U.S. patent application number 14/537153 was filed with the patent office on 2015-02-26 for method for manipulating magnetic particles in a liquid medium.
The applicant listed for this patent is Spinomix S.A.. Invention is credited to Amar Rida.
Application Number | 20150056611 14/537153 |
Document ID | / |
Family ID | 42266685 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150056611 |
Kind Code |
A1 |
Rida; Amar |
February 26, 2015 |
Method for Manipulating Magnetic Particles in a Liquid Medium
Abstract
A method of mixing magnetic particles (3) in a reaction chamber
(2) that is part of a microfluidic device and that contains the
said particles in suspension, comprises the steps: (a) providing an
electromagnetic means (1,1',6,7) to generate magnetic field
sequences having polarity and intensity that vary in time and a
magnetic field gradient that covers the whole space of the said
reaction chamber (2); (b) applying a first magnetic field sequence
to separate or confine the particles (3) so the particles occupy a
sub-volume in the volume of the reaction chamber (2); (c) injecting
a defined volume of the said reagent in the reaction chamber; and
(d) applying a second magnetic field sequence to leads the
particles (3) to be homogenously distributed and dynamically moving
over a substantial portion of the whole reaction chamber
volume.
Inventors: |
Rida; Amar;
(Chavannes-Renens, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Spinomix S.A. |
Lausanne |
|
CH |
|
|
Family ID: |
42266685 |
Appl. No.: |
14/537153 |
Filed: |
November 10, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12340018 |
Dec 19, 2008 |
|
|
|
14537153 |
|
|
|
|
PCT/IB2007/052409 |
Jun 21, 2007 |
|
|
|
12340018 |
|
|
|
|
PCT/IB2007/052410 |
Jun 21, 2007 |
|
|
|
PCT/IB2007/052409 |
|
|
|
|
Current U.S.
Class: |
435/5 ; 366/341;
435/30; 435/6.1 |
Current CPC
Class: |
B01F 3/1235 20130101;
B01F 13/0077 20130101; B01F 13/0809 20130101; B01F 3/1207 20130101;
B01F 2215/0037 20130101; C12N 15/1013 20130101; G01N 35/0098
20130101 |
Class at
Publication: |
435/5 ; 366/341;
435/30; 435/6.1 |
International
Class: |
B01F 13/08 20060101
B01F013/08; B01F 13/00 20060101 B01F013/00; B01F 3/12 20060101
B01F003/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2006 |
IB |
PCT/IB2006/052005 |
Nov 9, 2006 |
IB |
PCT/IB2006/054182 |
Claims
1. A method of mixing magnetic particles with a reagent in a
reaction chamber that is part of a microfluidic device and that
contains the particles in suspension, comprises the steps: a.
providing an electromagnetic means to generate magnetic field
sequences having polarity and intensity that vary in time and a
magnetic field gradient that covers the whole space of the reaction
chamber; b. applying a first magnetic field sequence to separate or
confine the particles so the particles occupy a sub-volume in the
volume of the reaction chamber; c. injecting a defined volume of
the said reagent in the reaction chamber; d. applying a second
magnetic field sequence to cause the particles to be homogenously
distributed and dynamically moving as a fog of particles occupying
a majority of the whole reaction chamber volume; e. leaving the fog
of particles in the homogenous state for a defined period of time
to allow a reaction to fake place between the particle surfaces and
the reagent injected in the said .reaction chamber, thereby forming
a complex; and f. repeating the steps (b)-(e) until a given reagent
volume has passed through the reaction chamber.
2. The method of according to claim 1, wherein the particles have a
surface coating designed to selectively bind the particle with at
least one target molecule in suspension within the reaction
chamber.
3. The method according to claim 1, wherein the reaction chamber
comprises a microchannel.
4. The method according to claim 1, wherein the reaction chamber
comprises a cavity that has an inlet port and an outlet port and at
least one segment with diverging/converging parts connected
respectively to inlet and outlet ports for delivering liquids into
end from the reaction chamber.
5. The method according to claim 1, wherein the magnetic means
comprise at least two electromagnetic poles facing each other
across the reaction chamber and electromagnetically actuatable
independently from each other.
6. The method of mixing magnetic particles according to claim 5,
wherein the said magnetic poles are geometrically arranged in a way
to be co-diverging/co-converging with diverging/converging parts of
the cavity.
7. The method according to claim 1 wherein the time-varied magnetic
sequence has a substantially rectangular, sinusoidal, saw-tooth,
asymmetrical triangular, or symmetric triangular form; or any
combination of said forms.
8. The method according to claim 7, wherein the oscillation
frequency of the magnetic field is between 0.1 to 1000 cycles per
second.
9. The method according to claim 1, wherein during the step (b) the
particles are separated or confined at the outer border of the
reaction chamber.
10. The method according to claim 1, wherein during the step (c)
the injected reagent volume is equal to or lower than the reaction
chamber volume.
11. The method according to claim 1, which further comprises the
steps of: g. applying a further magnetic field sequence to separate
or to confine the complex in a specific area of the reaction
chamber; h. evacuating the reagent from the chamber; i. injecting
another reagent info chamber; and j. repeating the steps (b)-(e) of
claim 1 until a given reagent volume has passed through the
reaction chamber.
12. A method to extract target molecule(s) that enter in a
composition of intracellular complexes in a sample volume, said
method comprises: a. providing a reaction chamber that is part of a
microfluidic device and that contains a first type of magnetic
particles in suspension; wherein the first type of particles have a
surface coating designed to selectively bind with the target
molecules; b. providing an electromagnetic, means to generate
magnetic field sequences having polarity and Intensify that vary in
time and a magnetic field gradient that covers the whole space of
the reaction chamber; c. applying a first magnetic field sequence
to separate or to confine the first type of particles so the
particles occupy a sub-volume in the volume of the reaction
chamber; d. injecting in the reaction chamber a defined volume of
the said sample, wherein the cells were previously bound to a
second type of magnetic particles; e. applying a second magnetic
field sequence to cause the first type of particles to be
homogenously distributed and dynamically moving as a fog of first
particles over a substantial portion of the whole reaction chamber
volume; f. leaving the fog of the first type of particles in the
homogenous state for a defined period of time to allow strong
contact between the surfaces of the first type of particles and the
said magnetically labelled cells injected in the reaction chamber,
thereby forming by means of dipolar interaction a complex composed
of the first type of particles and the magnetically labelled cells;
g. applying a further magnetic field sequence to separate or to
confine the complex in a specific area of the reaction chamber; h.
repeating the steps (c)-(g) until a given sample sub-volume is
passed through the reaction chamber; and i. lysing the complex to
release the target molecules in the reaction chamber to capture the
target molecules on the first particles types surfaces.
13. The method according to claim 12, wherein the said target
molecules are selected from nucleic acids, proteins and
peptides.
14. The method according to claim 12, wherein the said cells are
selected from viruses bacterial cells, human cells, animal cells
and plant cells.
15. The method according to claim 12, which further comprises the
stop of washing the target molecules captured on the particles from
a residual liquid medium.
16. The method according to claim 12, which further comprises the
step of eluting the captured molecules from the surfaces of the
particles.
17. The method according to claim 12, which further comprises the
step of detecting the target molecules.
18. The method according to claim 1, wherein the microfluidic
device further comprises a plurality of reagent sources fluidly
connected to said reaction chamber; and a computer controller for
controlling reagent flow and application of the magnetic field
sequences.
19. The method according to claim 1, which further comprises the
steps of: (k) applying magnetic field sequences having polarity and
intensity that vary in time to disperse the magnetic particles;
said varying magnetic field sequences being effective to break
particle claim aggregates and inhibit the formation of particle
claim aggregates and to maintain the particles in suspension as a
fog of particles in relative dynamic motion; and (l) combining
different magnetic field sequences to induce displacement of the
fog of particles across the reaction chamber whereby the fog of
particles occupies substantially the whole reaction chamber
volume.
20. The method according to claim 7, wherein during the step (b)
the particle size and the homogeneity of mixing the particles are
controlled by varying respectively the frequency and the amplitude
of the magnetic field.
21. The method according to claim 19, wherein the fog of particles
occupies substantially the whole reaction chamber volume
quasi-instantaneously.
22. The method according to claim 19, wherein the fog of particles
occupies substantially the whole reaction chamber volume over a
period of time.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of International Application
No. PCT/IB2007/052409 filed on 21 Jun. 2007, International
Application No. PCT/IB2007/052410 filed on 21 Jun. 2007,
International Application No. PCT/IB2006/052005 filed on 21 Jun.
2006, and International Application No. PCT/IB2006/054182 filed on
9 Nov. 2006, which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to a method of handling and mixing
magnetic particles within a reaction chamber that is a part of a
fluidic or microfluidic platform. More particularly, the invention
concerns a method of handling magnetic particles in a way to
improve the mixing of the particles with the surrounding liquids
medium and where the liquids are automatically handled in a fluidic
platform. Further, the invention relates to a method for conducting
assays on a test sample containing specific biological or chemical
substances using active biochemically surface magnetic particles
and where the particles are handled following the foregoing
method.
DESCRIPTION OF RELATED ARTS
[0003] Nowadays, magnetic particle (bead) is a standard technology
in biochemical assays and diagnostics. Magnetic particle technology
is indeed a robust technology that allows achieving high
performances (sensitivity and accuracy) and also opens the
possibility of easy automation of assay protocols. For many
applications, the surface of magnetic particles is coated with a
suitable ligand or receptor, such as antibodies, lectins,
oligonucleotides, or other bioreactive molecules, which can
selectively bind a target substance in a mixture with other
substances. Examples of small magnetic particles or beads are
disclosed in U.S. Pat. No. 4,230,685, U.S. Pat. No. 4,554,088 and
U.S. Pat. No. 4,628,037.
[0004] One key element in magnetic particles bio-separation and
handling technology is an efficient mixing to enhance the reaction
rate between the target substances and the particle surfaces.
Indeed, as for any surface-based assay the reaction is strongly
limited by the natural diffusion process, a strong steering and
mixing is necessary to promote the affinity binding reaction
between the ligand and the target substance.
[0005] A typical example of magnetic particles mixing apparatus in
test medium is disclosed in U.S. Pat. No. 6,231,760 and
commercially available by Sigris Research Inc under the name of
MIXSEP.TM. system. In this patent and system, the test medium with
the magnetic particles in a suitable container are placed in a
magnetic field gradient generated by an external magnet. The mixing
concept is based on either the movement of a magnet relative to a
stationary container or movement of the container relative to a
stationary magnet using mechanical means, therefore inducing a
"relative displacement" of the magnetic gradient position within
the container. This magnetic field gradient displacement will in
turn induce the magnetic particles to move continuously with the
change of the magnet (magnetic field gradient) position, thereby
effecting mixing. However, with this method the magnetic field
gradient will attract and confine the particles in a cavity region
close to the walls of the container. In such condition, the contact
between the particles and the test medium is limited to the said
cavity space which reduces the mixing efficiency. Although the
"mechanical movement" of magnets is claimed as a mixing means, also
described is the possibility of producing angular movement of the
particles by sequential actuation of electromagnets disposed around
the container. However, while electromagnets provide a much lower
magnetic field when compared with permanent magnets, as described
the magnetic coupling between adjacent electromagnets strongly
repel the magnetic flux outside the container resulting in a
further reduction of the magnetic field intensity and
intensification of the cavity effect. Under such condition, the
particles agitation (movement) and mixing will be strongly altered
leading the particles to slowly move, mostly, as aggregates at the
region close the walls border.
[0006] Within the same spirit, in the U.S. Pat. No. 6,764,859 a
method of mixing magnetic particles in a container is disclosed
based on relative "mechanical" movement between the container and
intervening array geometry of magnet. In such configuration the
adjacent magnets have opposite polarity which induces a change of
magnetic field polarity during the relative intervening movement
between the container and two adjacent magnets. In such conditions
indeed, the particles can be moved while relatively separated from
each other, which will potentially affect the mixing. However, in
this approach when one takes in consideration the whole duration of
the particles handling process, the time during which the particles
are relatively separated from each other is relatively short. As a
consequence, several mixing cycles are necessary to assure
effective mixing. Moreover, during the mixing process the particles
are not homogenously contacted with the sample volume in the test
tube, which will in turn strongly limit the mixing efficiency. This
issue is more pronounced as the sample volume is large.
[0007] Consequently when these mechanical mixing approaches are
compared with the "manual shaking of a test tube", the reaction
time and the performance are substantially similar if not lower,
indicating that diffusion is still an important limiting
factor.
[0008] Other aspects for magnetic particles separation and
resuspending are disclosed in E.P, Pat 0,504,192. This patent
discloses the use of sequential actuation of two magnetic field
sources (electromagnets) disposed opposite to each other at the
walls of a chamber. The proposed actuation concept of the said
electromagnets is based on sequential energizing (actuation) of the
electromagnets by "binary" (i.e., on and off) or "analog" in which
a first electromagnet is gradually fully energized, and then has
its power reduced, while the next electromagnet is gradually
energized, and so on. Through this actuation the particles will be
moved and drawn to the reaction chamber volume and thereby
resuspended. While the concept of using (at least) two
electromagnets with "sequential" actuation is conceptually an
evident manner for particles resuspension from an aggregate, during
their "movement" the particles remain mostly agglomerated due to
their dipolar interaction under the applied magnetic field. The
only way, after moving the "super-paramagnetic" particles to occupy
the chamber volume, to fully assure "homogenous" resuspension in
the chamber is to completely remove the external magnetic field and
leave the desegregation to Brownian and thermal agitations.
Additionally, the application discloses that alternately energizing
and de-energizing the two electromagnets at a sufficiently rapid
rate keeps the particles suspended in the center of the chamber.
This process limits the movement of the particles to a relatively
small distance, significantly reducing the mixing efficiency
between particles and the surrounding liquid medium.
[0009] In general, beyond the limited mixing capability of the
state of the art magnetic particles technologies, mainly based on
the concept of "bringing a magnet in the proximity of a test tube",
the integration and the automation of magnetic particles assay
procedures are very complex, necessitating bulky robotic systems.
These limitations become all the more critical as the assay
procedures are becoming more and more complex.
[0010] Microfluidics based technology is nowadays perceived as an
emerging technology with a great potential that can lead to easier
integration of complex bio-chemical assay procedures in an
easy-to-use and miniaturized automated system. Combining magnetic
particles technology with microfluidics Will certainly be of great
importance as the precise control of different reagents (allowed by
microfluidics) and handling of biological species and their
reactions (allowed by magnetic particles) will be integrated
together within a single system.
[0011] One approach of mixing magnetic particles in a microfluidics
channel is taught in the publication "Magnetic Force Driven Chaotic
Micro-Mixer", by Suzuki, H, in the proceedings of The Fifteenth
IEEE International Conference on Micro Electro Mechanical Systems,
2002. The approach consists in flow mixing of magnetic particles
injected in suspension in a microfluidic channel and where the
mixing with the surrounding medium is assured by a magnetic field
generated by embedded micro-electromagnets along the flow path. The
combination of the magnetic force induced by the
micro-electromagnets on the particles along with the flow driving
force in the microchannel induces a chaotic regime and thereby
mixing. A similar concept has been recently disclosed in U.S.
patent application number 2006/140,051 where the magnetic field is
generated by electromagnets disposed on the sidewalls in a
predetermined direction with respect to the direction of the flow.
By turning off/on the electromagnets in sequential operation, a
rotating magnetic force can be created leading to mixing of the
particles carried by the flow. The major limitation of this "in
flow mixing" approach is that the volume of the test medium that
can be mixed with the particles is very small and the reaction time
very short, limiting considerably the cases of its
applicability.
[0012] To overcome the limitations of the "in-flow mixing
approach", a solution consists in retaining the particles in a
given location of a fluidic channel or chamber using a magnetic
field gradient while the test medium is injected with a flow
through the retained magnetic particles. This approach has been
disclosed in the U.S. patent applications number 2005/032,051 and
2004/166,547 where the particles retained in a flow microchannel
have been used as a solid support for immunoassay procedures. Along
the same lines, a flow-through concept applied for DNA
hybridization and detection assay is described in the publication:
"Dynamic DNA hybridization on a Chip Using Paramagnetic Beads", by
Z. Hugh Fan & al., Analytical Chemistry, 71, 1999. However the
so described flow-through approach suffers from a serious physical
constraint since in order to be handled in an environment with
continuous fluidic processing, the particles must be continuously
exposed to a magnetic field. Under such conditions the particles
will stick together aid agglomerate thereby losing their main
advantage: the particle surface that is in active contact with the
fluid flow will be drastically reduced which will seriously
compromise the assay performance.
[0013] A solution to the agglomeration problem of magnetic
particles in the flow-through approach has been disclosed U.S.
patent application 2005/208,464. In this approach, the particles
are retained in a portion of a flow-channel to form a kind of a
filter that substantially homogenously covers the flow-channel
cross section. To obtain this filter, the magnetic particles are
manipulated using a time-varying field (amplitude, frequency and
polarity) to control the particles agglomeration. The efficiency of
this approach for microfluidic mixing of liquids has been
demonstrated in a publication from the same author group
"Manipulation of Self-Assembled Structures of Magnetic Beads for
Microfluidic Mixing and Assaying", by A. Rida & al. Analytical
Chemistry, 76, 2004. Even demonstrating an important development in
magnetic particles handling and mixing in a microfluidic
environment, the approach disclosed in U.S. patent application
2005/208,464 suffers however from many practical limiting
constraints. First, as the particles are kept stationary and fixed
in a narrow segment of the flow-through cell, the contact between
the particles and the target substance is limited to that narrow
region and for a very short time, which in practice makes such
process difficult to set up. Secondly, this approach is
specifically adapted for handling and mixing magnetic particles
under flow-through conditions in microfluidics environments, which
make it not fully adapted for different assay conditions.
[0014] The applicable known procedures and approaches have
shortcomings, including the requirement for handling and mixing
magnetic particles in various environments with more focus on
microfluidics, as well various process constraints, limiting
factors and inefficiencies.
SUMMARY OF THE INVENTION
[0015] The present invention provides devices and methods for
handling and efficiently mixing magnetic particles in microfluidics
environments. "Mixing" in the present context means in particular
contacting in a very efficient manner large particles surfaces with
the surrounding liquid medium, in such a way as to achieve: (1) an
effective binding of the particles to a certain target molecule(s)
and (2) further possibilities to wash, separate, elute and detect
the targets captured on the particles from the residual liquid
medium.
[0016] The proposed mixing mechanism provides a considerable and
perpetual increase of the active surface of particles per unit of
volume leading to an enhanced contact between the large surface of
particles and the target substances. Further, the proposed magnetic
particles handling process advantageously assures a homogenous
mixing covering substantially the whole reaction volume in a
fraction of time allowing thereby much more sample volume to be
effectively and rapidly contacted with the particle surfaces.
Moreover, during their manipulation the particles are in perpetual
effective movement covering the whole reaction chamber volume,
which is a key in enhancing particles mixing.
[0017] Further, the invention provides new devices and methods that
practically allow the integration of complex assay procedures in a
compact and easy to use system that can operate under flow-through
or, advantageously, under non flow-through conditions.
[0018] Accordingly, a main aspect of the invention concerns a
method for manipulating and mixing magnetic particle in
microfluidics environments, attained according to independent claim
1.
[0019] Accordingly, a main aspect of the invention concerns a
method for manipulating and mixing magnetic particle in
microfluidics to extract target molecule(s) that enter in the
composition of intracellular complexes in a sample volume, attained
according to independent claim 12.
[0020] Different embodiments are set out in the dependent
claims.
[0021] According to one embodiment of the present invention, a
method for reagents processing and mixing magnetic particles with
the said reagents in a reaction chamber that is part of
microfluidic device and that contains the said particles in
suspension, comprises the steps: [0022] a. providing an
electromagnetic means to generate magnetic field sequences having
polarity and intensity that vary in time and a magnetic field
gradient that covers the whole space of the said reaction chamber;
[0023] b. applying a first magnetic field sequence to separate or
confine the particles so the particles occupy a sub-volume in the
volume of the reaction chamber; [0024] c. injecting a defined
volume of the said reagent in the reaction chamber; [0025] d.
applying a second magnetic field sequence to lead the particles to
be homogenously distributed and dynamically moving as a fog of
particles occupying a substantial portion of the whole reaction
chamber volume; [0026] e. leaving the fog of particles in the
homogenous state for a defined period of time to allow a reaction
to take place between the particle surfaces and the reagent
injected in the said reaction chamber; and [0027] f. in the case of
a large reagent volume, repeating the steps (b)-(e) until a given
reagent volume has passed through the reaction chamber.
[0028] Another key aspect of the present invention concerns the
magnetic poles actuation mechanism which consists of: [0029] 1.
applying from the electromagnetic poles magnetic field sequences
having polarity and intensity that vary in time; said varying
magnetic field sequences being effective to break or inhibit
particle claim aggregates and to maintain the particles in
suspension as a fog of particles in relative dynamic motion; and
[0030] 2. combining the magnetic fields from different magnetic
poles in a sequence to induce displacement of the fog of particles
across the reaction chamber whereby the fog of particles occupies
substantially the whole reaction chamber volume
quasi-instantaneously or over a period of time.
[0031] Accordingly, the present invention concerns a method of
mixing magnetic particles in a microfluidic environment with
surrounding medium in a reaction chamber that is a part a
microfluidic network, wherein at least a couple of electromagnetic
poles face each other across the reaction chamber, arranged to
provide a magnetic field gradient over the whole volume of the said
reaction chamber. The key element of the invention is related to
the magnetic poles actuation mechanism which is based on the
application in each electromagnetic pole of magnetic field
sequences having polarity and intensity that vary in time. It has
been found that such magnetic poles actuation mechanism leads to
continuous time variations of the position (displacement) of the
magnetic field gradient maxima across the reaction chamber volume,
leading thereby the particles to be in perpetual relative
translational and rotational motion that can substantially cover
the whole reaction chamber volume.
[0032] Additionally, the desired effect obtained by the actuation
mechanism according to the invention is that during their motion
the particles do not displace as a compact aggregate but they are
rather moving in as a fog of particles resulting in a strong
enhancement of the contact between the particles surfaces and the
surrounding liquid medium.
[0033] Additionally, the desired effect obtained by the actuation
mechanism according to the invention is that the particles mixing
will cover substantially the whole reaction chamber volume and not
be limited to a narrow segment as in the disclosed prior art
concepts. This magnetic particles handling process advantageously
assures therefore a homogenous mixing allowing much more liquid
volume to be effectively contacted with the particle surfaces.
[0034] Additionally, the desired effect obtained by the actuation
mechanism according to the invention is the possibility of
selecting the magnetic field sequence to not only homogenously mix
the particles but also separate or confine the particles so the
particles occupy a sub-volume in the volume of the reaction chamber
as the outer borders of the reaction chamber. For instance one can
apply a first magnetic field sequence to homogenously displace and
therefore mix the particles in substantially the whole reaction
chamber volume; and then apply a second magnetic field sequence
that specifically selects the direction of the magnetic field
gradient leading the particles to be drawn to a sub-volume of the
reaction chamber determined by the direction of the said applied
magnetic field gradient. This flexibility in controlling the
particles is advantageously important as it allows to handle and
control the particles state in correspondence with the assay
process.
[0035] Another feature of the invention is that during their
perpetual motion (movement) the size of the particles aggregates
can be mainly controlled by the frequency of the magnetic field
polarity oscillations while the homogeneity of mixing is controlled
by the magnetic field amplitude. Accordingly, the magnetic field
(gradient) amplitude can be used as a switching parameter between,
for instance, a homogenous mixing state over the reaction chamber
volume and a separated state at the outer border of the reaction
chamber.
[0036] Additionally, the desired effect obtained by the device and
the actuation mechanism according to the invention, is the
extremely fast manipulation of the particles. For instance,
starting from a configuration where the particles are first
separated to the outer border of the reaction chamber using a
specific first actuation sequence, and a fraction of time (a second
or less) is sufficient to put the particles in a homogenous mixing
configuration using a second actuation sequence. The particles can
afterwards again be drawn in a fraction of time to the outer border
of the reaction chamber by applying the first actuation sequence.
This rapid manipulation process can be even reached in a complex
high viscous medium like blood lysate.
[0037] According to the previously described aspects and effects of
handling magnetic particles, the inventive method further includes
the steps of conducting an assay wherein: (1) the particles are
separated or confined in a sub-volume of the reaction chamber as
the reaction chamber external borders using a first magnetic poles
actuation sequence; (2) followed by the injection of a defined
volume of an assaying reagent that preferably will not exceed the
reaction chamber volume; (3) as the liquid flow is stopped, the
particles will be mixed to be substantially homogenously
distributed over the reaction chamber volume using an appropriate
actuation sequence; (4) after mixing for a defined time period to
allow the desired reaction between the particles surfaces and the
injected assay reagent to take place, the particles will be
attracted again to the reaction chamber walls and the new sample
volume injected to the reaction and then mixed; and (5) this
process will be repeated in sequential way until a defined volume
of the assay reagent is mixed with the magnetic particles.
[0038] One of the advantages of such "separation/injection/mixing"
mode is the assay process, performed in "discontinues batches" of a
volume equal to the reaction chamber volume. The small scale of the
reaction volume along with the rapid and effective mixing and
separating the magnetic particles according to the invention, allow
for an efficient and rapid conduction of the assay batches and
thereby the overall assay process.
[0039] To achieve the previously described method and the related
effects, the reaction chamber is preferably a cavity that has an
inlet port and an outlet port and at least one segment with
diverging/converging parts connected respectively to inlet and
outlet port for delivering liquids into and from the reaction
chamber. Moreover, the said reaction chamber can be inserted in the
air gap of electromagnetic poles that are geometrically arranged in
a way to be co-diverging/co-converging with diverging/converging
parts of the reaction chamber. The said electromagnets are arranged
to provide a magnetic field gradient over the whole volume of the
said reaction chamber
[0040] To achieve the previously described method and the related
effects, preferably the magnetic poles are each electromagnetically
actuatable independently from each other and wherein each couple of
magnetic poles form a closed magnetic circuit with a magnetic gap
in which the said reaction chamber is located.
[0041] To reach the desired effects, preferably the magnetic
particles are initially unmagnetized magnetic particles that
develop a specific ferromagnetic hysteresis response to an external
magnetic field. More specifically, the particles have a coercive
field between 200 to 1000 Oe.
[0042] To reach the desired effects, the time varied magnetic field
sequences can have a substantially rectangular, sinusoidal,
saw-tooth, asymmetrical triangular, or symmetric triangular form;
or any combination of such forms, with a frequency that is
preferably greater than 1 cycle per second and a maximum amplitude
that is lower than the coercive field of the magnetic particles in
use.
[0043] According to one embodiment of the present invention and
based on all of the previous aspects of mixing magnetic particles,
a method to extract target molecule(s) that enter in the
composition of intracellular complexes in a sample volume
comprises: [0044] a. providing a reaction chamber that is part of a
microfluidic device and that contains a first magnetic particles
type in suspension; wherein the said first type particles have a
surface coating designed to selectively bind with the said target
molecules; [0045] b. providing an electromagnetic means to generate
magnetic field sequences having polarity and intensity that vary in
time and a magnetic field gradient that covers the whole space of
the said reaction chamber; [0046] c. applying a first magnetic
field sequence to separate or to confine the said first particles
type so the particles occupy a sub-volume in the volume of the
reaction chamber; [0047] d. injecting in the reaction chamber a
defined volume of the said sample, wherein the cells are previously
labelled with a second type of magnetic particles; [0048] e.
applying a second magnetic field sequence to lead the said first
particles type to be homogenously distributed and dynamically
moving as a fog of first particles that occupies a substantial
portion of the whole reaction chamber volume; [0049] f. leaving the
fog of first particles type in the homogenous state for a defined
period of time to allow strong contact between the first particle
type surfaces and the said magnetically labelled cells injected in
the reaction chamber, thereby forming by means of dipolar
interaction a complex composed from the first particles type and
magnetically labelled cells; [0050] g. applying a further magnetic
field sequence to separate or to confine the said complex in a
specific area of the reaction chamber; [0051] h. in the case of a
large sample volume, repeating the steps (c)-(g) until a given
sample sub-volume is passed through the reaction chamber; and
[0052] i. lysing the so separated cells to release the said target
molecules in the reaction chamber in conditions that allows
specific capturing of the target molecules on the first particles
types surfaces.
[0053] Accordingly, the disclosed method of extracting a target
molecule(s) such as nucleic acids or proteins that enter in the
composition of cells such as bacteria or cancer cells in a sample
volume, allows to combine complex analytical procedures as cells
selection and separation followed by the cells lysis and target
molecules purification, in one reaction chamber.
[0054] In practice, this method consists first of the cell
selection using a first type of magnetic particles having a surface
coating designed to allow affinity recognition of the said cells.
The so-labelled cells are then magnetically separated in a
microfluidic chamber where second magnetic particles type are
manipulated according the inventive method of mixing magnetic
particles. The separation process is performed following the
"separation/injection/mixing" mode, wherein the separation process
is allowed by the efficient mixing to strongly contact the
magnetically labeled cells and the second magnetic particles type.
One aspect, of the cell separation method, indeed, is the formation
of a complex composed from the first particles type and
magnetically labelled cells by means of dipolar interaction during
the mixing process of the first particle type. As this complex is
formed, the separation of the magnetically labelled cells from the
supernatant can be easily performed during the separation of the
first magnetic particles type. Regarding a final objective of the
invention, the particles in use have a surface coating designed to
allow affinity recognition with at least one target molecule or
reaction with the surrounding liquid medium within the reaction
chamber. The said target molecules or reagents are carried in the
reaction chamber by a liquid flow. When combined together all
aspects of the current invention allow the processing with enhanced
performance of complex bio-chemical, synthesis and analysis
procedures using magnetic particles as a solid support. Typical
examples but without limitation of such procedures are
enzymes-linked assays, proteins and nucleic acids extractions, or
detection methods based on enzymatic signal amplification
methodologies such as chemioluminescence, NASBA, TMA or PCR.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] The objects and features of the present invention are set
forth with particularity in the appended claims. The present
invention, both as to its organization and manner of operation,
together with further objects and advantages, may best be
understood by reference to the following description, taken in
connection with the accompanying drawings, wherein
[0056] FIGS. 1(a) and (b) are schematic representations of the
concept of flow-through magnetic particle handling approaches as
described in the prior art.
[0057] FIG. 2(a) shows a schematic view of one preferred embodiment
of the invention which includes one couple of "diverging" magnetic
poles facing each other across a gap and a reaction chamber
(channel) placed in this gap. FIG. 2(b) is a cross-sectional view
of FIG. 2(a), showing in particular the electromagnetic circuit
that provides the magnetic field in the reaction chamber.
[0058] FIG. 3(a) shows a schematic representation of another
preferred embodiment of the invention which includes in particular
one couple of "diverging" magnetic poles and a reaction chamber
that has a diverging cavity arranged co-divergently with the gap
geometry. FIG. 3(b) shows the magnetic field variation profile
along the axis of the said magnetic poles.
[0059] FIGS. 4(a) and (b) show the flow velocity profile and
variation induced by the diverging reaction chamber geometry.
[0060] FIGS. (5)(a) and (b) are schematic representations of
magnetic particle handling and mixing according to a preferred
embodiment of the invention which includes in particular a change
in the direction of the polarity of the magnetic poles, whose
induced magnetic field has the effect of axially moving the
particles.
[0061] FIG. 6 shows a schematic view of yet another preferred
embodiment of the invention which includes a quadrupole
configuration of magnetic poles, co-diverging/co-converging with
the reaction chamber cavity.
[0062] FIGS. 7(a) to (d) schematically represent, for the preferred
embodiment of FIG. 6, the relative position and motion of the
particles across the reaction chamber volume as a consequence of
the actuation sequences of the electromagnetic poles using a
magnetic field having a polarity and amplitude that vary with
time.
[0063] FIG. 8 schematically represents the desired effect obtained
with the quadrupole embodiment according to the invention where the
particles mixing and movement homogenously cover the whole reaction
chamber volume.
[0064] FIGS. 9(a) and (b) show a perspective view of the
electromagnetic circuit according to a preferred embodiment of the
invention.
[0065] FIG. 10 show a layout of a microfluidic chip according to a
preferred embodiment of the invention.
[0066] FIGS. 11(a) to (c) schematically represent a process of
using the inventive mixing method and device for performing an
assay in general and an immunoassay in particular.
[0067] FIG. 12 schematically illustrates another embodiment of
handling and mixing magnetic particles with the surrounding medium
in a "pulsed-injection/mixing" mode.
[0068] FIG. 13 illustrates different behavior of the magnetic
particles under a rotating magnetic field.
[0069] FIG. 14 schematically represents another configuration of
the magnetic poles and their operation to obtain the desired
effects according to the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0070] The main attainable effect of the present invention is an
effective control of the magnetic particles that allows an enhanced
and homogenous mixing with the surrounding medium. In particular,
the mixing of the magnetic particles is realized in a reaction
chamber that is a part of a microfluidic network and where the
particles are handled using external magnetic poles with specific
configurations and geometries. Accordingly, the different reagents
are introduced to the reaction chamber using liquid flows and the
magnetic poles are specifically actuated to control the magnetic
particles in use inside the reaction chamber.
[0071] Another main attainable objective of the present invention
is a method of biological liquid samples and reagents processing in
a microfluidic environment in combination with their mixing with
magnetic particles.
[0072] In general, the microfluidic environment of the invention
concerns devices typically designed on a scale suitable to analyze
micro-volumes preferably in the range 0.1 ml to 500 .mu.l. However,
in one of major application of the invention large samples are used
to concentrate specific biomolecules in the device to a small
volume for subsequent analysis. The microscale flow channels and
wells have preferred depths and widths on the order of 0.05-1 mm.
The "reaction chamber" that is part of a microfluidic network as
used herein refers to chambers with a cavity that have a volume in
the range of 0.1 ml to 500 ml and preferably in the range 10 ml to
100 ml. However, for many applications, larger "mesoscale"
dimensions on the scale of millimetres may be used. Similarly,
chambers in the substrates often will have larger dimensions than
the microchannels, on the scale of 1-10 mm width and 1-5 mm
depth.
[0073] To illustrate the key advantage of the present invention,
FIG. 1 schematically shows the concept of a flow-through magnetic
particles handling approach as described in the prior art. In the
prior art indeed, the particles (3) are kept fixed in a narrow
region of a flow-through cell (2) delimited by magnetic pole tips
(1) and (1'). The particles can partially cover the flow-through
cell cross section (FIG. 1(a)) when a static field is applied as
described in U.S. patent application 2004/166,547, or homogenously
cover the flow cell cross section (FIG. 1(b)) when a time varied
magnetic field is applied as described in U.S. patent application
2005/208,464. The pole tip configurations used to generate a
magnetic field gradient confine the particles in a stationary fixed
and narrow segment of the flow-through cell, which limits the
contact between the particles and the target molecules to that
narrow region and for a very short time.
[0074] To overcome the limitations of the prior art, a new magnetic
device and magnetic pole geometry is disclosed. Accordingly, as
shown in FIG. 2, a device for manipulating and mixing magnetic
particles in a surrounding liquid medium, comprises: (i) at least
one couple of magnetic poles (1)-(1') facing each other across a
gap, the facing poles diverging from a narrow end of the gap to a
large end of the gap. The poles form part of an electromagnetic
circuit and are arranged to provide a magnetic field gradient (5)
in the gap region. In this gap region is placed a tubular reaction
chamber (2) that is a part of a fluidic network and in which the
magnetic particles in use will be manipulated. The magnetic circuit
is composed of a magnetic core (6) and coils (7) that when supplied
with an electric current produce a magnetic field in the gap region
through the magnetic poles (1), (1'). Moreover, each magnetic pole
(1) and (1') is preferably electromagnetically actuatable
independently from each other using two independently actuatable
coils (7).
[0075] The effect obtained by the described magnetic pole geometry
is that the magnetic field gradient will not be limited to a narrow
region but will cover the whole space region extending along the
axial X direction in the said poles air-gap.
[0076] To enhance the mixing effect, preferably the reaction
chamber (2) placed in the air gap region has a cavity shape that
varies in the same direction as the geometry of the magnetic poles.
As schematically represented in FIG. 3(a), rather than having a
flow channel with a uniform geometry (uniform cross-section) as in
FIG. 2(a), the reaction chamber (2) has preferably a variable
geometry that is substantially co-diverging in the diverging gap
between the poles. With such variation of the reaction chamber
geometry one will induce a transverse velocity gradient (8) leading
to more effective flow mixing (see FIG. 4).
[0077] In operation, the space-varied magnetic field generated by
the magnetic poles (1)-(1') provides a magnetic field gradient and
thus a magnetic force (5) along the X direction that will be used
to retain the magnetic particles (9) during the flow of a fluid in
the reaction chamber (2) (see FIG. 5). In order to be able to
retain the particles (9) in the reaction chamber (2), the generated
magnetic force (5) acting on the particles must be greater than the
flow drag force which tends to drive the particles away. Moreover,
since the magnetic force (5) and the flow drag force decrease in
the same way along the X direction, it will be possible to control
the generated magnetic force (5) so that it is substantially equal
to the flow drag force. When introduced in the reaction chamber (2)
and subjected to a static magnetic field (5), the magnetic
particles (9) tend to form magnetic chains along the magnetic field
flow line. Due to the magnetic field gradient generated in the
reaction chamber (2), the magnetic particle chains will coalesce to
form a strongly aggregated chain-like structure. Preferably the
amount of magnetic particles (9) used is such that the magnetic
aggregated structure mostly located near the magnetic poles in the
conical part of the reaction chamber (2), as shown in the left of
FIG. 5(a). A time varied magnetic field as an alternating magnetic
field is then applied to break down the aggregated chain-like
structures with the fluid flow through such magnetic particle
structures at a predefined flow rate (i.e. slightly increased, as
necessary). A low aggregated magnetic structure is obtained, as
illustrated in the right of FIG. 5, and controlled by adjusting the
magnetic field amplitude and frequency, the magnetic field gradient
provided by the pole geometry, and the fluid flow rate in the
reaction chamber (2).
[0078] The desired effect obtained by the so-described reaction
chamber/magnetic pole geometries is that the magnetic field
gradient variation profile corresponds to the same variation
profile as for the flow velocity gradient in the reaction chamber
(as shown in FIG. 3(b) and FIG. 4(b)). Such "co-variation" of the
flow velocity/magnetic field gradients (forces) allows to reach
more homogeneity in the mixing conditions (and therefore more
controlled and efficient mixing) of the magnetic particles with a
liquid flow.
[0079] The geometrical parameter of the device of FIGS. 2 to 5
according the invention, must be set in a way to reduce the
magnetic losses and assure a maximum focus of the magnetic flux in
the reaction chamber (2). Moreover, the adjustment of these
dimensions must be performed in a way that the generated magnetic
field gradient covers the whole reaction chamber and minimizes the
existence of zones inside the reaction chamber with a vanishing
magnetic field gradient. In this perspective, the ratio between the
depth (H) of the large end to the length (L) of the diverging part
of the reaction chamber is between 0.1 to 10 and preferably between
0.1 to 1. Typical values of the length (L) of the diverging parts
are between 50 .mu.m and 10 mm, preferably between 100 .mu.m and 5
mm. The dimensions of the microfluidic channel connected to the
narrow end of the reaction chamber are in the range of 50 .mu.m to
5 mm and preferably between 100 .mu.m and 1 mm.
[0080] Accordingly, a key aspect of the present invention concerns
the magnetic poles actuation mechanism which is based on the
application in each electromagnetic pole of magnetic field
sequences having polarity and intensity that vary in time.
[0081] A typical example of this actuation aspect according to the
invention is illustrated in FIG. 5. As schematically shown in FIG.
5(a), a "parallel oscillating" magnetic field (4)-(4') is applied
to the magnetic poles:
Poles 1 and 1': B=B.sub.0 sin(2.pi.f.sub.1t) (1)
[0082] under such conditions and due to the perpetual change in the
field polarity, the magnetic agglomeration (chains) will break down
to smaller particles chain-like structures with a size that
decreases with the field frequency (f.sub.1). Ultimately, the
particles will behave like a fog of particles in relative dynamic
motion. Another important phenomenon characterizing the use of
"oscillating" magnetic field, is the generation of negative dipolar
interaction between the particles (due to the fact that the
particles will not rotate at the same rate) that contribute further
in the particles agglomeration break-up. For instance, contrary to
the case of a static field where the particles will be mostly
attracted as an aggregated mass toward the magnetic poles (as shown
in FIG. 1(a)), in an oscillating magnetic field the particles
(while rotating) tend to be homogenously distributed over the
reaction chamber cross section (as shown in FIG. 5(a)). In other
words, under an oscillating magnetic field the particles will tend
to occupy a larger space due the development of repulsive magnetic
forces between the particles.
[0083] In summary, the use of a magnetic field that has a polarity
and amplitude that vary in time as a base actuation of the magnetic
poles according to the invention allows for an effective breaking
or inhibiting of particle aggregates and tends to maintain the
particles in suspension as a fog of particles in relative dynamic
motion.
[0084] However, as the manipulation of magnetic particles
necessitates the use of magnetic (force) gradient (5), the
particles will be attracted to the narrow segment of the reaction
chamber, which will confine and therefore tend to agglomerate the
particles. This agglomeration can be reduced by reducing the
applied field amplitude (B.sub.0) and thereby the magnetic force
gradient. If in fact, one reduces the force by reducing applied
field amplitude (B.sub.0), one observes that the "rotating"
particles structure (9) will expand radically along the X direction
due to the repulsive magnetic forces between the particles induced
by their relative rotation.
[0085] To overcome further agglomeration induced by the magnetic
field gradient, according to the invention as shown in FIG. 5(b),
the polarity of the magnetic field polarity (4)-(4') generated from
each of magnetic poles (1) and (1') is changed from parallel to
opposite (anti-parallel):
Pole 1: B=B.sub.0 sin(2.pi.f.sub.1t+.pi.)
Pole 1': B=B.sub.0 sin(2.pi.f.sub.1t) (2)
to cause a change in the direction of the magnetic force (5), which
will move the particles (9) axially in the X direction, following
the direction of the magnetic force (5), from the narrow segment to
the large segment of the reaction chamber.
[0086] Accordingly, continuous "switching" between the two
actuation schemes of the magnetic poles defined by equations (1)
and (2) leads to continuous time variations of the position of the
magnetic field gradient maxima from the narrow to the large
segments of the reaction chamber. These magnetic field gradient
maxima changes will in turn lead the particles to be in perpetual
axial movement between the narrow and the large segments of the
reaction chamber following the magnetic field gradient (5)
variations.
[0087] Accordingly, the actuation mechanism according to the
invention is based on the finding that by appropriate choice of the
switch frequency (f.sub.2) between the actuation scheme of the
magnetic poles defined by equation (1) and (2), one can reach a
state where the particles will substantially homogenously cover the
whole reaction volume, as schematically shown in FIG. 5(c).
[0088] The so described actuation mechanism, leads the particles to
be in perpetual relative translational and rotational motion that
can substantially cover the whole reaction chamber volume. Such
particles dynamics is the key factor in the disclosed particle
mixing according to the invention, as the mixing will cover
substantially the whole reaction chamber volume and not be limited
to a narrow segment as in the disclosed prior art concepts. This
magnetic particles handling process advantageously assures
therefore a homogenous mixing allowing much more liquid volume to
be effectively contacted with the particle surfaces.
[0089] Moreover, as when compared with the previous art magnetic
particles resuspension concept of E.P, Pat 0,504,192, the use of
sequential energizing (actuation) of the electromagnets by "binary"
(i.e., on and off) or "analog" with the disclosed magnetic device
of FIG. 2, leads the particles to move very slowly while remaining
mostly agglomerated. Moreover, the polarity alteration between the
two states of FIG. 5(a) and 5(b) will not substantially solve this
issue as suggested by the U.S. Pat. No. 6,231,760. Such
difficulties are a specificity of "microfluidics" where the
relatively "small" working volume leads to strong magnetic coupling
between the adjacent magnets.
[0090] For solving this issue, the key finding of the present
invention is to apply in each electromagnetic pole magnetic field
sequences having polarity and intensity that vary in time, the role
of which is to effectively break or control the particle aggregates
and to maintain the particles in suspension as a fog of particles
in relative dynamic motion; and then combining the magnetic fields
from different magnetic poles in a sequence to induce homogenous
mixing of the particles over substantially the whole reaction
chamber volume.
[0091] For clarity, and contrary to what one could as a first view
expect, the "arrows" representing the magnetic poles polarity in
FIGS. 5(a) and 5(b) (and in the other Figures), are not fixed. In
practice, these polarities are continuously changing direction in
time. The "fixed" narrow direction, "instead", represents the
"relative" polarization of the electromagnets during the particles
manipulation.
[0092] In another embodiment according to the invention and as
shown in FIG. 6, a device for manipulating and mixing magnetic
particles is provided where the magnetic poles form a quadrupole
comprising (i) a first couple of magnetic poles (1)-(1') facing
each other forming a diverging gap; and (ii) a second couple of
magnetic poles (10)-(10') facing each other and forming a diverging
gap, with the large ends of the diverging gaps of the first and
second couples of poles facing one another; and (iii) a reaction
chamber (2) that is a part of a fluidic network, having a cavity
with diverging parts of the reaction chamber that are arranged
co-divergently in the diverging gaps between the poles.
[0093] It is clear that the quadrupole configuration is a more
sophisticated version of the previous embodiments allowing more
enhanced effects. More specifically, the magnetic field gradient
(5), rather than being substantially axial (axial symmetry) as in
the case of the previously described two poles configuration, has a
substantially "spherical symmetry". The possibility of having a
"multi-directional" magnetic field gradient induced by more than a
couple of magnetic poles, offers the possibility to move the
position of the magnetic field gradient maxima following more
"rich" configurations as shown in FIG. 7(a)-(d). In particular, by
proper and sequential actuation of the magnetic field (4) induced
from each magnetic pole of the multi-poles (quadrupole)
configuration one can to move the position of the magnetic field
gradient maxima across the reaction chamber volume in way that the
sequential position of these maxima covers the whole reaction
chamber volume.
[0094] FIGS. 7(a)-(d) schematically represent the different
magnetic poles actuation (4) and the corresponding magnetic
particles configurations, which correspond indeed to the position
of the magnetic field gradient maxima. An effect obtained by such
magnetic handling process is that by sequentially moving the
particles, for instance, following the configuration of FIGS.
7(a)-(d), the particles movement will cover substantially the whole
volume of the reaction chamber as shown in FIG. 8, thereby assuring
a strong mixing with the surrounding liquid medium.
[0095] From what precedes, a first key element in the actuation
mechanism according the invention is a "base" magnetic field
actuation (4) of the magnetic poles which is a magnetic field with
a polarity and amplitude that vary with time. A typical example of
this actuation field is an oscillating magnetic field as the one of
equation (1). In general, such base magnetic actuation field has a
substantially rectangular, sinusoidal, saw-tooth, asymmetrical
triangular or symmetric triangular form or any combination of such
forms.
[0096] From what precedes, a second key element in the actuation
mechanism according to the invention is that the magnetic poles are
actuated following a certain sequence to induce continuous time
variations of the position of the magnetic field gradient maxima
across the whole reaction chamber volume, causing thereby the
particles in use to be in relative dynamic motion covering the
whole reaction chamber volume.
[0097] Accordingly, in the invention by "field sequences having
polarity and intensity that vary in time" one means the composition
of the "base" actuation field on each magnetic pole along with its
sequential variation to induce the particles movement across the
reaction chamber volume. In practice indeed, one can decompose the
field sequences actuating each magnetic pole in two main
components: (1) a base actuation field component that has a
polarity and amplitude that vary with time and (2) a sequential
variation of this base actuation field to induce the particles
displacement across the reaction chamber and thereby affecting
particles mixing.
[0098] Accordingly, in practice the base actuation field component
will have the role of breaking the particles chains aggregates and
thereby assure large surfaces of the particles to be in contact
with the surrounding liquid medium while the sequential variation
of this base actuation field will induce continuous move of the
particles "fog" over the whole reaction chamber assuring thereby an
homogenous exposure of the "disaggregated" particles over
substantially the whole volume of the reaction chamber.
[0099] Consequently, a desired effect obtained by the actuation
mechanism according to the invention is that during their motion
the particles do not displace as a compact aggregate but they are
rather moving as a fog of particles resulting in a strong
enhancement of the contact between the of particles surfaces and
the surrounding liquid medium.
[0100] In the previously described actuation mechanism, the time
variation of the base actuation field as well as the sequence
actuation of the magnetic poles is a non-periodic variation but it
is preferably a periodic variation. In the periodic case, the
frequencies of the base field (f.sub.1) and the actuation sequence
(f.sub.2) can be in practice different (f.sub.1.noteq.f.sub.2). To
reach the previously described particle mixing effects, the
actuation sequence frequency (f.sub.2) is lower than or at most
equal to the to base field frequency (f.sub.1). In general, to
reach the previously-described particle mixing effects the time
field variation of the base field (i.e. the time variations of the
amplitude and the polarity of in each magnetic pole) is preferably
higher or at least equal to the sequential time actuation of the
magnetic poles.
[0101] The time variations of the magnetic field in accordance with
the invention, defined by the frequencies f.sub.1 and f.sub.2, is
in the order of 0.1 Hz to 1000 Hz and preferably between 1 Hz and
500 Hz, or other time scales characterizing non-periodic
variations.
[0102] An advantageous effect obtained by the actuation mechanism
according to the invention is that particles will exhibit a
dynamics movement that substantially covers the whole reaction
volume over a certain period of time. According to the invention
indeed, the particles will homogenously cover at least 60% of the
reaction chamber volume and preferably between 80% and 99% of the
reaction chamber volume. This homogenous coverage will be achieved
in period of time that is determined by the sequence actuation time
(or frequency) of the magnetic poles. In practice, the homogenous
mixing is achieved in a period of time between 10 s and 10 ns and
preferable is and 10 ms. In preferred embodiments and depending on
the actuation field parameters the homogeneity of mixing will cover
99% of the reaction chamber over time.
[0103] To reach the desired effects, the magnetic particles in use
are preferably initially unmagnetized magnetic particles that
develop a specific ferromagnetic hysteresis response to an external
magnetic field. More specifically, the particles have a coercive
field between 200 to 1000 Oe. Contrary to what is reported in the
previous art where the particles in use are preferably
"superparamagnetic", it has been found that the fact that the
particles exhibit a specific (ferromagnetic) hysteresis response is
a key condition to achieve the mixing effects according to the
invention. In fact, as described before, the particles actuation
mechanism consists in the use of a preferably a high frequency
"oscillating" field as "base" actuation magnetic field component on
each magnetic pole to control and break down the particles
aggregates. At such high variation frequencies (f.sub.1>1 Hz),
the fact that the particles have hysteresis response allow them to
follow such "rapid" field variations by physically rotating with
the field oscillations. This particles rotation in a high frequency
oscillating magnetic field (field having polarity and intensity
that vary in time) is at the origin of the particles desegregation
process.
[0104] Moreover, to reach the desired effects, it has been found
that preferably the particles in use are manipulated with an
"oscillating" (field having polarity and intensity that vary in
time) magnetic field with an amplitude (maximum field strength)
that is lower then the coercive field of the particles in use.
[0105] Accordingly, the particles in use preferably are synthesized
with properties following the process disclosed in the patent
application WO2006/056579, herein incorporated entirely as a
reference.
[0106] In general the invention provides a method of integrating
all of the previously described magnetic particles handling and
mixing in microfluidic environment concepts. The method consists in
the use of a reaction chamber that is a part a microfluidic
network, wherein: at least one couple of electromagnetic poles face
each other across the reaction chamber, the method comprising: (a)
applying magnetic field sequences having polarity and intensity
that vary in time from each of the electromagnetic poles, (b)
combining the magnetic field from each magnetic pole to induce
continuous time variations of the position of the magnetic field
gradient maxima across the whole reaction chamber volume; and (c)
causing the particles to be in relative translational and
rotational motion covering the whole reaction chamber volume.
[0107] To obtain the desired effect, the magnetic poles are
preferably magnetically coupled one to each other by a "closed"
magnetic circuit. A typical example of such magnetic circuit is
illustrated in the perspectives views of FIG. 9. Indeed as shown in
FIG. 10(a), for the quadrupole configuration of FIG. 6, each
magnetic pole (1)-(1'), (10)-(10') is connected to an electromagnet
formed by a magnetic core (6) with a coil (7). Moreover, each
magnetic core (6) is in contact with a "base" magnetic core part
(6') in form of an "8". The "8" shape of the base magnetic core 6'
assures that each magnetic pole pair configuration forms a closed
magnetic circuit assuring thereby a stronger circulation of the
magnetic flux during the actuation process like the one described
by equation (1). Moreover, the fact that each pair of magnetic
poles form a "closed" magnetic circuit is essential to strongly
focus (concentrate) the magnetic flux and magnetic flux gradient in
the reaction chamber. Moreover, this condition is particularly
preferable to assure the mixing process and effects, in accordance
with invention, as previously described.
[0108] FIG. 9(b) shows a more sophisticated form of the quadrupole
configuration and magnetic circuit of FIG. 9(a), with an array of
quadrupole configurations to assure parallel actuation and handling
magnetic particles according to the invention in four different
adjacent reaction chambers. The same design and construction of a
quadrupole array can be extended for handling magnetic particles in
a larger number of reaction chambers.
[0109] Another aspect of the invention is related to a microfluidic
chip that integrates the different geometrical aspects of magnetic
particles manipulation and mixing described above. Accordingly, a
microfluidic chip comprises: (a) reaction chamber (2) that is a
part of a fluidic network containing the magnetic particles in use
in suspension and having at least one cavity with
diverging/converging parts, (b) inlet (12) and outlet (13)
channels, for delivering liquids into and from the reaction chamber
and connected respectively to the narrow segments of the
diverging/converging parts, (c) an entries structure (14) placed on
both sides of the reaction chamber (2) to receive magnetic poles
that are part of an external magnetic circuit and wherein the
magnetic poles are geometrically arranged in a way to be
co-diverging/co-converging with diverging-converging parts of the
reaction chamber.
[0110] In addition to the reaction chamber, the microfluidic chip
of the invention is configured to include one or more of a variety
of components that will be present on any given device depending on
its use. These components include, but are not limited to, sample
inlet ports; sample introduction or collection modules; cell
handling modules (for example, for cell lysis (including the
microwave lysis of cells as described herein), cell removal, cell
concentration, cell separation or capture, cell growth, etc.;
separation modules, for example, for electrophoresis, gel
filtration, ion exchange/affinity chromatography (capture and
release) etc.; reaction modules for chemical or biological
reactions or alteration of the sample, including amplification of
the target analyte (for example, when the target analyte is nucleic
acid, amplification techniques are useful.
[0111] All the previously described embodiments and aspects of the
present invention have as a main objective to enhance the reaction
rate between any target substances within a liquid medium and the
particle surfaces suspended in the said liquid. An effective
mixing, will indeed have a strong impact on the performance of any
biochemical process such as the extraction or (and) detection of
biomolecules for example (but not limited to) nucleic acids and
proteins. Moreover, one key element of the disclosed magnetic
particles handling concept is that the particles manipulation
procedure can be readapted or adjusted in correspondence with the
biochemical process in consideration.
[0112] Usually the surface of the magnetic particle is
biochemically functionalized by specific ligands for the probing
and manipulating of biomolecules and chemical substances using
well-known techniques. For this, the magnetic particle surface
comprises for example a functional group or a ligand that is
capable of binding to a target molecule or to class of target
molecules. Potential functional groups comprise but are not limited
to carboxylic acids, hydroxamic acids, non-adhesive compounds,
amines, isocyanates, and cyanides. Potential ligands comprise but
are not limited to proteins, DNA, RNA, enzymes, hydrophobic
materials, hydrophilic material, and antibodies. More generally,
examples of ligands suitable for use in the present invention
include, but are not limited to, molecules and macromolecules such
as proteins and fragments of proteins, peptides and polypeptides,
antibodies, receptors, aptamers, enzymes, substrates, substrate
analogs, ribozymes, structural proteins, nucleic acids such as DNA
and RNA and DNA/RNA hybrids, saccharides, lipids, various
hydrophobic or hydrophillic substances, lipophilic materials,
chemoattractants, enzymes, hormones, fibronectin. and the like.
Such molecules and macromolecules may be naturally occurring or
synthetic. The term ligand may also include larger entities such as
cells, tissues, entire microorganisms, viruses, etc.
[0113] Using the so functionalized particles, the mixing and
separation process of the present invention has particular utility
in various laboratory and clinical procedures involving biospecific
affinity binding reactions for separations. Such biospecific
affinity binding reactions may be employed for the determination or
isolation of a wide range of target substances in biological
samples. Examples of target substances are, cells, cell components,
cell subpopulations (both eukaryotic and prokaryotic), bacteria,
viruses, parasites, antigens, specific antibodies, nucleic acid
sequences and the like.
[0114] Moreover, the mixing and separation process of the present
invention have particular use in detection procedures including,
but not limited to polymerase chain reaction (PCR), real-time PCR,
ligase chain reaction (LCR), strand displacement amplification
(SDA), and nucleic acid sequence based amplification (NASBA).
[0115] An example of use of the disclosed magnetic particles
handling and mixing devices/method is illustrated in FIG. 11. This
Figure illustrates the different steps of a sandwich immunoassay
where: (a) In a first step (FIG. 11(a)) the particles coated with
specific capturing probes will be mixed to homogenously cover the
reaction chamber as previously described in FIGS. 7 and 8. In this
step the sample containing the target biomolecules is pushed with a
liquid flow through the reaction chamber. For that purpose reaction
chamber/magnetic pole "co-variation" geometries will assure (as
shown in FIG. 3 and FIG. 4) the homogeneity of the mixing
conditions of the magnetic particles with the liquid flow. All
these conditions when tidily adjusted allow a strong capturing
efficiency of the targets on the particles surfaces. (b) After a
washing step, as described in FIG. 11(b), a defined volume
(substantially equal to the volume of the reaction chamber) of a
reagent containing detection probes is injected in the reaction
chamber. In this case the particles can be again homogenously mixed
with the surrounding medium allowing efficient capturing of the
detection probe on the particle surfaces. (c) After a washing step,
as described in FIG. 11(c) a defined volume (substantially equal to
the volume of the reaction chamber) of a reagent detection
substrate is injected in the reaction chamber. In this case the
particles can be homogenously contacted and mixed with the
surrounding medium allowing efficient interaction between the
substrate and the detection probes on the particle surfaces.
Contrary to classical immunoassay tests where the detection signal
is mainly induced by diffusion, our mixing process allows a strong
interaction between the detection substrate and the particles
surfaces covering the whole reaction chamber volume. A large
enhancement of the detection signal can be therefore generated in
this way allowing the detection of low target molecules
concentration within the starting sample (as blood or plasma). As
shown in FIG. 11 (c), during the detection the particles can be
drawn (separated) to the reaction chamber borders following the
sequence of FIG. 7(b).
[0116] In a different embodiment of the use of magnetic particles
handling and mixing according to the invention, rather than having
a flow-through for capturing targets from a large sample volume (as
for instance described in the first step of the previous example
11(a)), a target concentration can be achieved in a more controlled
way under a static (no-flow) condition. This embodiment,
schematically illustrated in FIG. 12, is based on the use of the
concept of "pulsed injection" (instead of continuous flow) of the
sample in the reaction chamber followed by a homogenous mixing of
the particles. More specifically, in a first step (FIG. 12, top)
the particles are attracted to the reaction chamber walls (using
the actuation sequence of FIG. 7(b)) and retained while a defined
volume of the sample, that will not exceed the reaction chamber
volume, is injected. In a second step (FIG. 12 bottom), the
particles coated with specific capturing probe (surface chemistry)
will be mixed to homogenously cover the reaction chamber as
previously described in FIG. 8, but without any flow. After mixing
of a defined time period, the particles will be attracted again to
the reaction chamber walls and the new sample volume injected to
the reaction and than mixed. This process will be repeated in
sequential way until the full sample volume is mixed with the
magnetic particles.
[0117] One of the advantages of such "pulsed-injection/mixing" mode
is that one will avoid to deal with the constraints of handling
magnetic particles in a flow-through condition which is actually
very difficult to setup. Moreover, contrary to the flow-through
case where the contact time is still relatively extremely short,
the mixing time can be more easily controlled in a pulsed mode and
adapted in correspondence with the target molecules and the final
use.
[0118] Additionally, we have experimentally observed that it is
difficult to retain the particles in the reaction under
flow-through conditions when the particles are manipulated in a
homogenous fog at high field variation frequencies where the
particles are desagglomerated and therefore mixing conditions are
more favorable. In such more favorable mixing conditions, the
particles losses are very important and therefore the
"pulsed-injection/mixing" mode is practically more appropriate.
[0119] With a view to minimize and even completely avoid particle
losses during the assay process, the particles are separated in a
portion of the reaction chamber where the magnetic field gradient
is higher, to retain the particles in the reaction chamber during
the reagents injection step.
[0120] One favourable separation position of the particles is the
outer borders (as in FIG. 12, top). In this position and after
applying a "time varied magnetic field sequence", necessary to
effectively separate the particles, a static magnetic field can be
applied to agglomerate the particles and thereby strongly fix them
at the reaction chamber borders.
[0121] Another favourable separation position of the particles is
the narrow corner of the reaction chamber facing the injected flow
channel as previously described in FIGS. 2 to 5. At this position
indeed, the particles are strongly retained under a high flow rate
while at the same time assuring a strong mixing with the fluid
flow, which gives a clear advantage to this position when for
instance compared to the first one.
[0122] From what precedes, the "separation/injection/mixing" mode
can be considered as sequential (discontinuous) micro-reaction
process, where the reaction can be conducted in "small" reaction
volume in the presence of large particle "fog" surfaces provided by
the disclosed mixing method. When taking in consideration the rapid
switching (in a fraction of a second of time) between the full
mixing and separation states of the particles, each batch or
sequence can take typically between 1 to 5 seconds. This means that
for instance for a volume of the reaction chamber in the range of
10 .mu.l to 100 .mu.l, an effective high "flow-rate" of up to 3
ml/min can be achieved.
[0123] With the rapid processing of the
"separation/injection/mixing" mode, this method provides also
enhanced assay performance as the reaction takes places in a small
volume with enhanced particles mixing, as described herein.
[0124] In practice, the "separation/injection/mixing" mode can be
principally used advantageously to concentrate a target molecule
from a large volume (up to 10 ml or even more), but it can be also
used for potentially any reagent and assay process such as but not
limited to: washing, biomolecules labeling, detection of a target
and/or elution of the target from the particle surfaces.
[0125] In a different embodiment, the magnetic particles handling
and mixing according to the invention is used to concentrate
specific cells and then extract molecule(s) that enter in the
composition of that cells in a sample volume consists.
[0126] The cell concentration process consists in: (1) providing in
the reaction chamber a first type particle have a surface coating
designed to selectively bind with the said target molecules; (2)
These particles will be mixed and separated following the
previously described process and method using magnetic field
sequences having polarity and intensity that vary in time; (3) in a
first step the particles are separated or confined in a sub-volume
in the volume of the reaction chamber as for instance schematically
represented in FIG. 12, top; (4) as separated, a defined volume of
the assayed sample contains the cells that contain the targets as
part of its composition. At this stage and according to the
invention the "target" cells are previously labelled with a second
type of magnetic particles; (5) when introduced in the reaction
chamber, the magnetically labelled cells are separated from the
crude sample by mixing them with the first particles types. The
mixing process is as described before, and consists in the
application of an appropriate magnetic field sequence to lead the
said first particles type to be homogenously distributed and
dynamically moving as a fog of first particles that occupies a
substantial portion of the whole reaction chamber volume (as shown
in FIG. 12 bottom). By mixing the first magnetic type with the
injected labelled cells the objective is to promote the contact
between the two magnetic entities and thereby form by means of
dipolar interaction a complex composed from the first particles
type and magnetically labelled cells. As the complex between the
first particles type and magnetically labelled cells, the
separation of the magnetically labelled cells will be
"synthetically" assured; and (6) the "pulse-injection" can be
repeated until a given sample sub-volume is passed through the
reaction chamber and therefore the target cells separated.
[0127] As the so described cell concentration process, consists on
the use of the mixing and magnetic handling process to separate
magnetically labelled using "magnetic" interaction between the
cells and the mixed particles. Usually indeed the magnetic cell
separation necessitate the use of high magnetic gradient separator
with langue flow channels or magnetic columns. As proposed herein,
the cell separation method, indeed, is based on the formation of a
complex composed from the first particles type and magnetically
labelled cells by means of dipolar interaction during the mixing
process of the first particle type, facilitating thereby the
separation of the magnetically labelled cells from the supernatant
during the separation of the first magnetic particles type. When
compared with the previous art, this separation and concentration
process is performed in a small volume of the reaction chamber
(between 10-100 .mu.l), using relatively very low magnetic field
forces and in a fraction of time.
[0128] When the cells are concentrated in the reaction chamber, in
an additional step, the cells are lysed to release its contents and
particularly the desired target molecule(s) to be separated and
purified. To do so, the lysis step is preferably performed in
"buffer" conditions that allow specific capturing of the said
target molecules on the first particles types surfaces in the
reaction chamber. This targets capturing step is "naturally"
performed by homogenously mixing the particles over the reaction
chamber volume.
[0129] In practice, this method consists first of the cell
selection using a first type of magnetic particles having a surface
coating designed to allow affinity recognition of the said cells.
The so-labelled cells are then magnetically separated in a
microfluidic chamber where second magnetic particles type are
manipulated according the inventive method of mixing magnetic
particles. The separation process is performed following the
"separation/injection/mixing" mode, wherein the separation process
is allowed by the efficient mixing to strongly contact the
magnetically labeled cells and the second magnetic particles type.
As the cells are concentrated in the reaction chamber, in a second
step, the cells are lysed to release a least one target molecule
entering in its composition in conditions that allow specific
capturing of the said target molecules on the first particles types
surfaces in the reaction chamber.
[0130] Accordingly, the disclosed method of extracting a target
molecule(s) such as nucleic acids, peptides or proteins that enter
in the composition of cells such as bacteria, viruses or human body
fluids contained (circulating) cells in a sample volume, allows to
combine complex analytical procedures as cells selection and
separation followed by the cells lysis and target molecules
purification, in one reaction chamber.
[0131] The sizes of the said first and second magnetic particles
types are respectively in the range of 0.1 .mu.m to 500 um and 5 nm
to 5 .mu.m. Preferably with a size between 1 .mu.m to 10 .mu.m, the
first particles type are initially unmagnetized particles that
develop a ferromagnetic response with a hysteresis. Practically,
the second particles type are preferably selected with a size lower
and at most equal to the size of the first particles type.
Moreover, as the second particles type are used as a label for the
target cells, their size is preferably taken in the nanometre
range, more specifically between 10 nm and 500 nm. The second
particles type can be, however, superparamagnetic but initially
unmagnetized particles that develop a ferromagnetic response can be
used as well. In the latter case, can be advantageous to assure
strong dipolar interaction during the cells separation and
concentration step.
[0132] For the lysis step, one can use different means: chemical
lysis with guanidinium SCN, enzymatic lysis with proteinase K or
lysostaphin, thermal lysis, use of ultrasound as well as electrical
fields or electromagnetic radiations, a strong pH gradient inducted
by localized electrolysis and mechanical lysis, The lysis using
physical means like electromagnetic radiations or an electric field
as the lysis can be performed in a favourable environment and
conditions allowing the capture of the target molecules on the
particle surfaces. The said conditions can be performed by
adjusting for instance the pH or in the specific buffer conditions
such as in chaotropic or anti-chaotropic agents.
[0133] Regarding the final objective of the invention which is the
integration and automation of complex biochemical assays in an easy
to use device, and to control the different steps and aspects
described before, the microfluidic system with the chip and
magnetic device further comprises a plurality of reagent sources
fluidly connected to said reaction chamber; means for
driving/controlling liquids like pumps and valves; and a computer
controller for controlling reagent flow and application of the
magnetic field sequences.
[0134] Even though all of the described methods and aspects are
realized through the previously described features as magnetic
poles/reaction chamber, some of the effects disclosed here can be
obtained by other magnetic pole configurations. As described
before, the main conditions that must be satisfied are: (1) that
the magnetic field gradient generated by the electromagnets must
cover the whole reaction chamber with a defined localization of
magnetic field (gradient) maxima when the magnetic poles are
specifically actuated; (2) the magnetic field gradient must retain
and confine the particles in the reaction chamber during the
manipulation process; and (3) the magnetic poles are in a
configuration that tends to focus magnetic flux in the reaction
chamber in a way that each pair of magnetic poles facing the gap
can be magnetically coupled through the said gap. To further
enhance this effect, the poles are preferably part of a closed
magnetic circuit.
[0135] Typical example of such configuration is the one illustrated
by FIG. 14. The configuration comprises a quadrupole pole types
configuration (19) that can be actuated as the same way as the
converging/diverging magnetic poles previously described. The
quadrupole configuration (19) is preferably associated with two
couples of magnetic poles placed at the right (16) and the left
(17) of the quadrupole configuration (19). The magnetic pole
couples (16) and (17) are actuated (in opposite configuration) in a
way to generate a lateral magnetic field gradient (18)-(17) that
tends to confine the particles in the quadrupole air gap region. In
the case of the converging/diverging magnetic poles configuration,
the lateral confining magnetic field gradient is assured by the
magnetic poles geometry and therefore has the advantage to avoid
the need for additional magnetic poles. Further, to have the
desired magnetic field flux focus in the reaction chamber and the
magnetic field gradient covering the whole reaction chamber volume,
the magnetic poles must be in form of magnetic tips (of 1 to 5 mm)
located close to each other in a small gap of about 1 to 5 mm.
[0136] However, the magnetic poles configuration of FIG. 14, is
also limited by the fact that the volume of the region under which
the particles can be mixed and handled is very small.
[0137] The following examples further describe in detail the manner
and process of using the present invention. The examples are to be
considered as illustrative but not as limiting of this invention.
All manipulations given in the examples are at ambient temperature
unless otherwise indicated.
EXAMPLE 1 OF ACTUATION MECHANISMS
[0138] The actuation sequences of FIG. 7, is an illustration on how
the magnetic particles, while the magnetic poles are actuated using
a time varied (amplitude and polarity) magnetic field, is used as a
base sequence, can be moved following the combination of the
magnetic field from each magnetic pole. During this movement the
particles will substantially cover the whole reaction chamber
volume as a fog of particles thereby assuring mixing. Although
represented as "discontinuous" sequences, the sequences of the
particles as shown in FIG. 7 can be achieved with a "rotating
magnetic field" following the magnetic poles actuation
sequences:
Pole 1 and 10': B=B.sub.0 sin(ft)
Pole 1' and 10: B=B.sub.0 sin(ft+.pi./2) (3)
[0139] In equation (3) the base sequence actuation in each magnetic
pole is an oscillating field while the actuation process is assured
by a phase shift of .pi./2 between the diagonally coupled magnetic
poles. In this configuration the base and the sequence actuation
fields have the same frequency f.
[0140] In the actuation according to the sequences of equation (3),
two particles regimes can be distinguished: a low frequency and
high frequency regime.
[0141] At low frequency typically for f>5 Hz, the particles will
rotate relatively "slowly" and the particles will move across the
reaction volume producing typically the sequences as schematically
shown in FIG. 7. The particularity of this regime is that the
particles during their movement from one magnetic poles
configuration to the other (see FIG. 7), the particles will have
enough time to "aggregate" in longer magnetic chains. For
frequencies higher than 1 Hz, the particles will exhibit fast and
strong dynamics that covers substantially the whole (>90%)
reaction volume. However, at this regime of a rotating magnetic
field the particles still "relatively" aggregated.
[0142] More disaggregated particle sate will be ultimately obtained
at higher frequencies of the rotating field f>5 Hz. At this
regime instead the particles behaviour is drastically different as
the fast rotation of the magnetic particles will not give enough
time for chain formation leading the particles chains to break down
to smaller particles chain-like structures with a size that
decreases with the field frequency. As a difference with the
low-frequency regime, the sequence of FIG. 7 (b) will not be
observed as the particles will not have time to extend along the
diagonal of the reaction chamber. What happens at high frequency
indeed is that the particles will be attracted and confined at the
reaction chamber walls. FIG. 13(a) shows a video of the particles
behaviour at high frequencies.
[0143] To overcome this problem, a finding of this invention is to
reduce the amplitude while increasing the frequency of the applied
rotating field in combination with the use of ferromagnetic
particles. The reduction of the magnetic field amplitude indeed
allows to expand the particles more over the reaction chamber
volume due to reduction of the magnetic gradient forces and the
repulsive dipolar forces between the rotating particles. However,
as the reduction of the magnetic forces will slow down the
particles movement, a higher frequency field is required to further
propel the particles movement. At such high frequencies typically
between superior to 20 Hz and preferably in the range of 100 Hz to
500 Hz, the use of ferromagnetic particles is key as the "magnetic
anisotropy" of these particles leads them to move and follow the
field variations. FIG. 13(b), shows the homogenous coverage of the
particles in the reaction chamber obtained under a high rotating
frequencies (around 300 Hz). During this mixing, the particles
strongly move across the reaction chamber allowing thereby strong
and efficient mixing.
[0144] It is important to point out here that the frequencies
values given in this example are typical values just for
indication, obtained with specific particles used in experiments
(MagNA Pure LC particles from Roche Diagnostics). The use of other
particles types will certainly affect the frequencies limits of
different particles regimes and behaviours as described before.
EXAMPLE 2 OF ACTUATION MECHANISM
[0145] Equation (4) describes another actuation sequences to
achieve mixing according to the invention.
Pole 1 and 10': B=B.sub.0 sin(f.sub.1t)sin(f.sub.2t)
Pole 1' and 10: B=B.sub.0 sin(f.sub.1t)sin(f.sub.2t+.pi./2) (4)
[0146] In this sequence indeed the first oscillation component
(sin(f.sub.1t)) is nothing more than the base actuation field at a
frequency f.sub.1 of the magnetic poles while the second term
defines the actuation sequence that moves the "fog" of particles in
rotation form with a frequency f.sub.2. The sequence of equation
(4) allows in particular to solve the previously reported (in the
Example 1) agglomeration of particles in a low frequency rotating
field of equation (3). For instance by rotating the particles as a
frequency f.sub.2=1 Hz, the particles chains will break down due to
the fast oscillation of the base field f.sub.1>10 Hz.
EXAMPLE 3 OF ACTUATION MECHANISM
[0147] Equation (5) describes another actuation sequence to achieve
mixing according to the invention, where the frequency of the
rotating magnetic field of equation (1) of equation is
"modulated".
f=f.sub.0+f.sub.1 sin(.OMEGA.t) (5)
[0148] The finding is that modulating the frequency between a low
frequencies regime and the high frequencies regime assures thereby
efficient mixing. By appropriate choice of the modulating frequency
(.OMEGA.), when can balance between the two regimes: homogenous
mixing with agglomerations at lower frequencies and the
"inhomogeneous" mixing with fog particles structure at higher
frequencies. This way of "modulating" the frequency of the rotating
field is particularly important for highly viscous liquids where
homogenous mixing is difficult to achieve by only increasing the
oscillating frequency as described in Example 1.
[0149] It is obvious for skilled persons that the frequency
modulation can be done by other forms, as for instance a "square"
signal where one switch between one high frequency value and a low
frequency one. Each value can be maintained for a certain time that
depends essentially on the liquid viscosity, to assure an
homogenous mixing.
[0150] It is worth to emphasize here again that the particles in
use are preferably ferromagnetic to allow the particles to move and
rotate at high frequency.
EXAMPLE 4 OF ACTUATION MECHANISM
[0151] Although the previous examples are based on using "rotating
magnetic field", linear actuation sequence of particles fog can be
also used to mix and reach an homogenous state. Typical example of
that linear actuation mode can be achieved by first moving the
particles to the out border as shown in FIG. 7(b) using the
actuation sequence:
Pole 1 and 1': B=B.sub.0 sin(.omega.t)
Pole 10 and 10': B=B.sub.0 sin(.omega.t+.pi.) (6)
[0152] At this stage the particles can be moved to the left corner
(narrow part) of the reaction chamber by the sequence:
Pole 1 and 1': B=B.sub.0 sin(.omega.t)
Pole 10 and 10': B=B.sub.0 sin(.omega.t+.pi./2) (7)
[0153] By symmetry a displacement toward the right corner (narrow
part) of the reaction chamber can be achieved by the sequence:
Pole 1 and 1': B=B.sub.0 sin(.omega.t+.pi./2)
Pole 10 and 10': B=B.sub.0 sin(.omega.t) (8)
[0154] A sequential shift between the previous three configuration
following the sequences: (6).fwdarw.(7).fwdarw.(6).fwdarw.(8) at a
determined rate, one can achieve an homogenous mixing over the
time.
[0155] In practice, better mixing processes are achieved not
through only a rotating or a linear mode but usually a mix of both
modes is preferred.
[0156] Herein in these examples the choice of a "sinusoidal" field
as base actuation is only for it is practical analytical
formulation with an equation. Within the invention scope, more
complex actuation "base sequences" having polarity and intensity
that vary in time will lead to the same effects.
EXAMPLE 5 OF USE OF THE MIXING CONCEPT AND DEVICE
[0157] In this example the disclosed magnetic particles device and
method are used for DNA extraction from bacteria (E-coli) culture
with an inserted plasmid. For the extraction, MagNA Pure LC kit
from Roche Diagnostics (Switzerland) is used. A particularity of
this kit is that the magnetic particles exhibit a ferromagnetic
response with a coercive field of around 200 Oe. For the sample
preparation, 200 .mu.l of the bacteria culture in PBS with a
concentration of around 2.times.108 cells/ml are mixed with: (a)
400 .mu.l of lysis binding buffer, (b) 100 .mu.l of isopropanol,
and (c) 100 .mu.l of Proteinase-K. The total extraction volume is
therefore 800 .mu.l.
[0158] For the assay, a microfluidic chip with the layout of FIG.
11 is used. The reaction chamber in this chip has the following
dimensions: H=0.25 mm, L=0.5 mm and a depth of 1 mm. The total
volume of the reaction chamber is therefore around 25 .mu.l. In
this reaction chamber around 50 .mu.l of the glass particles from
the kit is separated and retained in the reaction chamber.
[0159] The samples and reagents processing through the chip is
performed following the previously described "pulse-injection" mode
and where the particles are homogenously mixed over the reaction
chamber over a period of 2 s followed by a separation and liquid
injection of around 1 s. Around 3 seconds are necessary to process
25 .mu.l of the sample volume witch is equivalent to processing
flow rate of 0.5 ml/min.
[0160] The washing step is performed using the three washing
reagents of the kit with 300 .mu.l volume of each. The washing is
performed by combining both the flow-through mode and
"pulse-injection" mode. Less than 2 minutes are necessary to
perform all the necessary washing steps. For the DNA elution, a
volume of the elution buffer from the kit substantially equal to
the reaction chamber volume (-30 .mu.l) and homogenously mixed for
around 3 minutes.
[0161] To determine the homogenous mixing benefits, the extraction
performance is compared with the standard manual extraction (as a
reference) and the non-homogenous mixing under a high frequency
rotating magnetic field as described in example 1 and shown in FIG.
13(a). For the performance of DNA extraction experiments we use the
optical absorbance, with the following results:
TABLE-US-00001 Total DNA amount (.mu.g) Purity (OD 260/280) Manual
extraction 6 1.7 Homogenous mixing 5.5 1.9 Non-homogenous mixing
1.2 1.6
[0162] From these results one can see the strong impact of the
proposed magnetic particles mixing effect in enhancing the affinity
binding between the particles and the target molecule (DNA) in the
sample. In fact, while the manual extraction takes around 20
minutes to be performed around 8 minutes are necessary for full
extraction using the disclosed homogenous mixing method and device.
Moreover, in the manual extraction around 100 .mu.l of particles
suspension is used while only 50 .mu.l is used in the microfluidic
homogenous mixing. Taking in consideration the relatively large
amount of DNA that can be purified (up to 10 .mu.g) in a small
reaction chamber volume (25 .mu.l) with the disclosed homogenous
mixing as disclosed herein, is a clear expression of the large
available surface of particles during the mixing demonstrating the
effective particles desegregation and mixing during the assay.
Another demonstration of the particles homogenous mixing is the low
performance obtained by non-homogenous mixing.
EXAMPLE 5 OF USE OF THE MIXING CONCEPT AND DEVICE
[0163] In this example the disclosed magnetic particles device and
method are used for DNA extraction from human whole blood. For the
extraction, MagNA Pure LC kit II from Roche Diagnostics
(Switzerland) is used with the same process and protocol as Example
4.
[0164] The extraction results show a yield between 4-5 .mu.g of DNA
with an OD value between >1.7. This example, demonstrate the
efficient DNA extraction of the disclosed mixing method from a
complex sample like whole blood.
[0165] Those skilled in the art will appreciate that various
adaptations and modifications of the just-described preferred
embodiments can be configured without departing from the scope and
spirit of the invention. Therefore, it is to be understood that,
within the scope of the appended claims, the invention may be
practiced otherwise than as specifically described herein.
* * * * *