U.S. patent application number 11/917922 was filed with the patent office on 2010-03-11 for magnetic particles with a closed ultrathin silica layer, method for the production thereof and their use.
This patent application is currently assigned to SIEMENS MEDICAL SOLUTIONS DIAGNOSTICS GMBH. Invention is credited to Karlheinz Brand, Guido Hennig.
Application Number | 20100063263 11/917922 |
Document ID | / |
Family ID | 36968634 |
Filed Date | 2010-03-11 |
United States Patent
Application |
20100063263 |
Kind Code |
A1 |
Hennig; Guido ; et
al. |
March 11, 2010 |
Magnetic particles with a closed ultrathin silica layer, method for
the production thereof and their use
Abstract
The invention relates to magnetic particles coated with silica
(Si02), whereby the silicate layer is closed and tight and is
characterized by having an extremely small thickness on the scale
of a few nanometers--hereafter also referred to as a silica
nanolayer. This invention also relates to an improved method for
producing these silicate-containing magnetic particles that, in
comparison to the prior art, lead to a product having a closed
silicate layer and thus entail a highly improved purity. In
addition, the novel method prevents an uncontrolled formation of
aggregates and clusters of silicates on the magnetite surface,
thereby having a positive influence on the properties and
biological applications cited below. The novel method also enables
the depletion of nanoparticulate solid substance particles on the
basis of a fractionated centrifugation. The inventive magnetic
particles exhibit an optimized magnetization and suspension
behavior as well as a very advantageous run-off behavior from
plastic surfaces. These highly pure magnetic particles coated with
silicon dioxide are preferably used for isolating nucleic acids
from cell and tissue samples, whereby the separating out from a
sample matrix ensues by means of magnetic fields. These particles
are particularly well suited for the automatic purification of
nucleic acids, mostly from biological body samples for the purpose
of analyzing them with different amplification methods.
Inventors: |
Hennig; Guido; (Koln,
DE) ; Brand; Karlheinz; (Krefeld, DE) |
Correspondence
Address: |
SIEMENS CORPORATION;INTELLECTUAL PROPERTY DEPARTMENT
170 WOOD AVENUE SOUTH
ISELIN
NJ
08830
US
|
Assignee: |
SIEMENS MEDICAL SOLUTIONS
DIAGNOSTICS GMBH
Erlangen
DE
|
Family ID: |
36968634 |
Appl. No.: |
11/917922 |
Filed: |
June 13, 2006 |
PCT Filed: |
June 13, 2006 |
PCT NO: |
PCT/EP2006/005677 |
371 Date: |
November 17, 2009 |
Current U.S.
Class: |
536/23.1 ;
252/62.51R; 427/127; 536/25.4 |
Current CPC
Class: |
H01F 1/01 20130101; C12N
15/1013 20130101; H01F 1/112 20130101; C12Q 1/6806 20130101; H01F
1/36 20130101; H01F 41/005 20130101 |
Class at
Publication: |
536/23.1 ;
252/62.51R; 427/127; 536/25.4 |
International
Class: |
C07H 21/04 20060101
C07H021/04; H01F 1/00 20060101 H01F001/00; B05D 5/12 20060101
B05D005/12; C07H 21/02 20060101 C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 23, 2005 |
EP |
05013522.7 |
Claims
1. Silica-coated magnetic particles, characterized in that they
have a closed surface coating with silicate with a maximum layer
thickness of 5 nm.
2. The silica-coated magnetic particles as claimed in claim 1 with
a maximum layer thickness of silicate of 2 nm.
3. The silica-coated magnetic particles as claimed in claim 1 with
a maximum layer thickness of silicate of 0.5 nm.
4. The silica-coated magnetic particles as claimed in claim 1, with
the magnetic material being iron oxide or magnetite.
5. The silica-coated magnetic particles as claimed in claim 4,
characterized in that the grain size distribution is between 0.1
and 1 .mu.m.
6. A method for the production of particles as claimed in claim 1,
characterized in that the initial silicate deposition from sodium
silicate or silica sol on the magnetic particles is triggered by
the surface properties of the iron particles and the surface is
then smoothed and sealed by slow, continuous dilution and reduction
of the pH value to neutral pH values.
7. The method for the purification of nucleic acids from biological
body samples using magnetic particles as claimed in claim 1.
8. The method as claimed in claim 7, characterized in that the
nucleic acids are RNA or DNA.
9. The method as claimed in claim 7, characterized in that it
relates to RNA of HCV or HIV.
10. The method as claimed in claim 7, characterized in that the RNA
or DNA is from fixed body samples.
Description
[0001] In recent times, molecular diagnostics have become
increasingly important. Molecular diagnostics have entered into the
clinical diagnosis of illnesses. This includes the measurement of
molecular markers to improve the diagnosis of a disease, early
detection, the monitoring of an illness during therapy, the
prognosis of illnesses and the prediction of effects or
side-effects of medicines (including the detection of infective
agents, detection of mutations of the genome, the prediction of
effects and side-effects of medicines on the basis of predetermined
genetic patterns or those acquired in the course of an illness,
detection of circulating tumor cells and the identification of risk
factors for predisposition to an illness). Methods of molecular
diagnosis have meanwhile also been used in veterinary medicine,
analysis of the environment and foodstuff testing. A further
application area is investigations by pathological/cytological
institutes or in the course of forensic investigations. Genetic
diagnosis has meanwhile also been used as part of healthcare (e.g.
investigation of banked blood for freedom from infective agents),
and legislation is planned to regulate such tests. Methods which
are also used in clinical molecular diagnosis (such as
hybridization or amplification techniques such as PCR (polymerase
chain reaction), TMA (transcription mediated amplification), LCR
(ligase chain reaction), bDNA (branched DNA) or NASBA (nucleic acid
sequence based amplification) also form part of routine procedures
in basic scientific work.
[0002] A precondition for performing an assay in molecular
diagnostics is generally the isolation of DNA or RNA from the
sample to be analyzed. There are of course analysis methods, such
as bDNA-based tests that enable nucleic acid isolation and
detection reaction to be carried out at the same time but PCR, as
the most widely used molecular biological method in molecular
diagnostics, almost always requires the use of previously purified
nucleic acids because of their capacity to be influenced by
exogenic factors.
[0003] The conventional preparation process for nucleic acids is in
this case based on a fluid-fluid extraction. An example of this is
the phenol-chloroform extraction of DNA from body samples. However,
the great effort required and the need to sometimes perhaps use
highly toxic substances means that this method has fallen
considerably into disfavor in recent years compared with
solid-phase based methods.
[0004] With the use of solid-phase based extraction methods for
nucleic acids, the sample preparation can be subdivided into the
actual analysis operation, largely independent of the particular
problem, into four basic steps: 1. Conditioning of the solid phase;
2. Selective or specific bonding of the analytes to the solid phase
and removal of the remaining sample matrix; 3. Washing out any
impurities from the solid phase and 4. Elution of the enriched and
purified analytes.
[0005] The well known property of nucleic acids to bond
specifically to silicate-containing adsorbents such as glass powder
[Proc. Natl. Acad. USA 76 (1979) 615-619, Anal. Biochem. 121 (1982)
382-387], diatomaceous earth [Methods Enzymol. 65 (1979) 176-182]
or native silicon dioxide [J. Clin. Microbiol. 28 (1990) 495-503,
EP 0 389 063 B1] under chaotropic or high-salt conditions, i.e. at
high concentrations of chaotropes or other salts, has long been
used for the selective and reversible bonding of nucleic acids.
With the aid of a buffer containing water-soluble organic solvent,
usually a low aliphatic alcohol, impurities are then washed from
the adsorbent, the carrier is dried and the adsorbed nucleic acids
are eluated with distilled water or a so-called low-salt buffer,
i.e. a buffer with a low ion strength.
[0006] In view of the complete and cost-effective automation of
nucleic acid isolation, methods with super-paramagnetic adsorbents
play an increasingly important role.
[0007] In the simplest example (WO 01/46404), commercially produced
magnetic particles produced for technical applications, such as
electrographic toners, are used directly for nucleic acid
preparation without further modification.
[0008] Products of this kind produced by technical mass production
do, however, meet one of the most important preconditions, such as
a specific nucleic acid absorption and magnetisability. On the
other hand, these commercially available products are unable to
meet important boundary conditions that are indispensable for
highly-sensitive and reproducible results. For example, it is of
decisive importance in the field of virus diagnostics (e.g. HCV or
HIV) to extract the viral nucleic acids quantitively from the serum
or plasma, i.e. with almost 100% yield, in order from this to
derive an accurate virus concentration in the serum/plasma and thus
make decisions with regard to therapy. The purity of the magnetic
particles also plays a decisive role with regard to optical
evaluation. Especially with magnetite particles that are frequently
still micro-porous, diffusion of iron atoms from the particles can
lead to colored solutions that can severely disturb the
transmission or reflection measurements.
[0009] For this reason, various developments of magnetic particles
for biological applications, particularly with regard to the manual
and automated isolation of nucleic acid, are described.
[0010] In this case, magnetic particles that support a high density
of SiOH groups on the surface play an outstanding role. It is known
that SiOH groups can form reversible bonds with nucleic acids.
Silica-modified magnetic particles are also the object of this
invention.
[0011] To obtain highly-sensitive, quantitative and reproducible
results, such magnetic particles must, in addition to
magnetizability and the capacity to bond with nucleic acid, fulfill
further boundary conditions, which are described in more detail in
the following.
[0012] Particle Size and Particle Size Distribution
[0013] It has been shown that magnetic particles of Fe.sub.30.sub.4
(magnetite), for electrographic toner with primary particle sizes
of approximately 0.1 to 1 .mu.m, e.g. available from the Lanxess
company under the name Bayoxide E, meet almost ideal preconditions
with regard to particle size. Such particle sizes enable the
important boundary condition of "suspension stability" important
for biological applications to be achieved. This must on the one
hand be sufficiently resistant to ensure that no significant
sedimentation occurs within a few minutes, for example ten to
fifteen minutes (adsorption of nucleic acids) after shaking,
whereas the magnetic particles loaded with nucleic acids must be
able to be completely separated as regards the shortest possible
analysis times within a few minutes, for example within one to five
minutes.
[0014] However, the available magnetic particles of Fe.sub.30.sub.4
(magnetite) unfortunately still have ongoing deficiencies in this
respect, in that small amounts of very fine magnetite particles in
the nanometer range are still present.
[0015] These unwanted by-products that because of their large
surface can bond considerable amounts of nucleic acids are
unfortunately not separated in the magnetic field within a few
minutes and thus the information content of these nucleic acids,
especially with regard to the quantitive measurement of nucleic
acids, can be lost.
[0016] In addition to these losses in yield, this also often leads
to unclear, often yellow-brown, supernatants that can not only
negatively influence the commercial marketing but can also
interfere with the photometric evaluation of the eluates.
[0017] It would therefore be of great advantage if this
"nanoparticle magnetite particulate component" could be separated
for the biological application described here.
[0018] Silicate Content:
[0019] As mentioned above, some magnetic particles produced on a
large technical scale, for example the Bayoxide E series from the
Lanxess company, still have a certain nucleic acid bonding capacity
even without special silica post-treatment, because they are
produced in bulk and therefore also support SiOH groups on the
surface in small amounts. Because of the low nucleic acid
adsorption capacity, such products require corresponding relatively
large amounts of magnetic particles, which means that the
preparation of small sample volumes is hampered.
[0020] Furthermore, such products have a wetting behavior of vessel
walls, such as glass or plastic walls of microtiter plates such as
are routinely used for nucleic acid purification, that is
unfavorable for the application described here. Therefore
substantial amounts of the unmodified, relatively hydrophobic
magnetic particles remain adsorbed in aqueous suspensions on the
microtiter plate walls and thus lead to inaccuracies in pipetting
and loss of yield.
[0021] Particles with a high density of SiOH surface groups, which
because of their hydrophilicity very advantageously roll off
plastic walls in particular, such as the aforementioned microtiter
plates, behave very favorably in this respect.
[0022] With many magnetic particle developments for the isolation
of nucleic acid, the silica proportion is accordingly dominant
compared with the magnetite proportion. As, for example, described
in WO 01/71732, silica particles that can be magnetized by the
magnetite inclusion are obtained by hydrolysis from reactive silica
compounds such as tetraethoxysilane (TEOS) in the presence of
magnetite particles. Because of the high density of SiOH groups on
the surface, such particles however show a high nucleic acid
bonding capacity and a favorable wetting behavior of the microtiter
plate walls, but on the other hand the magnetic properties are very
heavily reduced corresponding to the reduced magnetite content.
Furthermore, the magnetic silica particles produced in this way
have significantly more unfavorable morphological properties, such
as very heterogeneous particle sizes and particle size distribution
and it should be mentioned that large non-spherical particles can
lead to blockages during automatic pipetting.
[0023] Extractable Components:
[0024] The nucleic acids isolated using the magnetic particle
process are generally subject to further processes such as a PCR
(polymerase chain reaction), TMA (transcription mediated
amplification), LCR (ligase chain reaction) or NASBA (nucleic acid
sequence based amplification). These are highly-sensitive,
enzyme-controlled processes that can be disturbed by numerous
impurities and iron compounds, that, for example, can act as enzyme
toxins.
[0025] Therefore, the magnetic particles produced for the nucleic
acid purification must fulfill particular purity requirements. If
iron oxides, such as Bayoxide from the Lanxess company produced
using technical mass production, are used this problem is certainly
not insignificant because the magnetite particles have a certain
porosity and surface roughness. Therefore, impurities can become
included in the micropores both from the process of iron oxide
production and in the succeeding silica treatment that as enzyme
toxins or in the case of colored impurities can interfere with the
photometric evaluation during subsequent processes.
Object of this Invention
[0026] The object of this invention is to produce, on the basis of
commercially available magnetic particles, silica-modified magnetic
particles with a high density of SiOH surface groups and a closed
and tight surface layer of silicate. Neither the morphology nor the
very good magnetic properties of the initial products should be
substantially influenced by the silica modification. Equally, the
wetting behavior on plastic surfaces should be positively
influenced by the silica coating. Furthermore, the silica-modified
magnetic particles should be optimized with regard to extractable
impurities to the extent that the release of impurities or iron
compounds from the magnetite core is prevented and no interference
is possible with either the biological detection reactions or the
photometric evaluation.
Nearest Prior Art
[0027] On the basis of Bayoxide E magnetic particles from the
Lanxess company, WO 03/058649 describes a smart process for silica
deposition on the particle surface using sodium silicate solutions,
for example sodium silicate HK 30 from the Cognis company. By a
gradual dilution of the pH value in the Bayoxide E/sodium silicate,
equivalent to a gradual pH shift from strong alkaline (pH11.5) to
neutral (pH7), a careful deposition of silica on the magnetic
particle surface takes place. If, as mentioned in WO 03/058649, the
pH reduction takes place due to the addition of acids (WO
98/31840), uncontrolled conversion of sodium silicate into silica
(SiO.sub.2) can occur at the acid infusion point with magnetic
particles becoming stored in the structure of the silica, so that
the aforementioned controlled silica deposition on the magnetic
particle surface is by no means achieved. Nonetheless, the
formation of minute silica aggregates or clusters on the surface
cannot be completely prevented by the "batch method" described in
WO 03/058649.
[0028] Whereas the silica-modified magnetic particles described in
WO 03/058649 have good properties with regard to surface structure
and nucleic acid bonding behavior, very disadvantageous
yellow-brown supernatants can be observed in the long-term behavior
(after standing for a few weeks) of the relevant aqueous
suspensions. In biological assays during which surfactants are
generally used, this effect can be observed even after short stand
times. An analysis shows that, in addition to sodium silicate
components, traces of iron compounds and very fine magnetite
particles can be found in these colored supernatants. Clearly,
these impurities became locked into the porous magnetic particle
structure through the silica surface, from where they diffuse
outwards in the course of time. These observations also indicate
that the silicate layer is not completely closed or is irregularly
distributed by the batch method described in WO 03/058649 and
therefore cannot prevent the release of iron compounds.
DETAILED DESCRIPTION OF THIS INVENTION
[0029] In view of the elimination of the aforementioned extractable
impurities, the technical process described in the following was
optimized, with the progress compared with the method described in
WO 03/058649 being documented. However, with the tests described
here, in contrast to the examples of WO 03/058649, Bayoxide E 8707,
which is no longer available as a standard product, was replaced by
the very similar Bayoxide E 8706 type. In both cases it is
Fe.sub.30.sub.4 magnetite that has a low Si content due to its
production, with type 8707 having an Fe/Si content of 99.1/0.9 and
Bayoxide E 8706 having 99.4/0.4. The surface quality, particularly
the pH value of the Fe.sub.30.sub.4 magnetite, is important for the
method according to the invention. Whereas Bayoxide E 8707 with a
pH of 6.5 has a slightly acid surface, a neutral pH value, or
depending on the batch even a slightly alkali value (pH 7.5), is
found with the Bayoxide E 8706 now used. Surprisingly, it was found
that even these slightly alkali surface properties can induce
sodium silicate deposition. Normally, the silica deposition takes
place from the very alkali sodium silicate solutions by the
addition of acids.
[0030] Comparison tests then surprisingly showed that distinctly
better results could be obtained with regard to extractable
components if instead of the gradual pH reduction described in WO
03/058649 a continuous method, such as a membrane method was used.
In this case, as described in more detail in the examples, the
aqueous sodium silicate/magnetic particle suspension was purified
after a reaction time of one hour using "cross flow
microfiltration". Cross flow microfiltration, which is carried out
at a slight negative pressure, is, as described in "Basic
Principles of Membrane Technology" by M. Mulder, a known separation
or purification method. In this case the work is carried out at
constant volumes, i.e. the permeate volume flow containing the
impurities is replaced by the same volume flow of incoming fresh
water. In contrast to the dialysis method known in biology,
depending on the pore diameter not only low-molecular salts but
also particulate impurities are separated during microfiltration.
This continuous cleaning process was continued until the quality of
the outflowing permeate quality corresponded to the degree of
purity of the incoming fresh water, which took approximately 12 to
15 hours depending on the size of the preparation.
[0031] During the analytical surface characterization using ESCA it
was very surprising to find that the silica-modified magnetic
particles produced in this way have a novel, i.e. ultrathin, silica
structure on the silica surface, with which the improved
purification or increased purity can be correlated. This silica
nanolayer is characterized by a silica layer of up to 5 nm
distributed uniformly over the complete particle surface.
Furthermore, the method according to the invention however also
describes a layer thickness of 2 nm and also, quite particularly
preferred, layer thicknesses of 0.5 nm to 0.2 nm. The particles
coated in this way have a surface coating which is characterized in
that it, for example, prevents the escape of irons into the
surrounding solution.
[0032] The production of magnetic particles with a silica layer
thickness of 0.2 nm is described in example 3.
[0033] Furthermore, the inventive method is characterized by a
closed and tight silica layer, which is also associated with the
improved purity or reduced observed contamination effect in the
supernatant. The purity of these silica-coated magnetic particles
produced according to the inventive method is substantially better
compared with the method described in WO 03/058649. Thus, visible
discoloration of the supernatant after production and washing no
longer occurs (see examples 2 and 3). In particular, the tight and
closed silica layer prevents the escape of visible, or also
invisible, impurities, for example iron ions, which can disturb the
amplification methods or the optical evaluation of biological
experiments (see examples 4 and 5).
[0034] Furthermore, it was surprising to find that the formation of
aggregates and clusters of silicates on the magnetite surface was
almost completely prevented due to the slow and continuous dilution
and thus reduction of the pH value to neutral values in the
described membrane filtration process and/or again strongly reduced
compared to the "batch method" described in WO 03/058649. This well
defined nanolayer of silicon positively influences the properties
and biological applications described in the following.
[0035] Furthermore, it was also found that additional product
optimization with respect to clear supernatants could be achieved
by carrying out a fractionated centrifugation, which enabled a
separation of slowly sedimenting iron oxide particles, after the
membrane process.
[0036] With the samples produced in this way, which are treated as
aqueous suspensions, all criteria such as a magnetisability
absolutely identical to the initial product, unchanged morphology,
high nucleic acid bonding capacity, favorable roll-off from the
walls of the microtiter plates and outstanding stability of the
suspension with trouble-free separation of the magnetic particles
in the magnetic field within a few minutes without significant
impurities in the supernatant are achieved.
[0037] The expression "magnetic particles coated with silica"
includes magnetite cores that are coated with a nanolayer of
silica.
[0038] The expression "closed and tight silica layer" includes a
uniform, homogenous single to multiple molecular silica layer in a
range of less than 5 nm, with a layer thickness of 2 nm being
particularly preferred and a layer thickness of 0.5 to 0.2 nm being
quite particularly preferred. This closed silica layer particularly
prevents the release of iron compounds and iron ions to the
environment of the silica-coated magnetic particle.
[0039] The expression "improved methods of production" includes a
washing process with the aid of a micro- or ultra-filtration unit
that is easy to perform but is very intensive and leads to extreme
purity of the silica-coated magnetic particle. With this method, a
slow, controlled and continuous dilution, and therefore a reduction
of the pH value to neutral pH values in the reaction solution,
occurs after an initial precipitation of the nanolayer of silicate
onto the particle surface, thus forming an extremely uniform,
tight, closed and homogenous layer of silicate on the surface of
the magnetite. Furthermore, unwanted formations of aggregates or
clusters of silicates are prevented or largely reduced.
[0040] The expression "depletion of nano particulate components
with the aid of the centrifugation technique" includes the
application of centrifugation techniques or simple gravitational
techniques. This produces sedimentation of the required fractions,
with it being possible to reject the unwanted nano particulate
components by removing the supernatant. By determining the particle
size distribution using ultra-centrifugation, this effect can be
detected by means of the depleted minute fractions. With the
centrifugation technique, the initial suspension is centrifuged for
fifteen minutes at approximately 3000 g, the supernatant is removed
and an equal amount of water or buffer is added and then
re-suspended and this step is repeated several times (up to ten
times). The gravitation technique simply means that instead of the
centrifugation a long time is allowed to elapse until a large
proportion of the particles has settled on the bottom of the vessel
and the aqueous supernatant is then replaced.
[0041] The expression "optimum magnetization behavior" includes the
property of the inventive particles to have the largest possible
amount of magnetite and thus be completely separated from the
sample matrix during the purification within a few minutes, for
example within one to five minutes, when a magnetic field is
applied from outside to a reaction vessel. This is particularly
noteworthy with respect to the shortest possible purification times
in an automated process using a pipetting robot and for the use of
the cheapest possible magnets with a limited magnetic field
strength as hardware components.
[0042] The expression "suspension behavior" includes the property
of the inventive particles to behave in such a way that due to an
optimum grain size distribution no significant sedimentation occurs
within a few minutes, for example ten to fifteen minutes
(adsorption phase of the nucleic acids) after shaking during the
purification phase.
[0043] The expression "optimum run-off behavior from plastic
surfaces" includes the property the inventive particles have of a
low affinity to the plastic articles used in biological
purification processes due to a hydrophilic surface quality. The
plastic articles used mainly include polystyrene, polyethylene and
polypropylene vessels or "microtiter" plates of comparable plastics
of any shape or size. The specific silica layer of the inventive
magnetic particles enables a repelling interaction with these
plastic surfaces, so that the coated magnetic particles roll off
these surfaces and undergo no great interactions, which in the end
could lead to a loss of yield during a biological purification
process of nucleic acids.
[0044] The expression "isolation" means the purification of nucleic
acids from a biological sample using the aforementioned
silica-coated magnetic particles and is divided into the following
steps. [0045] a) Dissolving the sample in a reaction vessel with a
lysis buffer and, after incubation, adding a bonding buffer, which
preferably contains chaotropic salts, with
guanidin(ium)isothiocyanate being particularly preferred, of high
molarity [0046] b) Adding silicate-coated magnetic particles [0047]
c) Incubating at a temperature at which the nucleic acid bonds to
the magnetic particles [0048] d) Removing constituents that are not
bonded from the reaction preparation by applying a magnetic field,
which separates the magnetic particles from the surrounding fluid
[0049] e) Applying a washing buffer several times followed by the
removal of said buffer with magnetization of the particles for
cleaning unspecifically bonded molecules from the nucleic acid
[0050] f) Adding an elution buffer under conditions in which the
nucleic acid is separated from the magnetic particles [0051] g)
Separating the eluate with the nucleic acid after re-application of
a magnetic field.
[0052] The expression "automated purification" includes variations
of these processes in which the manual labour by humans is replaced
either completely or only partially in steps, especially with the
biological body sample being dissolved with a special buffer during
the steps, the addition of magnetic particles, the incubation at a
specific temperature, the removal of non-absorbed sample
constituents, the washing steps, the elution of bonded nucleic
acids from the particles at a specific temperature and the
separation of the eluate from the particle suspension.
[0053] The expression "nucleic acids" includes oligomer and polymer
ribonucleotides or 2'-desoxy-ribonucleotides with a chain length of
more than 10 monomer units. The monomer units in nucleic acids are
linked by phosphoric acid diester compounds between 3'- and
5'-hydroxyl groups of adjacent monomer units and the 1'-atom of the
respective carbohydrate component is glycosidically bonded to a
heterocyclic base. Nucleic acids can form double and triple strands
due to the development of intermolecular hydrogen bridge bonds.
[0054] This also includes protein/nucleic acid complexes and
nucleic acids with synthetic nucleotides such as morpholinos or
PNAs (peptide-nucleic acids).
[0055] The expression "biological body sample" includes biological
material containing nucleic acid, such as whole blood, blood serum
or blood plasma, especially serum or plasma containing a virus,
very particularly serum samples infected with HIV and HCV, "Buffy
Coat" (white blood cell fraction of the blood), faeces, ascites,
swabs, sputum, organ aspirates, biopsies, tissue sections, in this
case very particularly differently fixed tissue sections,
especially those fixed with fixing agents containing formalin, and
paraffin-embedded tissue sections, secretions, liquor, bile,
lymphatic fluid, urine, stool, sperm, cells and cell cultures. This
can also include nucleic acids that originate from biochemical
processes and are then to be purified.
[0056] The expression "detection with various amplification
methods" includes the duplication of purified nucleic acids using
various molecular-biological technologies, especially PCR,
transcription-mediated amplification (TMA), LCA or also NASBA and
the succeeding or simultaneous detection of the amplification
products. This also includes detection using signal amplification
methods such as of bDNA, i.e. without nucleic acid amplification.
Detection of the PCR in particular can be carried out by the
application of kinetic methods with the aid of fluorescence
technology under real-time conditions or can be carried out using a
conventional agarose gel. The real-time PCR in particular enables a
very good quantitive determination of nucleic acids by using
suitable calibrators. What is critical and limiting for clinical
sensitivity (avoidance of false negative results) in this case is
the efficient purification of the nucleic acids (i.e. efficient
bonding to the magnetic particle and the reversible release under
PCR-compatible conditions).
[0057] A further object of the invention is a kit for performing a
method according to the invention that contains the following
components: [0058] (a) Reagents for dissolving the sample [0059]
(b) Magnetic particles containing silica or a suspension of
magnetic particles containing silica [0060] (c) Washing buffer
[0061] (d) Elution buffer
[0062] The above lists and the following examples are applicable
for the individual components. Single or several components of the
kit can also be used in a modified form.
[0063] With this invention it is possible by using specially
produced silica-coated magnetic particles to detect nucleic acids
particularly efficiently, automatically and quantitively from
biological body sample purifications using appropriate
amplification techniques.
[0064] This invention thus represents an important contribution to
nucleic acid diagnostics.
EXAMPLES
[0065] The following are examples of protocols for performing the
described invention. Exact reaction conditions for the respective
nucleic acids to be purified are given in these examples, but
nevertheless various parameters such as magnetic particle quantity,
incubation temperature and washing temperature, incubation and
washing times and the concentration of lysis buffer, washing buffer
and elution buffer can vary depending on the particular nucleic
acid to be purified.
Example 1
Production of Silicate-Coated Magnetite Particles from Bayoxide E
8706 Using Sodium Silicate 37/40 by the Gradual Reduction of the pH
Value (Similar to the Method in WO 03/058649 A1)
[0066] Reaction Part:
[0067] 4000 g of sodium silicate solution 37/40 (Cognis GmbH) is
placed in a 6 l three-neck flask with a KPG stirrer. 2000 g of
Bayoxide 8706 (Bayer AG) is added within ten minutes whilst
stirring. Stirring then continues for one hour at room
temperature.
[0068] Purification:
[0069] After the stirrer is switched off, the silica-coated
magnetite beads settle. This process can be accelerated if
necessary by applying a magnetic field. After a waiting time of one
hour, the supernatant is drawn off. For purification, 4 l of water
is added whilst stirring for approximately ten minutes. The
supernatant is again drawn off. This washing process is repeated at
least four times until the last wash water has achieved a pH value
of 7.5-7.0.
[0070] Properties of the Silica-Magnetic Particles:
TABLE-US-00001 Zeta potential: -50.2 Silica content according to
ESCA 7.0 atom % Si
[0071] Purity: The supernatant was colored yellow/brown after
standing ten days at room temperature.
Example 2
Production of Super-Pure Silica-Coated Magnetite Particles from
Bayoxide E 8706 Using Sodium Silicate 37/40 with a Continuous
Reduction of the pH Value by Cross Flow Microfiltration
[0072] The reaction part described in Example 1 was repeated but
the processing took place not gradually or batchwise but instead
with the aid of the "Centramate.RTM." micro filtration unit from
PALL with a 0.2 .mu.m Supor.RTM. membrane cassette.
[0073] For this purpose, the magnetic particle suspension was drawn
off via a hose by means of a pump and passed through the membrane
cassette, with the permeate being rejected but the retentate being
fed back into the reaction vessel. The amount equivalent to the
permeate was then resupplied to the particle suspension.
[0074] After a filtration time of 12 h, the pH and conductivity of
the permeate had achieved the quality of the original water and the
cleaning process was ended.
[0075] Properties of the End Product:
TABLE-US-00002 Zeta potential: -41 mV Si content: 4.9 atom % Si
determined according to ESCA Silica content of the starting product
Bayoxide 8706: 2.4 atom % Si
[0076] The differential amount, 2.5 atom % Si, was accordingly
deposited on the surface of the particles by silica treatment using
sodium silicate. This produces a silica layer thickness of 0.4
nm.
[0077] Purity: The particle suspension purified by ultrafiltration
showed no discoloration in the supernatant even after standing for
several months at room temperature.
Example 3
Production of Super-Pure Silicate-Coated Magnetite Particles from
Bayoxide E 8706 and Sodium Silicate 37/40 with a Continuous
Reduction of the pH Value by Cross Flow Microfiltration Followed by
Fractionated Centrifugation
[0078] The end product described in Example 2 was centrifuged for
seven minutes at 3225g with the aid of a centrifuge (Eppendorf
5810). Whereas the main part (>98%) of the product was
sedimented, a dark brown colored supernatant remained that was
discarded.
[0079] The residue was again added to water, centrifuged and
separated from the colored supernatant. This fractionated
centrifugation was repeated eight times until the supernatant
became colorless.
[0080] Properties of the End Product (Ninth Centrifugate):
TABLE-US-00003 Zeta potential: -35 mV Si content: 3.0% Si.
[0081] The differential amount, 0.6 atom %, was accordingly
deposited on the particle surface by silica treatment with sodium
silicate. This resulted in a silicon layer thickness of 0.2 nm.
[0082] Purity: The supernatant of the magnetic particle suspension
produced in this way remained completely colorless even after
storing for several months.
[0083] This product quality showed outstanding values particularly
with regard to magnetic separation. Thus, after applying a magnet
an absolutely clear supernatant was observed after less than twenty
seconds.
Example 4
Optical Measurement of Aqueous Supernatants from Silica-Coated
Magnetic Particle Suspensions
[0084] In this experiment, the absorption spectra of two aqueous
supernatants of the silica-coated magnetic particles with lot
designation HIE13266 (originating from the inventive method of
Example 2) and 3) and lot designation HIE12106R2 (originating from
the method from WO 03/058649 A1, based on Bayoxide E 8707) were
recorded in a range of 221-750 nm using a spectrometer from the
Nanodrop company (see FIG. 1).
[0085] A water spectrum drawn from these spectra was used as a
reference. A water spectrum was again taken as a sample for control
purposes (zero line).
[0086] From the spectra, it could be seen that the aqueous
supernatants of the silica-coated magnetic particles HIE13266 had
an absorption behavior similar to water. On the other hand, the
absorption lines of the supernatants of HIE12106R2 showed a clearly
changed and elevated absorption behavior up to a range of
approximately 500 nm.
[0087] From this it can be seen that the new inventive production
method with continuous washing (particles HIE13266) in a
microfiltration unit led to reduced contamination effects or the
occurrence of iron compounds in the supernatant compared to
particles HIE12106R2 with sequential multiple washing or gradual
reduction of the pH value (see also WO 03/058649 A1). These
contamination effects with particles HIE12106R2 manifest themselves
by visible discoloration of the supernatants over time and also
increased absorption behavior. Furthermore, these reduced
contamination effects in the supernatant from the method according
to the invention indicate a closed silica layer on the
particles.
Example 5
Behavior of Aqueous Supernatants of Silica-Coated Magnetic
Particles Using RT-PCR
[0088] The aqueous supernatants of the two differently
silica-coated magnetic particles (lot designations HIE13266 and
HIE12106R2) were processed using magnetization. Particle lot
HIE13266 was produced using the inventive production method with
continuous washing in a microfiltration unit (see Examples 2 and
3). Particle lot HIE12106R2 was produced by repeated sequential
washing (see WO 03/058649 A1) based on Bayoxide E 8707. Both
supernatants were then subjected to a quantitive RT-PCR
intervention:
[0089] The so-called quantitative RT (reverse transcription)-PCR
intervention was carried out on the MX 4000 from Stratagene. As
part of this, 5 .mu.l of the supernatants of both of the particle
supernatants, and 5 .mu.l of water as a control, was added to 20
.mu.l of Mastermix. This contains the following components: 400 nM
Primer A, 400 nM Primer B, 10 ng MCF-7 RNA (Ambion), Taqman Primer
200 nM, 1.times. Buffer A, 5 mM MgCl.sub.2; 1.2 mM dNTPs, 8 U
RNaseInhibitor, 20 U MuLV Reverse Transcriptase, 1.25 U Taq Gold
(all from Applied Biosystems). The PCR program was: 30 min at
45.degree. C., 10 min at 95.degree. C., 45 cycles of 15 seconds at
96.degree. C., 60 seconds at 63.degree. C. and 30 sec at 72.degree.
C.
[0090] The preparations were placed in a 96-well microtiter plate
(Stratagene), sealed and placed in the analysis device. On
completion of the run and using device software, an individual
C.sub.1 value (number of cycle at which the selected base value
intersects the amplification curve) was assigned to each sample at
a selected basic value (fluorescence intensity) in the exponential
amplification range of the signal curves.
[0091] As can be seen from FIG. 2, the amplification curves with
supernatants of particles HIE13266 are comparable with the
amplification curves with water as a sample. On the other hand, a
shift of the amplification curves of approximately 3 C.sub.1 values
with supernatants from HIE12106R2 can be seen on the right-hand
side, which indicates interference or negative influence on the
efficiency of the RT-PCR.
* * * * *