In
converse fashion, they may deplete a cell type by means of
magnetic-particle-based separation in order, by its absence, to study
its influence on biological processes.
For the most part, cell separations are performed by using an
antibody– magnetic-particle complex; however, antigens can also be
attached for the capture of antigen-specific cells such as
antibody-producing cells from hybridoma cultures and
antigen-specific B-cells, and also for the biopanning of antibody
phage-display libraries.6,7 Lectins as well can be attached to the
surfaces of magnetic particles and used as capture molecules for
polysaccharides and glycoproteins expressed on the surfaces of
eukaryotic and prokaryotic cells. In the reverse approach, magnetic
particles with attached oligosaccharides can be used to isolate
lectin-expressing cells.7
Cell separation has been used in clinical applications for the
identification and isolation of circulating tumor cells in peripheral
blood.8 Another use has been the depletion of T-cells from donor
bone marrow in order to combat graft-versus-host disease in
patients receiving bone marrow transplants.9
Magnetic particles are also employed extensively to purify nucleic
acids, proteins, and cellular organelles. While these types of
separations can be affinity based, like cell separations, they can also
rely largely on the principles of conventional chromatographic
separations. Magnetic-particle-based separations of nucleic acids
have been performed by means of ion-exchange functionalized
particles, silica-coated particles, nonspecific absorption, and
sequence-specific oligonucleotide capture. Several manufacturers
provide products suitable for protein purification via magnetic
particles functionalized with capture molecules. Particles
functionalized with nickel or glutathione molecules can be used to
purify recombinant proteins containing 6X His or GST affinity tags;
protein A or protein G for immunoglobulin purification; and particles
containing secondary or primary antibodies for magnetic
immunoprecipitation.
A Mobile Surface Area
Magnetic separations are somewhat analogous to chromatographic
separations in that an increase in particle surface area usually
results in larger binding capacity. The major difference, of course, is
that, in the former, the solid phase is mobile and responsive to
magnetic fields.
One manufacturer, Polysciences Inc. (Warrington, PA), has
developed superparamagnetic particles that have an irregular,
bumpy surface, which dramatically increases the surface area of the
particle. Trade named BioMag, these particles are made of a
crystalline magnetite formulation encapsulated with aminosilane
(see Figure 1). Their irregular surface provides 20 to 30 times more
surface area than similar-sized spherical particles. An irregular
magnetic particle 1 µm in breadth has a surface area in excess of
100 m2/g, as opposed to 4–8 m2/g for a 1-µm-diameter spherical
particle.
The greater surface area of the irregular particles translates into
greater binding capacity. When compared with a similar-sized
spherical particle in experiments, BioMag oligo (dT) particles bound
significantly larger amounts of polyadenylated messenger RNA
(mRNA) (see Figure 2). In another experiment, BioMag streptavidin
particles and two other magnetic streptavidin particles that were
spherical were used to bind radiolabeled biotinylated
oligonucleotides. The irregular particles bound approximately twice
as much as one type of spherical particle and more than eight times
the amount bound by the other spherical particle (see Figure 3).
Particle size can influence cell separation results, as well.
Experiments were conducted to compare equal masses of 1-µm
irregular magnetic particles with 1.8-µm irregular magnetic particles
for depletion of CD45 positive white blood cells. Use of the smaller
particles resulted in levels of depletion approximately 15% greater
at the highest volume of particles tested (see Figure 4). Again, the
higher capture rates of the smaller (BioMagPlus) particles are
attributable to their higher ratio of surface area to mass.
The size of magnetic particles used in biological separations can
vary dramatically. As might be expected, the size of the particle in
large part determines how it will behave in solution and what type of
magnetic separation device will have to be used. Magnetic particles
can be broadly grouped as large particles (0.75 to about 5 µm) and
small particles (less than 0.75 µm, and mainly 10–200 nm).
All three sizes are used in cell and antigen capture applications. In
the case of the colloidal labels, cell labeling kinetics are quite rapid;
little or no mixing is required.7 The effects of Brownian motion on
larger particles is minimal, making mixing of these particles
necessary for efficient antigen capture.
Size is a factor to consider not only in regard to mixing kinetics; the
effect of magnetic-particle size on magnetic responsiveness also is
important. While there are some exceptions, in general, the smaller
the magnetic particle, the stronger the magnetic field needed for
effective separation. This can be confounded, however, by
differences in the amount of magnetite or maghemite contained in
the particles. For example, when larger particles containing small
amounts of magnetically responsive material are used, longer
separation times or more-powerful magnetic fields are required.
Shape, Density, and Hydrodynamics
Figure 2. The isolation of polyadenylated mRNA by irregularly
shaped magnetic oligo (dT)20 (BioMag) and by spherical
(Competitor D) magnetic oligo (dT)20 particles was compared
experimentally over a range of particle amounts. The irregular
particles isolated significantly more mRNA at all levels, this greater
binding capacity reflecting the greater surface area (click to
enlarge).
The relationship of the hydrodynamic motion of magnetic particles
to the capture of bacteria has been recently described.1,2 In these
studies, the motion of the particles in an aqueous suspension was
considered to be controlled by gravity, buoyancy, and friction. The
authors used the mass of the particle, the density of the solution, the
density of the particle, gravitational acceleration, the viscosity of
the solution, and the radius of the particle to predict mathematically
the total volume of solution traversed by the particles.
An important distinction must be noted: whereas the hydrodynamic
radius of spheres is the same as the radius of the sphere, the
hydrodynamic radius of nonspherical particles is determined
primarily by their shape.1 The quantitative expression developed by
the researchers predicts that particles with larger mass, higher
density, and a nonspherical shape favor bacterial capture.
Figure 3. Irregularly shaped streptavidin-conjugated BioMag
particles and two different spherical polymer
streptavidin-conjugated magnetic particles were compared for their
ability to bind radiolabeled biotinylated oligonucleotides. The
maximum binding for the irregular particles was just under 200
pmol, compared with approximately 100 and 15 pmol for the other
particles. These results reflect, in part, differences in surface area
among the three types of particles (click to enlarge).
Streptavidin-conjugated irregular particles with a surface area of
1000 cm2/mg and a density of 2.5 g/ml were compared with an equal
mass of spherical magnetic streptavidin-conjugated polymer
particles having a surface area of 80 cm2/g and density of 1.3 g/ml,
using biotinylated antibodies to E. coli O157. In this comparison,
capture of E. coli was two to three times greater when particles of
higher density were used, and capture required less mixing time with
these higher-density particles.1 Additionally, when the capture
efficiencies of high-density smaller particles (1-µm particles) and
high-density larger particles (18-µm particles) with the same
antibody content were compared, the larger particles exhibited more
than 40 times the capture of E. coli O157 after 60 minutes of
incubation.2 This agrees with the mathematical model the
researchers developed, which predicted that the larger particles
would have a total sedimentation volume more than 100,000 times
that of the smaller particles.
In other words, the larger and more dense the particle, the more
volume the particle will be exposed to during the mixing and capture
phase of the assay. Therefore, according to these studies, when
antibody concentration is held constant, the rate-limiting step
controlling bacterial capture is the frequency of the
bacteria-particle collision, which is favored when particles of greater
density and size are used.
Applications
Many reviews have been published on the use of magnetic-particle
technology in genomics and proteomics, drug discovery, biomedicine,
and clinical applications.10-13 Biological and biomedical applications
of magnetic-particle technology have been employed widely for
DNA, RNA, protein, and cell separations in genomic, proteomic, and
immunological research. The ability to automate magnetic-particle
separations and obtain high-purity products while reducing reagent
costs, eliminating laborious and time-intensive steps, and minimizing
the mechanical stress to which products are exposed makes the
technology attractive for high-throughput applications such as
sample preparation. And in drug discovery, the screening of large
numbers of compounds to identify potential drug candidates is
another application for which automated high-throughput
magnetic-particle technology is well suited.
Figure 4. Results of the experimental depletion of CD45 positive
white blood cells by irregularly shaped BioMag (1.8-µm) and
BioMagPlus (1-µm) amine particles conjugated to anti-CD45
antibodies. For both particles, the greater the volume of particles,
the greater the depletion percentage. BioMag Plus, with a smaller
average diameter, presents an increased surface area, which is
reflected in greater capture rates (click to enlarge).
Magnetic-particle-based technology has also been used for clinical
screening and diagnostic applications. Their ability to covalently
couple with proteins, enzymes, antibodies, and other ligands makes
magnetic particles suitable for direct use in bioassays or as affinity
ligands for the capture of target molecules and cells.10 The ability of
these particles to bind protein molecules without significantly
changing the biochemical activity of the proteins is the basis for
magnetic-particle-based immunoassays. Magnetic particles coated
with leukocyte-specific antibodies have been used to detect and
remove tumor cells from whole blood samples, bone marrow, and
prepared mononuclear cells, and have enabled subsequent
diagnosis.11
In addition to the analysis of clinical samples, magnetic particles
have been used for the detection of pathogenic organisms in food
and environmental samples. Particles are coated with antibodies
targeted to surface-specific proteins on the microorganism, then
testing to identify the organism is conducted.
The relative simplicity and speed of procedures based on
magnetic-particle technology well suits the design of rapid diagnostic
tests. Because target molecules can be isolated from fairly crude
mixtures, sample preparation is minimized. Also,
magnetic-particle-based technology allows target molecules to be
concentrated in order to increase signal intensities, thus enhancing
detection.
In addition to magnetic-particle technology lending itself well to
automation for high-throughput sample preparation for biological
and biomedical research, the use of magnetic particles in drug
targeting and delivery for clinical applications is also being actively
investigated. Magnetic particles bound with established drugs can be
employed for site-specific drug targeting in the human body using a
magnetic field.13 Site-specific targeting can increase the amount of
drug delivered directly to the target area, while at the same time
minimizing systemic distribution or exposure to normal cells.
More-localized targeting of a drug may also make possible reduction
in the dosage.12 Many types of targeted drug delivery systems are
being investigated, including magnetoliposomes, magnetic
nanoparticles, and biodegradable magnetic particles.13
For magnetic particles to be effective for drug targeting, they must
be smaller than, or comparable in size to, a cell, virus, protein, or
gene so that they can get near the target of interest.12 In
performing targeted drug delivery, biocompatible ferrofluids or
colloidal suspensions of magnetic particles in a liquid carrier are
injected into the body. A strong magnetic field gradient is produced
over the target site outside the body, drawing the particles to the
target region.
The first reports of magnetic-particle targeting of sarcoma tumors
in rats with a cytotoxic drug (doxorubicin) were encouraging.12 This
has led the way to continued investigation and, more recently,
preliminary human trials. In addition to treating tumors and
cancerous cells, magnetically controlled drug targeting may be used
to deliver other types of drugs, such as antiinflammatories, steroids,
antibiotics, and any others that can be bound reversibly to
ferrofluids.
The future holds promise of increased use of magnetic-particle
technology and delivery systems. One potential application is the
transfer of genes into cultured cells or bacteria for the production of
pharmacological biological products.12 Magnetic particles might also
serve as detection probes to replace current radioactive,
fluorescent, or chemiluminescent modes of detection.10 And
specifically targeted magnetic particles could be used to initiate a
biochemical reaction within a cell.12
Conclusion
The various uses of magnetic particles for biological separations
speak to the many benefits they confer. Biomagnetic separations are
simple, robust, scalable, and amenable to automation. Furthermore,
many options exist with regard to magnetic-particle characteristics,
allowing selection of a type of particle that suits an application well.
Success in biomagnetic separation procedures is highly dependent on
the specificity and avidity of the capture molecule, the abundance of
target, and other factors. Particle morphology, size, and density are
important for the significant influence they can have on separation
effectiveness. For example, particle diameter being constant,
particles having irregular shape will present more surface area to
the separation and demonstrate better capture of target analytes.
Additionally, recent studies have shown that factors that influence
the hydrodynamics of mixing can strongly affect the efficiency of
pathogen capture, with particles of greater density and larger
diameter exhibiting higher levels of pathogen capture.
The wide use of magnetic particles in biological separations has led
to interest and research in their potential for clinical applications.
The ability to couple specific drugs to magnetic microparticles small
enough to enter the body has led to investigation into the use of
magnetic particles as targeted drug-delivery systems, as well as
studies of their utility for gene transfer and in vivo tracking. The
specificity and sensitivity of magnetic-particle technology promises
to stimulate the development of a range of future applications.
References
1. SI Tu et al., “The Capture of Escherichia coli O157:H7 for Light
Addressable Potentiometric Sensor (LAPS) Using Two Different
Types of Magnetic Beads,” Journal of Rapid Methods and
Automation in Microbiology 10 (2002): 185–196.
2. SI Tu et al., “Factors Affecting the Bacterial Capture Efficiency
of Immuno Beads: A Comparison between Beads with Different Size
and Density,” Journal of Rapid Methods and Automation in
Microbiology 11 (2003): 35–46.
3. MJ Payne, S Campbell, and RG Kroll, “Lectin-Magnetic
Separation Can Enhance Methods for the Detection of
Staphylococcus aureus, Salmonella enteritidis, and Listeria
monocytogenes,” Food Microbiology 10 (1993): 75–83.
4. A Thiel, A Scheffold, and A Radbruch, “Immunomagnetic Cell
Sorting—Pushing the Limits,” Immunotechnology 4 (1998): 89–96.
5. JT Kemshead and J Ugelstad, “Magnetic Separation Techniques:
Their Application to Medicine,” Molecular and Cellular
Biochemistry 67, no. 1 (1985): 11–18.
6. C Sawyer, J Embleton, and C Dean, “Methodology for Selection
of Human Antibodies to Membrane Proteins from a Phage-Display
Library,” Journal of Immunological Methods 204 (1997): 193–203.
7. I Safarik and M Safarikova, “Use of Magnetic Techniques for the
Isolation of Cells,” Journal of Chromatography B722 (1999): 33–53.
8. U Bilkenroth et al., “Detection and Enrichment of Disseminated
Renal Carcinoma Cells from Peripheral Blood by Immunomagnetic
Cell Separation,” International Journal of Cancer 92 (2001):
577–582.
9. F Vartdal et al., “Depletion of T Lymphocytes from Human Bone
Marrow. Use of Magnetic Monosized Polymer Microspheres Coated
with T Lymphocyte Specific Monoclonal Antibodies,”
Transplantation 43 (1987): 366–371.
10. ZM Saiyed, SD Telang, and CN Ramchand, “Application of
Magnetic Techniques in the Field of Drug Discovery and
Biomedicine,” Biomagnetic Research and Technology 1, no. 2
[online] (2003); available from Internet:
http://www.biomagres.com/content/1/1/2.
11. MB Meza, Designing Microsphere-Based Tests and Assays (The
Latex Course) (Fishers, IN: Bangs Laboratories, 2002).
12. QA Pankhurst, J Connolly, SK Jones, and J Dobson,
“Applications of Magnetic Nanoparticles in Biomedicine,” Journal of
Physics D: Applied Physics 36 (2003): R167–R181.
13. AS Lübbe, C Alexiou, and C Bergemann, “Clinical Applications
of Magnetic Drug Targeting,” Journal of Surgical Research 95
(2001): 200–206.
Debra A. Sesholtz is laboratory products business manager at
Polysciences Inc. (Warrington, PA). Mitchell T. Gore, PhD, formerly
of Polysciences, is now regional manager, mid-Atlantic, at
Integrated DNA Technologies Inc. (Coralville, IA). Robert L.
Frescatore is a senior chemist, and Kim M. Stever is an application
specialist at Polysciences. The authors can be reached at
sesholtz@polysciences.com, mgore@idtdna.com,
rfrescatore@polysciences.com, and kstever@polysciences. com,
respectively.
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Page 1
192MAGNETIC SOLID-PHASE EXTRACTION OF TARGET ANALYTES FROM LARGE VOLUMES OF URINEM. Safarikova & I. SafarikLaboratory
of Biochemistry and Microbiology, Institute of Landscape Ecology, Na Sadkach 7, 370 05 Ceske Budejovice, Czech RepublicINTRODUCTION:
Analysis of urine is often used to detect the presence or determine theconcentration of various biologically activecompounds
or xenobiotics. Urine has become thepreferred specimen since many compounds can bedetected for longer time periods in urine
than inblood. Furthermore, urine collection does notrequire phlebotomy, and urine is typically an ample and stable sample.
In most cases the concentrationof target analytes is low and thus the analytes haveto be preconcentrated before the application
of anappropriate analytical procedure. There is a wide range of preconcentrationprocedures available that can be used individuallyor
sequentially according to the complexity of thesamples, the nature of the matrix, the analytes, and the instrumental techniques
available. The use of an extraction technique is common in the pretreatment of most types of sample. Both liquid-liquid andsolid-phase
extraction procedures are widely usedfor the urine analytes analysis.A great increase in the use of solid-phase extraction
(SPE) as a preconcentration step has occurredrecently. Solid-phase extraction can effectivelyhandle small samples using only
small volumes oforganic solvents and very simple equipment(usually a small column and a syringe). In some cases, however,
it is needed to extract very lowamounts of the target analytes from larger volumes of samples. In this case standard SPE requiresadditional
equipment such as solid-phase extraction vacuum manifolds.Recently a new extraction procedure, calledmagnetic solid-phase
extraction (MSPE), has alsobeen developed [1]. This procedure based on theadsorption of the target analyte on relatively smallamount
of magnetic specific adsorbent enables tohandle liter volumes of samples. Using MSPE with subsequent elution very low concentrations
in ppb(µg/L) range of some compounds can be detected.There are two main advantages of this procedurewhen compared with standard
column SPE. Atfirst, it is the possibility to perform the extractionsteps in a very simple way, without the need ofexpensive
equipment, even using larger amount ofsample (up to 1000 mL of liquid can bemanipulated without problems). At second, MSPEcan
be performed not only in solutions, but also insuspensions. This is a general advantage ofmagnetic separation techniques due
to the fact thatmajority of accompanying impurities arediamagnetic and do not interfere with magneticparticles during the
magnetic separation step. The use of MSPE in urine analysis was tested using silanized magnetite particles with immobilizedreactive
copper phthalocyanine dye (C.I. ReactiveBlue 21; see Fig. 1) as an affinity ligand. Thisligand interacts specifically with
polyaromatichydrocarbons having three or more conjugated rings [2] and with diamino- and triaminotriphenylmethane dyes [3].
This adsorbent can thus be used for thepreconcentration of various compounds with proven or suspected carcinogenic properties
[2]. Fig. 1: Possible structure of the C.I. Reactive Blue 21. R – reactive linker arms of the followingstructure:
SO2NHC6H4SO2CH2CH2OSO3HFor experiments crystal violet (Basic Violet 3, CI42555; also known as gentian violet orhexamethylpararosaniline
chloride; chemicalstructure is shown in Fig. 2) was used as a modelanalyte. This dye belongs into the letter group ofcompounds
interacting with copper phthalocyaninederivative. Because of its potential cancerousEuropean Cells and Materials Vol. 3. Suppl.
2, 2002 (pages 192-195) ISSN 1473-2262
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193property FDA put this dye on the Food and DrugAdministration’s (FDA’s) priority list for drugs
that need analytical methods development.Fig. 2: Chemical structure of crystal violet Concern about the health risks and carcinogenicityassociated
with the use and contact with variousdyes and other xenobiotics requires a routinelaboratory method to be developed to monitor
thepresence of target dyes and dye metabolites in body fluids. In this paper we demonstrate the use ofMSPE for the preconcentration
of a model dye(crystal violet) from human urine. Due to the factthat this dye exhibits high extinction coefficient(112,000
M-1cm-1at 590 nm) subsequent spectrophotometric detection can easily beperformed. METHODS: In model experiments fine magnetiteparticles
with immobilized reactive copperphthalocyanine dye (blue magnetite) were used asan affinity adsorbent. Blue magnetite was
preparedin a similar way as described recently [3]. Iron(II,III) oxide (10 g; Aldrich, USA) wassuspended in 5 % nitric acid
and boiled in aclosed vessel at 100 ºC for 60 minutes. Afterthorough washing with distilled water, 40 mL of10 % water solution
(pH 4.0) of 3-aminopropyl-triethoxysilane (Sigma, USA) were added to thesedimented magnetite. The suspension was mixed in
a water bath at 80 ºC for 4 h. Then the silanizedmagnetite was thoroughly washed with water,suspended in 200 mL of water and
mixed with 4 gof Ostazin turquoise V-G (C.I. Reactive Blue 21;produced by Spolek pro chemickou a hutni vyrobu, Usti nad Labem,
Czech Republic) and 12 g ofsodium chloride. The suspension was warmed to 70 ºC and 15 min later 10 g of anhydrous sodiumcarbonate
were added. The suspension was stirred at 70 ºC for 4 h and then the mixture was left overnight at ambient temperature without
mixing. The blue magnetite particles were thoroughlywashed with water and the remaining free dye wasremoved using an extraction
with methanol in aSoxhlet apparatus. The extracted particles werethen repeatedly washed with methanol – 2 % acetic
acid (50:1, v/v). The washed blue magnetiteparticles were stored in water at 4 ºC. The dryweight of 1 mL of the settled blue
magnetite was322 mg. The copper phthalocyanine content of theblue magnetite was ca 80 µmol per g of the dryadsorbent (determined
from elemental analysis forcopper using a PU 7450 ICP spectrometer (Pye-Unicam, England) after mineralization of bluemagnetite
with concentrated nitric acid).Various volumes of human urine (usually 100 mLor more; obtained from healthy male donors) werespiked
with various amounts of crystal violet andsubsequently 400 µL of water suspension ofmagnetic adsorbent (corresponding to 100
µL ofsedimented adsorbent) was added. The suspensionwas stirred for 3 – 4 hours at room temperature.After that magnetic
particles were captured to thebottom of the beaker or flask using an appropriatemagnet or flat magnetic separator and urine
waspoured off taking care not to lose the adsorbent.The adsorbent was washed several times with water and transferred into
a test tube (test tube magneticseparators MPC-1 or MPC-6 from Dynal, Norway were used for magnetic separation). After washingand
removing all water 1.5 mL of methanol – 2 %acetic acid (50:1, v/v) solution was added to theadsorbent. The elution
proceeded for ca 20 minutes. The eluate was used for the spectrophotometric measurement when the position of the crystal violet
peak was verified using a crystal violet standard. RESULTS: Crystal violet was used as a model dye in order to test a new
preconcentration procedureMSPE (magnetic solid-phase extraction) for thedetection and determination of low concentration of
target analytes in urine. The procedure wasdeveloped for standard spectrophotometers havingthe possibility to record spectra
in visible region oflight and for use of standard semi-microcuvettes (working volume ca 1.5 ml). In previous experiments magnetic
carrier withimmobilized copper phthalocyanine dye (bluemagnetite) efficiently adsorbed chromatic form ofcrystal violet from
water solutions [3]. Nevertheless this dye can also be separated from other liquidsamples, such as urine. It was shown in
preliminary experiments that majority of the tested dye (added
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194in amounts 0.25 - 1 µg into 100 mL of urine) was adsorbed on blue magnetite in approximately 120min; further prolongation
of the incubation time did not improve the dye adsorption and subsequentrecovery (data not shown). This incubation period(ca
2 hours) was used in all further experiments. Fig. 3 shows an example of eluates spectra obtained after MSPE of crystal violet
from male urine. It can be seen that very low amounts of the dye can bedetected spectrophotometrically. After MSPEclearly
visible peak of crystal violet can be found at the concentration 0.5 µg/100 mL urine (i.e., 5 ppb) and even at the half concentration
(2.5 ppb). Itcorresponds to the concentration of crystal violet 6 - 12 nmol/L. The samples showing a peakcorresponding to
that one of crystal violet (with themaximum at ca 585 - 590 nm) can be considered as presumptive positive and the presence
of crystalviolet can be verified e.g. using a HPLC procedure. Fig. 3: Spectra of a standard and eluates (1.5 mL) after magnetic
solid-phase extraction of crystalviolet from 100 mL of male urine. A – controlsample (unspiked urine); B –
urine spiked with0.25 µg of crystal violet; C – urine spiked with 0.5 µg of crystal violet; D – urine
spiked with 1.0 µg of crystal violet; E – crystal violet (1.0 µg per 1.5 mL of the elution solution).The recovery
of crystal violet was ca 60 – 75 %depending mainly on the batch of magnetic affinityadsorbent used for the MSPE.
The reproducibilityof the technique was determined from five replicatemeasurements for one concentration (1.0 µg per100 mL
of urine) of crystal violet. The typicalrelative standard deviation of the adsorption andelution efficiencies (measured as
the absorbance ofthe eluate at the peak maximum) was 9.3 %. DISCUSSION & CONCLUSIONS: Isolation and preconcentration of
compounds present in solutions or suspensions in very low concentrations belongsto the basic operations in analytical, bioanalyticaland
environmental chemistry. The aim of this work was to develop a simple and ready to use procedure for the preconcentration
and subsequent detectionor determination of low concentrations of targetanalytes in urine. Crystal violet was chosen as amodel
compound because it belongs (together withe.g. malachite green) to a group of compounds that are linked to an increased risk
of cancer [4]. Thisdye is commonly used for many purposes (e.g. such as an antimicrobial or antifungal agent)nevertheless
its use has already been controlled insome individual member states within the European Community.The results confirmed the
applicability of MSPEprocedure for a treatment of biological samples,specifically urine. Using MSPE low concentrations (5
ppb) of the native dye (in a chromatic form) can be detected spectrophotometrically. Of course,different situation can be
expected if the dyes (orother xenobiotic compounds) enter the human body and their metabolic modification takes place. Rapid
metabolic conversion of crystal violet to thecolorless leuco form can be expected followed by its slow release into urine.
The leuco form cannot besimply separated using the described procedure. An appropriate oxidizing process may lead to theconversion
of leuco form into the chromatic form of crystal violet, which could then be separated andsubsequently detected using the
describedprocedure. The optimized oxidation /preconcentration procedure for the detection ofmetabolically modified crystal
violet will be studied soon. It can be supposed that MSPE can also be used for the preconcentration of other xenobiotics from
urine provided that suitable affinity ligands (e.g.,antibodies) will be available. MSPE could thus beefficiently used as a
simple screening andpreconcentration procedure that can be followed by a more precise chromatography analysis in casesuspected
urine samples are found. Currently wetest other types of magnetic adsorbents (bothspecific and non-specific ones) which could
be used for magnetic solid-phase extraction of variousxenobiotics from human urine.
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Page 4
195It can also be expected that MSPE could be usedfor the isolation and preconcentration of importantminority biologically
active compounds directly from urine and other body fluids such as blood and milk. REFERENCES: 1 M. Safarikova and I. Safarik(1999)
J Magn Magn Mater 194: 108-112.2 H. Hayatsu (1992) J. Chromatogr 597: 37-56. 3I. Safarik, M. Safarikova and N. Vrchotova(1995)
Collect Czech Chem Commun 60: 34-42. 4J.J. MacDonald and C.E. Cerniglia (1984) Drug Metab Dispos 12: 330-336.ACKNOWLEDGEMENTS:
The research is apart of ILE Research Intention No. AV0Z6087904. The experimental work was supported by theNATO Collaborative
Linkage Grant No.LST.CLG.977500, Ministry of Education of theCzech Republic (Project No. OC 523.80) and Grant Agency of the
Czech Academy of Sciences (Project No. S6087204).
_________________________________________________________
particles are not attracted to each other
Since there is usually no magnetic re-manence the particles are not
attracted to each otherand therefore they can be easily suspended
into a ho-mogeneous mixture in the absence of any externalmagnetic
Reld.
--------------------------------------------------------------------------------
Page 1
theories and technologies that will be needed to bringthese ideas to
fruition. The tasks are challenging, evenformidable, and require the
commitment of scientistsand engineers as well as the provision of
vast fundsfor research, development and capital investment
bygovernments and industry.Further ReadingCarberry JJ (1976)
Chemical and Catalytic Reaction Engin-eering. New York:
McGraw-Hill.Choudry VR and Doraiswamy R (1971) Industrial
Engine-ering and Chemical Product Research and Development10:
218.Durand JP, Boscher Y and Dorbon M (1990) Journal
ofChromatography 404: 49.Emmett PH (1957) Advances in
Catalysis, vol. 9, p. 645.New York: Academic Press.Hall WK,
MacIver DS and Weber HP (1979) Industrial andEngineering
Chemistry 52: 425.Guichon G and Gonnard F (1979) Analytical
Chemistry51: 379.Mile B, Adlard ER, Roberts GM and Sewell PA
(1988)Catalysis Today 2: 685.Mile B, Ryan TA, Tribeck TD,
Zammitt MA and HughesGA (1994) Topics in Catalysis 1: 153.Mile
B, Howard JA, Tomietto M, Joly HA and Sayari(1996) Journal of
Materials Science 31: 3080.Petroff N, Hoscheitt A and Durand D
(1993) HydrocarbonProcessing (International Edition) 72:
103.Rideal EK and Taylor HS (1919) Catalysis: Theory andPractice.
London: Macmillan.Rooney JJ (1985) Journal of Molecular
Catalysis 31:147.Somorjai GA (1984) Chemical Society Review 13:
321.Zelter MS (1993) Hydrocarbon Processing
(InternationalEdition) 72: 103.CATALYST STUDIES:
CHROMATOGRAPHYIsolation: Magnetic TechniquesI. S[ afar\
eHk and M. S[ afar\ eHkovaH , Institute of LandscapeEcology,
Academy of Sciences,C[ eske& Bude\ jovice, Czech
RepublicCopyright^2000 Academic PressThe Rrst experiments with
magnetic separation ofcells date from the 1950s when lymphocytes
weremagnetically separated after in vitro phagocytosis ofiron
granules. The real boom in the application ofmagnetic labels for cell
isolation came in the 1980sand since then an enormous amount of
work has beendone in both the development and application of
thistechnique.Magnetic separation of cells has several advantagesin
comparison with other techniques. In general, themagnetic
separation procedure is gentle, facilitatingthe rapid handling of
delicate cells in an unfriendlyenvironment. It permits the cells of
interest to beisolated directly from crude samples such as blood,bone
marrow, tissue homogenates, stools, food, culti-vation media, soil
and water. The cells isolatedby magnetic separation are usually
pure, viable andunaltered. Magnetic separation is a simple, fast
andefRcient procedure and the whole separation processcan be
performed in the same tube. Large differencesbetween magnetic
permeabilities of the magnetic andnonmagnetic materials can be
exploited in developinghighly selective separation methods. The
separationprocedure can easily be scaled up if large quantities
ofliving cells are required.Principles of Magnetic Separationof
CellsTwo types of magnetic separation can be distin-guished when
working with cells. In the Rrst type,cells to be separated
demonstrate sufRcient intrinsicmagnetic moment so that magnetic
separations canbe performed without any modiRcation. There
areonly two types of such cells in nature: magnetotacticbacteria
containing small magnetic particles withintheir cells and red blood
cells (erythrocytes) contain-ing high concentrations of paramagnetic
haemoglo-bin. In the second type, cells of interest have to betagged
by a magnetic label to achieve the requiredcontrast in magnetic
susceptibility between the label-led and unlabelled cells. The
attachment of magneticlabels is usually attained by the use of
afRnity ligandsof various types, which can interact with
targetstructures on the cell surface. Usually
antibodies2260III/CATALYST STUDIES: CHROMATOGRAPHY/
Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
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against speciRc cells surface epitopes are used, butother speciRc
ligands can also be employed. Thenewly formed complexes have
magnetic propertiesand can be manipulated using an appropriate
mag-netic separator.The magnetic separation process for the
puriRca-tion of target cells usually consists of the followingthree
fundamentals steps:1. The suspension containing cells of interest
ismixed with magnetic labels. Incubation time isusually not longer
than 30}60 min. Then the mag-netic complex formed is separated
using a mag-netic separator and the supernatant is discarded orused
for another application.2. The magnetic complex is washed several
times toremove unwanted contaminants. The selected cellswith
attached magnetic labels can be used directly,e.g. for cultivation
experiments. Alternatively,cells can be disrupted and the cell
content analysedusing various methods.3. If necessary, a variety of
detachment procedurescan be used to remove the magnetic labels
fromthe separated cells. After detachment, the mag-netic label is
removed from the suspension ina separator and free cells are ready
for furtherapplications and analyses.Necessary EquipmentMagnetic
labels and magnetic separators are neces-sary to be able to perform
efRcient cell separation.Typical examples are given below.Magnetic
LabelsWith the exception of erythrocytes and
magnetotacticbacteria, the cells to be isolated have to be
magneti-cally labelled in order to be amenable to
magnetictreatment. Magnetic labelling can be performed
withmagnetic and superparamagnetic particles (c. 1 mand more in
diameter), magnetic colloids (c.50}200 nm), magnetoliposomes or
with molecularmagnetic labels. In most cases the magnetic
propertiesof the labels are caused by the presence of smallparticles
of magnetite (Fe3O4) or maghemite ( -Fe2O3); in some cases,
chromium dioxide particles orferrite particles have been
used.Magnetic and superparamagnetic particles Mag-netic and
superparamagnetic particles (typical dia-meter c. 1}5 m) attached to
the target cells can easilybe removed from a suspensionwith a simple
magneticseparator. Since there is usually no magnetic re-manence
the particles are not attracted to each otherand therefore they can
be easily suspended into a ho-mogeneous mixture in the absence of
any externalmagnetic Reld. Magnetic particles typically
compriseRne grains of iron oxides dispersed throughout theinterior
of a polymer particle (in many cases ofa monosized type), the
surface chemistry of which canbe modiRed to provide a range of
different linkingmethods. Alternatively, silanized particles of
mag-netic iron oxides or magnetic porous glass can be usedfor the
same purpose.A number of particulate magnetic labels can
bepurchased commercially. Up to now, in most applica-tions,
monosized polymer particles marketed asDynabeads (Dynal, Oslo,
Norway) in various formshave been used. Dynabeads are prepared
from mono-sized macroporous polystyrene particles which
aremagnetized by an in situ formation of ferromagneticmaterial
inside the pores. Other commercially avail-able magnetic particles
can be successfully used forcell separation. A selection of these
products can befound in Table 1.Colloidal magnetic labels Colloidal
magnetic labels(typical size c. 50}200 nm) are prepared by a
varietyof methods which result in Socks composed of poly-mer
(typically dextran, starch or protein) and mag-netite and/or other
iron oxide crystals. A standardprocedure for the synthesis of
superparamagneticdextran nanospheres is performed by
precipitationof iron oxide in the presence of the polysaccharide.To
such materials, ligands (usually antibodies, lec-tins, streptavidin or
biotin) are coupled so they canbe used for cell separation. Using high
gradientmagnetic columns, the labelled cells are easily separ-ated.
Magnetic particles isolated from magnetotacticbacteria (50}100 nm)
composed of magnetitecovered by a stable lipid membrane can also
be usedsuccessfully.Magnetoliposomes Magnetoliposomes are
mag-netic derivatives of ordinary liposomes prepared
byincorporation of colloidal magnetic particles into thelipid vesicles.
When magnetoliposomes are associatedwith antibodies they can
label and/or selectively con-centrate the target cells.Molecular
magnetic labels Molecular magneticlabels are usually lanthanides
(especially erbium), fer-ritin and magnetoferritin. Erbium in the
form of er-bium chloride (ErCl3) has been used for
magneticlabelling of a variety of cells. Different Er3#bindingsites,
such as carboxyl groups in glycoproteins or theCa2#receptor sites on
the cell wall are responsible forthe binding of erbium
ions.III/CATALYST STUDIES: CHROMATOGRAPHY/ Isolation:
Magnetic Techniques2261
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Page 3
Table 1 Selected examples of commercially available magnetic
particles and colloidal magnetic labels used or usable for
magneticseparation of cellsNameDiameter ( m)
Polymercomposition/surfacemodificationEnd groups andactivation
possibilityImmobilizedcompoundsManufacturer/supplierBioMag&1S
ilanized iron oxides }COOH}NH2Secondary Abs,anti-CD
Abs,anti-fluorescein Ab,protein A, protein G,streptavidin,
biotinPerseptiveBiosystems,Framingham, MA, USADynabeads
M-280Dynabeads M-450Dynabeads
M-5002.84.55PolystyreneTosyl-activatedSecondary Abs,anti-CD
Abs, Absagainst Escherichiacoli O157, Salmonella,Listeria,
Crypto-sporidium, streptavidin,oligo(dT)Dynal, Oslo,
NorwayEstapor&1Polystyrene}COOH}NH2Prolabo,Fontenay-sous-
Bois,FranceFerrofluids0.1350.175Modified
hydrophilicprotein}COOH}NH2Secondary Abs,protein
A,streptavidinImmunicon, HuntingdonValley, PA, USAM 100M
104M 1081}10Cellulose}OHScigen,
Sittingbourne,UKMACSMicrobeads0.05Dextran}OHSecondary
Abs,anti-CD Abs,streptavidin, biotinMiltenyi Biotec,Bergisch
Gladbach,GermanyMagneticmicroparticles1}2Polystyrene,cellulose,
polyacrolein}COOH}NH2Secondary Abs,protein A,protein
G,streptavidinPolysciences,Warrington, PA,
USAMagneticnanoparticles0.09}0.6Starch, dextran,chitosan}OH,
}COOHStreptavidin,biotin, protein AMicro caps,
Rostock,GermanyMagNIM0.050.250.5}COOH}NH2Secondary
Abs,Ab against E. coliO157, streptavidin,protein ACardinal
Associates,Santa Fe, NM,
USAMagneticparticles&1Polystyrene}COOH}NH2Secondary
Abs,Protein A,streptavidin,Bangs Laboratories,Fishers, IN,
USAMPG5Porous glass}NH2,
hydrazide,glycerylStreptavidin,avidinCPG, Lincoln Park,
NJ,USAXM200Microsphere3.5Polystyrene}COOHSecondary
Abs,protein AAdvancedBiotechnologies,Epsom, UKFerritin is a
naturally occurring, soluble iron stor-age protein in mammals. For
magnetic modiRcationof cells cationized horse spleen ferritin
(ferritincoupled with N,N-dimethyl-1,3-propanediamine)ex-hibiting a
net positive charge at pH 7.5 is usuallyused. Under these conditions
the cationized ferritinreadily forms ionic bonds with the anionic sites
on thecell membrane. Magnetic derivatives of ferritin
calledmagnetoferritin, prepared by controlled reconstitu-tion
conditions, have also been used for cell labelling.Magnetic
SeparatorsA variety of magnetic separators is available on
themarket, starting with very simple concentrators
for2262III/CATALYST STUDIES: CHROMATOGRAPHY/
Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
Page 4
Figure 1 (See Colour Plate 63). Example of a magnetic separ-ator
(Dynal MPC-M) for work with microcentrifuge tubes of
theEppendorf type, with a removable magnet plate to facilitate
easywashing of magnetic particles. Courtesy of Dynal, Oslo,
Norway.Figure 2 (See Colour Plate 64). Examples of
laboratory-scalehigh gradient magnetic separators. Left:
MiniMACS separationunit; right: MidiMACS separation unit, both
with inserted columns.Courtesy of Miltenyi Biotec, Bergisch
Gladbach, Germany.Figure 3 (See Colour Plate 65).
Computer-controlled magneticcell sorter CliniMACS. Courtesy of
Miltenyi Biotec, Bergisch Glad-bach, Germany.one test tube and
ending with complicated fully auto-mated devices. In many cases,
especially when work-ing with larger labels, very cheap
home-mademagnetic separators can be used successfully.Batch
magnetic separators Batch magnetic separ-ators are usually made
from strong rare-earth per-manent magnets embedded in
disinfectant-proofmaterial. The racks are designed to hold various
sizesand numbers of tubes. Some of the separators havea removable
magnet plate to facilitate easy washingof magnetic particles (Figure
1). Test tube magneticseparators enable separation of magnetic
particlesfrom volumes ranging between about 5 L and50 mL. It is
also possible to separate cells from thewells of standard
microtitration plates. Magneticcomplexes from larger volumes of
suspensions (up toapproximately 500}1000 mL) can be separated
usingSat magnetic separators. More sophisticated mag-netic
separators are available, e.g. those based on thequadrupole and
hexapole magnetic conRguration.Flow-through magnetic separators
Flow-throughmagnetic separators are characterized by the Sow
ofthe liquid and suspended cells through the separationsystem.
These systems are usually more expensive andmore complicated in
comparison with batch separ-ators, but for preliminary experiments
simple devicescan also be used.Laboratory-scale high gradient
magnetic separtors(HGMS; Figure 2) are composed of small
columnsloosely packed with Rne magnetic-grade stainlesssteel wool
which are placed between the poles ofstrong permanent magnets or
electromagnets. Mag-netically labelled cells are pumped through the
col-umn, labelled cells are retained on the steel wool, theReld is
removed and cells are retrieved by Sow andusually by gentle
vibration of the column. Automa-tion of the separation process has
led to the develop-ment of computer-controlled magnetic cell
sorters,such as CliniMACS (Figure 3) and AutoMACS,
bothproduced by Miltenyi Biotec, Germany.III/CATALYST
STUDIES: CHROMATOGRAPHY/ Isolation: Magnetic
Techniques2263
--------------------------------------------------------------------------------
Page 5
Figure 4 (See Colour Plate 66). The Isolex 300i Cell
SelectionSystem. Courtesy of Nexell Therapeutics, USA.A
continuous magnetic sorter based on an elec-trophoresis
counter-Sow chamber has been de-veloped. The injected magnetic
particles are deviatedin the inhomogeneous magnetic Reld and
focused intoa stream that is completely separated from thestreams
of the undeviated particles.The Isolex 300i Magnetic Cell Separator,
producedby Nexell Therapeutics, USA, is intended for theisolation of
stem cells from blood or bone marrow.The whole process is done
automatically and thedevice represents a Sexible platform for future
ap-plications in cell separation (Figure 4).Procedures for Performing
MagneticSeparation of CellsMagnetic separation of cells is usually
performed inone of the following formats:1. Direct method. The
afRnity ligand is coupled tothe magnetic particles, which are then
added dir-ectly to the cell sample. During incubation themagnetic
particles will bind the target cells whichcan be then recovered using
a magnet.2. Indirect method. Target cells are Rrst sensitizedwith a
suitable primary afRnity ligand. After incu-bation, excess unbound
afRnity ligand is removedby washing the cells and then magnetic
particleswith an immobilized secondary afRnity ligandwith afRnity
for the Rrst ligand are added. Themagnetic particles will bind the
target cells, whichcan then be recovered using a magnetic
separator.Another differentiation of magnetic separationtechniques
is based on the selection of the magneti-cally labelled cells.1.
Negative selection. Negative selection is a methodby which a
cellular subset is puriRed by removingall other cell types from the
sample. Both the directand indirect method are applied for negative
selec-tion. The advantage is that the puriRcation processdoes not
involve any direct contact of magneticlabels with the cells to be
isolated.2. Positive selection. The target cells are isolatedfrom the
cell suspension. Both the direct and in-direct method can be used.
The separated magneti-cally labelled cell complexes can be further
charac-terized directly, but in many cases it is necessary toremove
larger magnetic particles from the posit-ively selected cells after
their isolation.3. Depletion of cells. Depletion is a method by
whichone or more unwanted cellular subsets is removedfrom a cell
suspension. Both the direct and theindirect procedure can be applied
for this purpose.Immunomagnetic SeparationImmunomagnetic
separation (IMS) is the most oftenused approach for the isolation of
cells. Most often,a monoclonal antibody is used for IMS, but
alsopolyclonal antibodies are used successfully. IMS canbe
performed in all the formats mentioned above.In the direct method
the appropriate antibody iscoupled to the magnetic particles and
colloids, whichare then added directly to the sample. Ideally,
theantibody should be oriented with its Fc part towardsthe magnetic
particle so that the Fab region is point-ing outwards from the
particle. Several proceduresare available for direct binding of
antibodies(Table 2).The indirect method is also used very often.
Thecell suspension is Rrst incubated with primary anti-bodies which
bind to the target cells. Not only puri-Red primary antibodies have
to be used, crudeantibody preparations or serum can also be
used.After incubation, the unbound antibodies are usuallyremoved
by washing. Then magnetic particles withimmobilized secondary
antibody are added to bindthe labelled cells. Target cells } primary
antibodycomplexes } can be also captured by protein A orprotein G
immobilized on magnetic carriers. Alterna-tively, biotinylated or
Suorescein-labelled primaryantibodies and magnetic particles with
immobilizedstreptavidin or anti-Suorescein antibody are used
tocapture the target cells.The indirect method is generally more
efRcient inremoving target cells from a suspension because
freeantibodies will Rnd their target antigen more
easily2264III/CATALYST STUDIES: CHROMATOGRAPHY/
Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
Page 6
Table 2 Selected procedures for binding of antibodies on magnetic
particles and colloidsAdsorption of antibodies on hydrophobic
magnetic particles (especially those made of polystyrene)Covalent
binding of antibodies on activated magnetic particles (e.g.
tosyl-activated), or on magnetic particles carrying
appropriatefunctional groups (e.g. carboxy, amino, hydroxy,
hydrazide) using standard immobilization proceduresImmobilization
of secondary antibodies (i.e. antibodies against primary antibodies)
on magnetic particles followed by binding of
primaryantibodiesImmobilization of biotinylated antibodies on
magnetic carriers with immobilized streptavidinImmobilization of
antibodies on magnetic particles with immobilized protein A and
protein GImmobilization of antibodies tagged with oligo dA on
magnetic particles with immobilized oligo dTImmobilization of
antibodies on magnetic carriers with immobilized boronic acid
derivative via their carbohydrate units on the Fc partTable 3
Selected typical procedures for detachment of cells after
immunomagnetic separationIncubation of rosetted cells overnight in
cell culture medium with subsequent mechanical forces such as firm
pipetting, flushing thesuspension 5}10 times through a narrow-tipped
pipetteTrypsin, chymotrypsin and pronase have general applicability
for proteolytic detachment of isolated cellsDetachment with a
polyclonal antibody that reacts with the Fab fragments of primary
monoclonal antibodies on magnetic beads. Thisprinciple is
commercialized by Dynal, Norway (DETACHaBEAD)Using
synthetic peptides which bind specifically to the antigen-bindingsite
of primary antibodies(Baxter Healthcare, Deerfield, IL,
USA)Antibodies immobilized via carbohydrate units on the Fc part
to the magnetic particles with immobilized }B(OH)3groups are
dissociatedwith sorbitolA complex primary antibody}DNA linker can
be split enzymatically using DNaseCryptosporidium oocysts were
successfully released from the immunomagnetic particles by
decreasing the pH of the suspension(adding HCl)than antibodies
bound to magnetic particles. Theindirect technique is recommended
when the targetcell has a low surface antigen density or a cocktail
ofmonoclonal antibodies is used. The direct method isusually faster
and requires less antibody than theindirect method. Also, the direct
method is advant-ageous when one does not want to cover all
antigensites with antibody.Typically, 95}99% viability and purity of
the pos-itively isolated cells can be achieved with a typicalyield of
60}99%. Depletion efRciency often reaches99.9% and leaves
remaining cells untouched. Sequen-tial depletions are markedly
more efRcient.Magnetic particles usually do not have any nega-tive
effect on the viability of the attached cells. Manytypes of magnetic
particles are usually compatiblewith subsequent analytical
techniques such as Sowcytometry, electron and Suorescence
microscopy,polymerase chain reaction (PCR), Suorescence in
situhybridization (FISH) or cultivation in appropriatenutrient media.
In some cases, however, it is neces-sary to remove larger
immunomagnetic particles fromthe cells after their isolation. The
detachment processcan be performed in several ways (Table
3).Incubation time for cell separation is usually5}60 min while the
binding of primary antibodies tosecondary coated magnetic particles
usually takes 30 minor less. In positive isolation, the purity of cells
gener-ally decreases with time, although the yield
increases.NonspeciRc interactions of nontarget cells with
hy-drophobic magnetic particles can be expected. Theseinteractions
can be partially eliminated using bovineor human serum albumin,
casein and nonionic ten-sides such as Tween 20.Magnetic
Separations using other LabelsAntigens Antigens immobilized on
magnetic par-ticles can be used for the isolation of antibody
ex-pressing or antigen-speciRc cells. This approach hasbeen
successfully used for selection of antigen-speciRchybridoma cells or
human antibody-producing celllines.Lectins Lectins immobilized on
magnetic carrierscan interact with saccharide residues on the cell
surfa-ces. A typical example of this approach is the applica-tion of
immobilized Ulex europaeus I lectin whichbinds to terminalL-fucosyl
residues present on thesurface of human endothelial cells. Magnetic
beadscan be released from the isolated cells using a freecompeting
sugar.Oligosaccharides Oligosaccharides immobilized onmagnetic
particles can be used for the rapid isolationof speciRc
lectin-expressing cells. Target cells boundto the magnetic particles
can be released using a freecompeting saccharide
structure.Bacteriophage
Salmonella-speciRcbacteriophageimmobilized to a magnetic solid
phase has been usedfor the separation and concentration of
Salmonellafrom food materials.III/CATALYST STUDIES:
CHROMATOGRAPHY/ Isolation: Magnetic Techniques2265
--------------------------------------------------------------------------------
Page 7
Erbium ions, ferritin and magnetoferritin havebeen used for
magnetic labelling of both prokaryoticand eukaryotic cells.
Magnetotactic bacteria can beintroduced into granulocytes and
monocytes byphagocytosis which enables their magnetic separ-ation.
Submicron magnetic particles of -Fe2O3adhere to the surface of
Saccharomyces cerevisiae,making the cells magnetic and amenable
to magneticseparation.Magnetotactic bacteria, due to the presence
of fer-romagnetic material in their cells, can be
magneticallyseparated without any labelling. Erythrocytes can
beseparated by the high gradient magnetic separationtechnique
after conversion of diamagnetic eryth-rocytes containing
oxyferrohaemoglobin into para-magnetic red blood cells by the
oxidation of the ironatoms in the cell haemoglobin to the ferric
state(methaemoglobin). Erythrocytes, infected by Plas-modium,
contain paramagnetic hemozoin, that isa component of malarial
pigment. The paramagneticmoment of hemozoin is of sufRcient
magnitude toenable the separation of malaria-infected
(hemozoin-bearing) erythrocytes.Magnetic Separations in
Microbiology,Cell Biology, Medicine andParasitologyIMS and, in
some cases, lectin-magnetic separationsare often used in the
above-mentioned disciplines. Inmicrobiology they are especially used
for the detec-tion of pathogenic microorganisms. IMS enables
thetime necessary for detection of the target pathogen tobe
shortened. Target cells are magnetically separateddirectly from the
sample or the pre-enrichment me-dium. Isolated cells can than be
identiRed by stan-dard, speciRc microbiological procedures. IMS is
notonly faster but also usually gives a higher number ofpositive
samples. Also sublethally injured and stressedmicrobial cells can be
very efRciently isolated usingIMS. The most important microbial
pathogens can bedetected using commercially available speciRc
im-munomagnetic particles; they are used for the detec-tion of
Salmonella, Listeria and Escherichia coli O157.New
immunomagnetic particles for the detection ofother microbial
pathogens are under development.Removal of cancer cells is one of
the most impor-tant applications of IMS in the area of cell
biologyand medicine. The Rrst experiments were performedin the
1970s and since then an enormous number ofapplications have been
described. Cancer cells areusually removed from bone marrow prior
to itsautologous transplantation and using IMS they aredetected in
blood. Elimination of graft-versus-hostdisease (GvHD) in allogenic
bone marrow transplan-tation requires an effective removal of T
cells fromthe bone marrow of the donor. A direct methodenabled a
103times depletion of T cells.Magnetic particles are being
increasingly used forisolation of human cell subsets directly from
bloodand other cell sources. B lymphocytes, endothelialcells,
granulocytes, haematopoietic progenitor cells,Langerhans cells,
leukocytes, monocytes, naturalkiller cells, reticulocytes, T
lymphocytes, spermato-zoa and many others may serve as examples.
Cellsfrom other animal and plant species have been suc-cessfully
separated, too.Not only whole cells, but also cell organelles can
beisolated from crude cellular fractions. Dynal (Oslo,Norway) has
developed Dynabeads M-500 Subcellu-lar, which are able to isolate
rapidly more than 99%of target organelles.In the area of
parasitology Cryptosporidium andGiardia are the parasites where
IMS is of interest.Two commercially available kits can be used for
thispurpose. Both products are used in the method
1622:Cryptosporidium in Water by Filtration/IMS/FA(December
1997 Draft) of the US EnvironmentalProtection Agency. In very low
turbidity samples(clean waters), IMS has demonstrated
signiRcantlybetter results than the standard procedures. Whenwater
samples were turbid, the recovery efRciency ofIMS
diminished.Future DevelopmentsMagnetic separation of cells is a
simple, rapid, speci-Rc and relatively inexpensive procedure, which
en-ables the target cells to be isolated directly from crudesamples
containing a large amount of nontarget cellsor cell fragments. Many
ready-to-use products areavailable and the basic equipment for
standard workis relatively inexpensive. The separation process
canbe relatively easily scaled up and thus large amount ofcells can
be isolated. New processes for detachmentof larger magnetic
particles from isolated cells enableuse of free cells for in vivo
applications. Moderninstrumentation is available on the market,
enablingall the process to run automatically. Such devicesrepresent
a Sexible platform for future applications incell separation.IMS play
a dominant role at present but otherspeciRc afRnity ligands such as
lectins, carbohydratesor antigens will probably be used more often in
thenear future. There are also many possibilities tocombine the
process of cell magnetic separationwith other techniques, such as
PCR, enabling theelimination of compounds possibly inhibiting
DNApolymerase. New applications can be expected, espe-cially in
microbiology (isolation and detection of2266III/CATALYST
STUDIES: CHROMATOGRAPHY/ Isolation: Magnetic Techniques
--------------------------------------------------------------------------------
Page 8
microbial pathogens) and parasitology (isolation anddetection of
protozoan parasites). No doubt manynew processes and applications
in other Relds of bio-sciences and biotechnologies will be developed
in thenear future.See Colour Plates 63, 64, 65, 66.See also:
II/Centrifugation: Analytical Centrifugation;Large-Scale
Centrifugation. III /Cells and Cell Or-ganelles: Field Flow
Fractionation.Further ReadingCell Separation and Protein
PuriTcation (1996) Oslo,Norway: Dynal. 165 pp.HaKfeli U, SchuKtt
W, Teller J and Zborowski M (eds) (1997)ScientiTc and Clinical
Applications of Magnetic Car-riers. New York: Plenum Press.Olsvik
", Popovic T, Skjerve E et al. (1994) Magneticseparation techniques
in diagnostic microbiology. Clini-cal Microbiology Reviews 7:
43d54.Recktenwald D and Radbruch A (eds) (1998) Cell Separ-ation
Methods and Applications. New York: MarcelDekker. 352
pp.S[afar\mHk I and S[afar\mHkovaH M (1999) Use of magnetic
tech-niques for the isolation of cells. Journal of Chromatog-raphy B
722: 33d53.S[afar\mHk I, S[afar\mHkovaH M and Forsythe SJ
(1995) The applica-tion of magnetic separations in applied
microbiology.Journal of Applied Bacteriology 78: 575d585.Ugelstad
J, Olsvik ", Schmid R et al. (1993) ImmunoafRn-ity separation of
cells using monosized magnetic poly-mer beads. In: Ngo TT (eds)
Molecular Interactionsin Bioseparations, pp. 229d244. New York:
PlenumPress.Ugelstad J, Prestvik WS, Stenstad P et al. (1998)
Selectivecell separation with monosized magnetizable
polymerbeads. In: AndraK W and Nowak H (eds) Magnetism
inMedicine: A Handbook, pp. 471d488. Berlin: Wiley-VCH
Verlag.UhleHn M, Hornes E and Olsvik " (eds) (1994) Advances
inBiomagnetic Separations. Natick: Eaton Publishing.CELLS AND
CELL ORGANELLES:FIELD FLOW FRACTIONATIONP. J. P.
Cardot, S. Battu,T. Chianea and S. Rasouli,Universite& de Limoges,
Limoges, FranceCopyright^2000 Academic
PressIntroductionAnalysis and sorting of living cells and puriRcation
ofcell organelles are important procedures in the lifesciences. There
is a wide range of techniques andmethodologies available, which can
be divided intothree main groups. The techniques in the Rrst
groupsare based on physical criteria such as species size,density and
shape, and include centrifugation, elutri-ation and Reld Sow
fractionation. Those in the sec-ond group are linked to cell surface
characteristics,while Sow cytometry techniques make up the
thirdgroup. At a fundamental level, Reld Sow fractiona-tion (FFF)
exploits the physical characteristics of thecells or cell organelles.
However, cells or cell or-ganelles exhibit some speciRc
characteristics that canbe described by a multipolydispersity matrix.
Thedifferent physical characteristics of these biologicalmaterials
require different FFF techniques and modesof operation. Special
care must be taken if biologicalintegrity and viability are to be
preserved.Speci\c Cell CharacteristicsCellular materials range in
size from 1 m to 50 m.Cell populations are classiRed by a set of
morphologi-cal, functional and biophysical characteristics.
Thebiophysical characteristics are of particular interestin FFF.
Usually, separations in FFF are inSuenced(but not directed) by
surface properties of the samplecomponents (to avoid
particle}particle or particle}separator interactions). These
properties can bemodulated by the use of appropriate
carrier-phasemodiRers (surfactants). In terms of FFF
separations,mass, size and density appear to be the major Rrstorder
parameters. However, size is generally deRnedby the radius or the
diameter of a sphere whosevolume is identical to that of the cell.
Size can there-fore be deduced accurately if the cell of interest
isperfectly spherical. However, this is not usually thecase, and the
sphericity index, I, is then used:I"4.84(V2/3)SIn this equation V is
the cell volume and S is itssurface area can be difRcult to determine.
In terms ofcell population, these dimensions are averages andshould
be associated with a variance. These generalIII/ CELLS AND
CELL ORGANELLES: FIELD FLOW FRACTIONATION2267
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CHAPTER 4.
PRODUCT HARVESTING AND FORMULATION
OF MICROBIAL INSECTICIDES
Contents - Previous - Next
4.1 Product Harvesting
Harvesting microorganisms from submerged fermentation is often
difficult due to the low concentration of the products, their
thermolabile nature and in some cases their poor stability.
Stabilizing adjuvants may have to be incorporated immediately
post-harvest to prevent spore death and/or germination. Rapid
drying or the addition of specific biocidal chemicals may be required
to prevent growth of microbial contamination in the broth or
centrifuge slurry (Soper and Ward, 1981).
Spore-forming Bacillus thuringiensis are usually concentrated prior
to drying by centrifugation or filtration. Centrifugation using a
continuous centrifuge concentrates the product from 2-3 %
suspended solids to 15-20 %. Centrifugation may result in some loss
of suspended solid as well as loss of dissolved materials. Such losses
may not be acceptable and concentration using this technique can
often be omitted. Following concentration, one of the technique
mixes the crystal/spores slurry with lactose, adjuvants such as
wetting agents, spreader-stickers or dispersing agents, and the
whole product is spray-dried at 175oC (Dulmage, 1981). The dry
product is blended and/or mixed with additional formulation
adjuvants before packaging and/or use. The lactose added may act
as a cryoprotectant or it may help to prevent clumping Dulmage and
Rhodes, (1971). Dulmage at al. (1970) (see chapter 2) developed an
alternative drying technique for laboratory preparations where
spray drying facilities are not available; this technique of recovery
of B. thuringiensis is based on the lactose-acetone processing (see
2.1.1). Many patents exist such as a foam flotation process for
separating B.t. sporulation products.
Fungal blastospores obtained in submerged culture are much less
stable than conidia and are consequently difficult to process after
harvesting. Laboratory cultures are frequently freeze-dried with or
without protectants, but even dried product may have short viability.
Verticillium lecanii freeze-dried blastospores, for example, have a
half-life at 5, 20 and 30oC of 11, 4 and 2 days, respectively. Blachere
et al.(1973) harvested Beauveria brongniartii by centrifugation
before mixing with silica powder, osmotically active materials (such
as sucrose and sodium glutamate), anti-oxidizing agents (sodium
ascorbate) and a mixture of liquid paraffin-polyoxyethylene glycerin
oleate. The resultant paste was then dried at 4oC in ventilated
drying closet. Blastospores dried in this fashion were viable for 8
months at 4oC (Blachere et al., 1973).
Belova (1978) dried Beauveria bassiana product in five different
ways: vacuum, freeze, spray-drying, drying by mixing with an inert
filler, and in a fluidized bed with an inlet temperature of 40oC and
an outlet of 30oC. The virulence of the fluidized bed-dried material
is enhanced and the process accelerated by precipitation using a
calcium carbonate, surfactant, silica gel mixture. In addition, a
sulphite liquor is mixed in. Drying in a vacuum desiccator produced
samples of high viability and virulence, some remaining active for 1
year at 4oC storage. Spray-dried B. bassiana spores together with
the culture media led to a complete loss of viability. However, using
a 2 % molasses mixture as a protective medium permitted retention
of viability and efficacy, although to a lesser extent than for the
freeze-dried product.
Globa (1980) recovered Beauveria bassiana from fermenter broth
by precipitation with calcium carbonate. Fargues et al. (1979)
spray-dried B. brongniartii conidiospores coated with bentonite clay,
yielding 50-70 % viable spores. However, blastospores were too
sensitive for this technique and these were lyophilized with
powdered milk and glycerin. The spray-dried conidia showed no loss
of viability after 18 months storage at 5oC: lyophilized blastospores
were still viable after 8 months.
4.2 Formulation
Angus and Luthy (1971), Couch and Ignoffo (1981) mentioned that
the development of microbial insecticide formulation closely
paralleled that of chemical insecticides. Pesticide formulation is the
process of transforming a pesticide chemical into a product which
can be applied by practical methods to permit its effective, safe and
economic use. Important specific differences, of course, do exist
because microbial insecticides do not directly depend on the effect
of a poisonous chemical but exploit the activity of living (or
self-replicating) entities. An exception is the enterotoxinosis caused
by Bacillus thuringiensis where a pre-formed toxic glycoprotein is
essential for infection to occur.
The aim of the formulator then is to avoid practices that might
inhibit or harm the pathogen and wherever possible to enhance the
possibility of infection. Thus, not only must one avoid agents in any
way antimicrobial but also, with B. thuringiensis, any compounds
capable of denaturing the glycoprotein comprising the toxic crystal.
Any attempts to utilize a particular species of micro-organism in an
insecticide formulation should be based on an intimate knowledge of
the host-parasite relationships. Generally, however it is the
multi-plication of a microorganism in the host tissues that leads to
disease and death.
4.2.1 Definitions
A microbial pesticide formulation is a physical mixture of living
entities with inert ingredients which provides effective and economic
control of pests.
Formulation of a pathogen product with an extensive shelf-life (>18
month) is critical to industrialization.
In commercial development of a basic formulation of an
entomopathogen, technology concerns maintaining pathogen
viability and virulence during the production process and developing
a product form which preserves or enhances these properties. To do
this, knowledge of the biology of pathogen and target insect is
essential. Effect of temperature, humidity and media (inert carrier)
on the entomopathogen can turn out to be the most important.
4.2.2 Additives
Spreaders
Spreaders or wetting agents are added to the water diluent to
ensure "wetting" of the surface to be sprayed. Many materials have
been used including dried milk, powdered casein, gelatin, saponins,
soaps etc. In so far as microbial insecticides are concerned, it is
essential that the compound used should encourage premature
growth or germination and that it should not inhibit successful
establishment of the pathogen. Some factors are likely to be rather
subtle, e.g. although detergents such as sodium dodecyl sulphate do
not inactive the crystal toxin of B.t, they open up the structure of
the crystal and make it more sensitive to destruction by other
means.
Table Additives that have been added to preparations of microbial
insecticides.
Diluent (dust) pathogen Adhesives and Stickers pathogen
Talc B.t., Tung oil B.t.
Celite B.t., Molasses B.t.
Starch B.t., Powdered skin milk B.t., V
Synthetic silicates B.t., F Methocel B.t., V
Bentonite B.t., Corn syrup B.t., V
Lactose B.t., V Latex D B.t.
Microcell B.t., Casein B.t.
Attagel B.t., Neosil A B.t., F
Pyrax B.t., Folicote B.t.
Wetting agents and spreaders Emulsifiers
Alkyl fenols B.t., Tween 80 B.t., V, F
Sandovit B.t., 9 D 207 B.t., V
Novémol B.t., Pinolene 1882 B.t.
Petro AG B.t., Span 80 B.t., V
Colloidal X77 B.t., V Triton N60 B.t., F
Triton X100 V Triton GR7M B.t., F
Triton 155 B.t., Atlox 848 B.t.
Tween 20 F Atlox 849 B.t.
Tween 80 F Atlox 3404/849 F
Triton X45 V Atplus 448 F
Triton X114 B.t., Atplus 300 F, V
Liquid Vehicles Botanicals
Water Citrus pulp
Preformed oil in water emulsion Corn cob
Preformed water in oil emulsion Corn meal
Edible oil, Wheat bran
Corn oil, Grape pomace
Crude sorbitol Apple pomace
Aromatic spray oils, Rice hulls
Emulsified cottonseed oil, Cracked corn
Suspending agents for B.t.
Bentone 38
CAB-O-SIL
SOLOID
4.3 Oil Suspension Formulation
Uniform suspension is prepared by preliminary wetting of the dry
microbial insecticide with an emulsifer water mixture before final
dispersion in oil carrier. Edible oil is suitable for microbial pesticide
formulation. The storage of fungal spores under edible oil preserve
its viability for sufficient time. Advantage of edible oil is its
nonphytotoxicity and its residua might be acceptable.
4.4 Dusts or Wettable Powder
In the case of one B.t., Formulation, the pathogen is cultured on a
semi-solid medium so that it is preferable to process it as a dust or
wettable powder rather than attempt to separate the spores and
crystals from the medium solids. When grinding and mixing material
containing the pathogens to obtain a sufficiently fine powder, care
should be taken to avoid increase in temperature or physical damage
that would harm the pathogen.
The choice of the filler for use in a dust formulation is subject to the
general provision that it is nontoxic to the pathogen being used.
The decision as to the most desirable form (spray or dust) varies
with the crop being protected, the target insect, climatic conditions
and, of course, the particular pathogen being used.
4.5 Suspension Concentrates (sc)
Are also known as flowable emulsions, colloidal suspensions or
dispersions. Biological agents are not soluble and dispersing medium
is edible oil, which is not aggressive to biological agents.
A flowable must have a satisfactory viscosity for handling purposes;
it should disperse spontaneously or with slight stirring when poured
into water before application.
4.5.1 Processing
Simplest method to produce flowables is by adding thickeners and
thixotropic agents to biological agent in powder form. The use of
thixotropic agents enables the production of flowables which at the
application point do not exceed 2,000 MPa`s. Commercially
available flowables have viscosities ranging from 200 to 2,000
MPa`s. This results then in an excellent self dispersibility. Flowables
should disperse spontaneously when poured into water, but roping
my be tolerated if the flowable disperses rapidly upon agitation.
The coarse and wide range of particle sizes produced by this
technique may cause sedimentation and ultimately claying or
cauling, due to the absence of interfacial forces preventing close
packing.
In commercial flowable production, "wet milling" techniques are
preferred as they provide the most economical and direct means of
producing the desired average particle size of 1-5 microns in
diameter. Possible mills include attritors, sand mills, and ball (roller)
mills for batch or continuous process. The use of an attritor or
sand-mill requires the preparation of a premixture, which usually
consists of all the ingredients (such as siloxyl) at their required
concentrations.
4.5.2 Function of the Surfactants
Surfactants play an important role as basic component in flowable
formulations. They are responsible for wetting of the pesticide
particles before and during the milling process. They help to liquefy
the unmilled premixture in the milling chamber. They help to
stabilize the micronised particles in the dispersing medium. The role
of the surfactant in every step of the production process can be
described as follows:
4.5.3 Wetting
When a dry pesticide is mixed with water during flowable production
process, the surfaces of individual particles must be wet and the air
between the particles must be displaced to make efficient wet
milling possible. As little air entrainment as possible is desired by the
formulator, because the air remaining on the surfaces after milling,
causes flocculating when bubbles of the neighbouring particles
coalesce. This coalescence reduces the dispersion stability.
The most widely used wetting agents are ethoxylated alcohols and
ethoxylated nonylphenols, which should have very low foaming
properties. Foam formulation should always be avoided during the
production process and also during spraying on the field. Foaming
can reduce the uniformity and the effectiveness of the pesticide in
the field.
The concentration on wetting agent varies usually from 0.5 % to 3
% depending on the concentration, the morphology and the surface
properties of the active ingredient.
4.5.4 Milling Aid
During the milling process, surfactants help to rewet and disperse
the newly formed particles. Bad rewetting will result in paste
formulation and the whole milling chamber will be blocked. During
the milling process, the temperature can easily rise up to 60oC. As
known, desorption of the surfactant from the particles take place at
the cloud point. Therefore it is necessary to use wetting and
dispersing agents with a sufficiently high cloud point. Usually,
nonionic surfactants are selected with a cloud point at least 10oC
above the milling temperature and the maximum required storage
temperature.
4.5.4.1 Stabilization
Because of the high loading and the small particle size of the active
ingredient, the dispersed particles have an inherent tendency to
irreversibility flocculate due to the London-Van der Waals forces of
attraction. Adsorption of a dispersing agent on the particles will
generate repulsive forces between the particles or eliminate the
attractive forces between the particles.
Electrostatic repulsion occurs when ionic surfactants are adsorbed
onto particles. The charge imparted by the surfactant causes
particles to repel each other. Electrostatic repulsion is most
important in aqueous flowables.
Steric hindrance results from the adsorption on nonionic surfuctants
having long chains which are soluble in the dispersing medium. When
two particles approach, the solvated chains interact to prevent
irreversible agglomeration. This type of repulsion is important in
both aqueous and oil-based flowables.
4.5.4.2 Milling Conditions
PREMIXTURE (wet grinding mills) Ideally, the premixture should
consist of all the ingredients at the required concentration and yet
be pumpable. The ingredients are:
1) dispersing medium (water, edible oil)
2) active biological agent (conidia, blastospores, spores and crystals)
3) wetting agent (Atlox 4862)
4) thickening or viscosity modifying agents (Atlox 1086 or Bentone
38 [0.1-1.0 % w/v] or Rhodopol 23 [0.05-0.3 w/v] or other type of
Atlox (1096, 4868 B)
5) stabilizer (if necessary)
The active biological agents
B.t. - spores and crystals
Fungi - Metarhizium anisopliae, conidia strong hydrophobic
- Beauveria bassiana, conidia medium hydrophobic
- Verticillium lecanii, conidia or blastospores hydrofility
These particles are under 20 microns but all of them aggregate and
must be milled. Polyethylene glycol preserves aggregation and can
be used during wet milling.
For oil based flowables can used:
Renex 702 / Atlox 1045 A Atlox 4884 / Atlox 1045 A
Atlox 4856 B / Atlox 4885 Atlox 4856 B / Atlox 3386 B
Atplus 300 F
These pairs provide:
The good emulsification of the oil when added to the water.
The required stability of the emulsion in the spray tank.
Furthermore the combination ATPLUS 300F/oil improves the
efficiency and the selectivity of the applied pesticide. As a thickener
"BENTONE 38" (NL Industries USA) can be used. This thickener is
activated by a polar solvent to ensure a good thickening effect. The
polar solvent is generally added whilst preparing the master solution.
Example of formulation of microbial insecticide:
250 - 100 g powder mix of spores and crystal B.t. (or conidia)
30 g Atplus 300F
68 g Bentone 38
to 1 litre of water or oil
4.6 Suggested Evaluation Techique of Flowables
Test of mechanical stability
The mechanical stability is tested by shaking the flowable on shaker
for half an hour. A good flowable will not thicken or gel under the
influence of shaking.
4.6.1 Suspensibility
Suspensibility, or dilution stability is the degree to which a flowable
stays suspended when diluted in water. It is expressed as the volume
percent of setting in a dilution mixture at various time intervals. The
degree of flocculating is noted by the number of inversions of the
test cylinder required to redisperse the sediment.
4.6.2 Storage Stability
Samples are stored in sealed bottles at -10oC, room temperature,
40oC for several months. Afterwards, they are periodically
inspected for:
a) bleeding; the amount of liquid separation on top of the flowable is
expressed as a percent of the total sample depth. The bleeding
should be minimal.
b) thickening; the absence of thickening is checked by probing with
a glass rod or by measuring the viscosity
c) sedimentation; the absence of packing is checked by inserting into
the flowable a glass rod to check the bottom of the container for any
sedimentation. Packing should be absent and any non-uniformity of
the suspension should be easily correctable with mild agitation.
4.6.3 Viscosity
The viscosity is measured at one day, one week and one month
intervals. A minimum variation in viscosity is tolerated. The main
criterion is to maintain pourability.
4.6.4 Bloom
While conducting the suspensibility test, the bloom is observed and
rated qualitatively on a three point scale: good, fair and poor I.O.U..
The scale is ranging from total spontaneity to no spontaneous
dispersion.
4.6.5 Biological Activity
The biological activity is measured at one day, one week and every
month intervals during storage under evaluated temperature to way:
Test of germination of spores, LD50 on target pest in lab.
4.7 Evaluation of Separation Process "Recovery"
Each process for the isolation and purification of bioproduct from
microorganisms consists of a sequence of individual process steps.
The choice of the individual process steps is governed by the
properties of the by-product to be isolated.
The yield of the step: The yield of the step, given in amounts (e.g., g
or kg), or in units (or mega units = millions of units, documents the
scale of the process step and the batch size and permits conclusions
concerning the equipment used.
The percentage yield of a step:
units (or amount) after purification
100 . --------------------------------------------- =% of efficiency
units (or amount) before purification
The common evaluation of enrichment factor and percentage yield
of the step permits a statement concerning the efficiency of the
technology used in the process step. The product of purification
factor times percentage yield of the step is termed the efficiency.
Economic aspects must also be included, especially the
manufacturing costs of the bioproduct. For calculating these, all
types of costs, such as:
- material costs - costs for waste treatment,
- personnel costs - overheads
- energy costs - costs of repair
- depreciations must be added and the result divided by the yield of
the step.
The manufacturing costs are given in
total costs total costs
.------------------ or -------------------------------
kg of product mega units of bioproduct
and they integrate the economic and technical parameters of the
process step.
The evaluation of the total process results from the evaluation of
the individual process steps. Thus, the percentage total yield may be
obtained by multiplying all the percentage step yield, and the
manufacturing costs of the end product by adding all the types of
costs of all process steps and dividing by the overall yield.
The technical and economic classification of a process into process
steps does not only permit an optimization of the process but also an
immediate adaptation to necessary changes in the process even
when they are independent of the process itself.
References:
ANGUS, T.A., and LUTHY, P. 1973: Formulation of microbial
insecticides. In: Burges H.D. and N.W. Hussey: Microbial control of
insects and mites. Acad. Press, London and New York., pp.623-636.
BELOVA, R.N., 1978: Proc. 1st Joint US/USSR Conf. Prod. Selec
Stand. Entomopath. Fungi, Jurmula (Riga) Latvia SSR, 20-21 May,
pp. 102-119.
BLACHERE, H., CLAVEZ, J., FERRON, P., CORRIE, G. and
PERINGER, P. 1973: Ann. Zool. Ecol. Anim. 5, 69-79.
DULMAGE, H.T. and Cooperators, 1981: In: Microbial control of
pests and plant diseases. Ed. H.D. Burges,pp. 191-220. Acad. Press,
London and New York.
DULMAGE, H.T. and RHODES, R.A. 1971: In:Microbial control of
insects and mites. Ed. H.D. Burges and N.W. Hussey, pp. 507-540.
Acad. Press, London and New York.
FARQUES J., ROBERT, P.H., and REISINGER, O. 1979: Ann.
Zool.
Ecol. Anim. 11 (2) 247-257.Ferron P.,1978: Annu. Rev. Entomol. 23:
409-442.
GLOBA, L., 1980: U.S.S.R. Patent SU 73 85 71.
SOPER, R.S. and WARD, M.G. 1981: Beltsville Symposia in
Agricultural Research. Vol.5: Biological Control in Crop Production,
pp. 161-180.
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