U.S. patent application number 12/299781 was filed with the patent office on 2009-07-02 for rapid magnetic biosensor.
This patent application is currently assigned to KONINKLIJKE PHILIPS ELECTRONICS N.V.. Invention is credited to Menno Willem Jose Prins, Richard Joseph Marinus Schroeders, Thea Van Der Wijk.
Application Number | 20090170212 12/299781 |
Document ID | / |
Family ID | 38617430 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090170212 |
Kind Code |
A1 |
Van Der Wijk; Thea ; et
al. |
July 2, 2009 |
RAPID MAGNETIC BIOSENSOR
Abstract
The present invention relates to methods and (bio)sensor
systems. Herein, magnetic fields are applied in order to transport
magnetic particles laterally over a sensor surface with analyte
specific probes. The methods of the invention allow the specific
binding of magnetic particles to the sensor surface, while
aspecific and unbound particles are removed.
Inventors: |
Van Der Wijk; Thea;
(Eindhoven, NL) ; Prins; Menno Willem Jose;
(Eindhoven, NL) ; Schroeders; Richard Joseph Marinus;
(Eindhoven, NL) |
Correspondence
Address: |
PHILIPS INTELLECTUAL PROPERTY & STANDARDS
P.O. BOX 3001
BRIARCLIFF MANOR
NY
10510
US
|
Assignee: |
KONINKLIJKE PHILIPS ELECTRONICS
N.V.
EINDHOVEN
NL
|
Family ID: |
38617430 |
Appl. No.: |
12/299781 |
Filed: |
May 7, 2007 |
PCT Filed: |
May 7, 2007 |
PCT NO: |
PCT/IB07/51698 |
371 Date: |
November 6, 2008 |
Current U.S.
Class: |
436/149 ;
422/82.01 |
Current CPC
Class: |
G01N 35/0098 20130101;
B82Y 25/00 20130101; G01R 33/02 20130101; G01R 33/1269 20130101;
G01N 33/54333 20130101; G01N 27/745 20130101; G01R 33/0213
20130101; G01R 33/093 20130101; G01R 33/12 20130101 |
Class at
Publication: |
436/149 ;
422/82.01 |
International
Class: |
G01R 33/12 20060101
G01R033/12 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2006 |
EP |
06113763.4 |
Claims
1. A system (1) comprising a reaction chamber (2) having a sensor
surface (5) and at least one region outside said sensor surface (6)
and further comprising at least one means for generating one or
more magnetic fields (3), wherein the at least one means for a
generating one or more magnetic fields are placed such that one of
said magnetic fields has a gradient with a component that is
parallel to said sensor surface (5) and spans said sensor surface
(5) and said at least one region outside the sensor surface
(6).
2. The system according to claim 1 wherein the one of said magnetic
fields has a gradient with a component perpendicular to said sensor
surface (5) and wherein said component is directed from the
reaction chamber (2) to the sensor surface.
3. The system according to claim 1, wherein the magnetic field is
arranged to move magnetic particles (7) laterally over the sensor
surface (5) to a first region outside the sensor surface (6).
4. The system according to claim 1, wherein the magnetic field is
capable of moving magnetic particles (7) from a first region
outside the sensor surface (6), laterally over the sensor surface
(5) to a second region outside the sensor surface (6).
5. The system according to claim 4 wherein said magnetic field is
applied in one direction.
6. The system according to claim 4 wherein said magnetic field is
applied once.
7. The system according to claim 4, wherein said first and second
regions outside the sensor surface (6) are the same.
8. The system according to claim 1 wherein a dedicated area is
provided on a region outside the sensor surface for the application
of magnetic particles.
9. The system according to claim 1, further comprising an inlet
means for introducing said magnetic particles into the reaction
chamber.
10. The system according to claim 1, wherein said one or more means
for generating a magnetic field comprises two magnets (3 and 3')
which are located at opposite sides of the sensor surface (5).
11. The system according to claim 1, wherein the magnetic field
applied during a given time period on the magnetic particles
transports said particles further that the distance obtained by
Brownian motion.
12. The system according to claim 1, wherein the at least one means
for generating one or more magnetic fields is a magnet generating
an out of plane field with smaller in-plane components and being
located below the sensor surface.
13. The system according to claim 1, wherein said sensor surface
(5) comprises an analyte-specific probe (8).
14. The system according to claim 1, wherein the dimension of the
sensor surface (5) located between two regions (6) outside the
sensor surface is at least between 10 and 30 micrometer.
15. The system according to claim 1 wherein current carrying
conductors suitable for attracting particles to the sensor surface
(5) or to a region outside the sensor surface (6) are absent.
16. The system according to claim 13, wherein said analyte-specific
probe (8) is selected from the group consisting of an
oligonucleotide, an antibody or fragment thereof, a lectin, a
pharmaceutical compound, a peptide or a protein.
17. The system according to claim 1, further comprising a
magnetic-particle detection means for detection of a magnetic
property of at least one magnetic particle on the sensor
surface.
18. The system according to claim 17 wherein said magnetic-particle
detection means detects the magnetic field from the at least one
particle, or detects the magnetizability of the at least one
particle when subjected to a magnetic detection field.
19. The system according to claim 17 wherein said magnetic-particle
detection means is a magneto-resistive sensor or a Hall sensor.
20. A disposable cartridge comprising a reaction chamber (2), a
sensor surface (5) having an analyte-specific probe or an
analyte-analogue (8), said reaction chamber comprising at least one
region outside the sensor surface (6) and at least one means for
generating one or more magnetic fields.
21. A method for quantifying and/or detecting an analyte,
comprising the steps of: a) providing magnetic particles (7) to a
reaction chamber with a surface sensor (5) having an
analyte-specific probe or an analyte-analogue (8) and a region
outside said sensor surface (6), b) applying a magnetic field
gradient within said reaction chamber having a component
essentially parallel to said sensor surface, whereby said magnetic
field moves said magnetic particles laterally over said sensor
surface (5) toward said region outside said sensor surface, and, c)
detecting said magnetic particles bound to said sensor surface.
22-28. (canceled)
Description
[0001] The present invention relates to systems, apparatus and
methods for detecting and/or quantifying molecules in a sample
using magnetic particles, including disposable cartridges for use
with such systems as well as to systems and methods for moving such
magnetic particles.
[0002] The use of magnetic particles in bioassays and biosensors
has several attractive aspects. Magnetic particles with analytes
can be stirred using magnetic fields which shortens reaction times
between probes and analytes. Magnetic fields can be used to remove
aspecific bound analytes from a probe, to concentrate magnetic
particles in a part of a reaction chamber, or to move particles in
and/or out of a reaction chamber. The magnetic properties of
magnetic particles themselves can be used for detection
purposes.
[0003] The type of magnetic field gradients that are generally used
are applied perpendicular to a sensor surface to move particles to
or from the sensor surface. Also conical fields have been applied
to concentrate particles to one particular point, e.g. the
objective a microscope. Circular or random magnetic fields are used
to create a stirring effect. Examples hereof are described in U.S.
Pat. No. 5,445,971, U.S. Pat. No. 6,548,311, U.S. Pat. No.
6,180,418, Perrin et al. (1999), J. Immun. Meth. 224, 77-87; Luxton
et al. (2004) Anal. Chem. 76, 1715-1719; and Ferreira et al. (2005)
Appl. Phys Lett. 87, 013901.
[0004] Nevertheless, devices which would allow the different assay
steps to be performed in a faster and more efficient way are highly
desirable, more particularly in the context of high throughput
screening and on site analysis.
[0005] It is an object of the present invention provide alternative
or improved systems and methods for detecting and/or quantifying
molecules in a sample using magnetic particles as well as
disposable cartridges for use with such systems and systems and
methods for moving such magnetic particles.
[0006] In one aspect, the present invention relates to a system
comprising a reaction chamber having a sensor surface and at least
one region outside said sensor surface and further comprising at
least one means for generating one or more magnetic fields, wherein
the at least one means for generating one or more magnetic fields
are placed such that one of said magnetic fields has a gradient
with a component that is parallel to said sensor surface and spans
said sensor surface and said at least one region outside the sensor
surface.
[0007] In addition the one of said magnetic fields can have a
gradient with a component perpendicular to said sensor surface
wherein said perpendicular component is directed from the reaction
chamber to the sensor surface. In the system the magnetic field can
be arranged to move magnetic particles laterally over the sensor
surface, i.e. along the surface, to a first region outside the
sensor surface. In the system, the magnetic field can move magnetic
particles from a first region outside the sensor surface, laterally
over the sensor surface to a second region outside the sensor
surface.
[0008] It is possible to have a magnetic field applied only in one
direction whereby the particles also move in one direction under
the magnetic field. It its simplest version the magnetic field is
applied once.
[0009] Optionally this process can be repeated by alternating the
application of the magnetic field with for example a fluid flow
which redirects the magnetic particles to their initial position,
i.e. the particles move or flow in two directions, one being the
reverse of the other. In this case the magnetic field is applied
once followed by a flow of the particles. Alternatively, the
magnetic field may be reversed and/or cycled in this manner to move
particles back and forth.
[0010] By reversing the magnetic field or combining magnetic fields
with other forces the first and second regions outside the sensor
surface as describe above are the same.
[0011] A system can be provided wherein a dedicated area is
provided on a region outside the sensor surface for the application
of magnetic particles. When these particles are applied in the same
plane of the sensor surface, there is no need for a magnetic field
that attracts magnetic particles perpendicularly to the sensor
surface. Thus, it is possible to design systems wherein magnetic
field generators such as current carrying conductors suitable for
attracting particles to the sensor surface or to a region outside
the sensor surface (6) are absent.
[0012] The system of the current invention can comprise inlet means
for introducing said magnetic particles into the reaction
chamber.
[0013] In particular embodiments one or more means for generating a
magnetic field comprise two magnets which are located at opposite
sides of the sensor surface. In any such system according to the
present invention magnetic fields can be applied during a given
time period wherein the transport of the magnetic particles
transports is larger that the distance obtained by Brownian
motion.
[0014] In certain embodiments the at least one means for generating
one or more magnetic fields is a magnet generating an out of plane
field with smaller in-plane components and being located below the
sensor surface.
[0015] The sensor surface of the system can comprise an
analyte-specific probe or an analyte-analogue.
[0016] The dimension of the sensor surface located between two
regions outside the sensor surface can be at least 10, 20, 30, 50
or 100 micrometer.
[0017] The analyte-specific probe which can be attached to the
sensor surface can be any suitable probe of which an
oligonucleotide, an antibody or fragment thereof, a lectin, a
pharmaceutical compound, a peptide or a protein are only
examples.
[0018] The system can also comprise magnetic-particle detection
means for detection of a magnetic property of at least one magnetic
particle on the sensor surface. The magnetic-particle detection
means can detect the magnetic field from the at least one particle,
or detects the magnetizability of the at least one particle when
subjected to a magnetic detection field. Examples thereof are a
magneto-resistive sensor or a Hall sensor.
[0019] Another aspect of the invention relates to a disposable
cartridge comprising a reaction chamber, a sensor surface having an
analyte-specific probe or an analyte-analogue, said reaction
chamber comprising at least one region outside the sensor surface
and at least one means for generating one or more magnetic
fields.
[0020] Another aspect of the invention relates to a method for
quantifying and/or detecting an analyte, comprising the steps
of:
a) providing magnetic particles to a reaction chamber with a
surface sensor having an analyte-specific probe and a region
outside said sensor surface (all or a part of the magnetic
particles can carry an analyte on their surface), b) applying a
magnetic field within said reaction chamber having a gradient
component parallel to said sensor surface, whereby said magnetic
field moves said magnetic particles laterally over said sensor
surface toward said region outside said sensor surface, and c)
detecting said magnetic particles bound to said sensor surface.
[0021] In this method said magnetic field gradient is reversed one
or more times before step (c). In such an embodiment the frequency
of reversal can be set at any suitable value, e.g. 0.01, 0.05, 0.1,
0.5 or 1 Hz.
[0022] In an alternative embodiment the magnetic field gradient is
not reversed.
[0023] In any embodiment of the method of the present invention one
or more magnetic fields can be arranged to move the magnetic
particles from a first region outside said sensor surface, to said
sensor surface, laterally over said sensor surface, and from said
sensor surface to a second region outside said sensor surface.
[0024] The first region outside said sensor surface and said second
region outside said sensor surface can be the same.
[0025] According to another embodiment, said magnetic particles are
labeled.
[0026] The methods of the present invention can be performed
essentially in the absence of the movement of liquid in the
reaction chamber.
[0027] The present invention also provides methods and sensor
systems wherein the step of concentrating magnetic particles
comprising an analyte, such as a ligand or a bio-active molecule,
on the sensor surface and the step of washing to remove unbound
magnetic particles and aspecifically bound particles, are merged.
An advantage is to significantly reduce assay time.
[0028] It is an advantage of the methods, systems and devices of
the present invention that it requires minimal use of reagents.
[0029] It is a further advantage of the methods, systems and
devices of the present invention that they do not necessarily
require a horizontal sensor surface, which can be of interest in
the space-management of the devices or laboratories involved.
[0030] The present invention discloses a sensing system suitable
for the use of magnetic particles (wherein all or a part of the
magnetic particles can carry an analyte on their surface),
comprising a biochemically active sensor surface with a probe,
means to generate a magnetic field that induces a movement of
particles over the sensor surface, wherein the induced lateral
travel distance of a magnetic particle, with respect to the surface
of the sensor is (i) larger than the width of the sensor surface,
and (ii) larger than the distance of diffusional transport in
absence of a magnetic field. The reaction chamber of the system
contains a fluid medium in which the magnetic particles move, can
bind to the probes on the sensor surface in case of specific
binding and can unbind from the probes on sensor surface in case of
aspecific binding.
[0031] The present invention permits the design of improved methods
and apparatus for the detection of analytes in samples.
[0032] The following Figures show embodiments of the present
invention in schematic form.
[0033] FIG. 1 shows a schematic representation of a magnetic sensor
with using one magnet. (MPs: magnetic particles). The arrow
indicates the lateral movement of the magnetic particles.
[0034] FIG. 2 shows a schematic representation of a magnetic sensor
using two magnets. (MPs: magnetic particles) The arrow indicates
the lateral movement of the magnetic particles.
[0035] FIG. 3 shows the magnetic movement towards a magnet (M) of
uncoated magnetic particles (white circles) and antibody coated
magnetic particles (black circles) over an antigen coated (grey
part) sensor surface.
[0036] FIG. 4 shows the magnetic movement of magnetic particles
with anti-morphine antibody over a morphine coated sensor
surface.
[0037] FIG. 5 shows a flowchart of the method for laterally moving
magnetic particles over a sensor surface.
[0038] The present invention will be described with respect to
particular embodiments and with reference to certain drawings but
the invention is not limited thereto but only by the claims. Any
reference signs in the claims shall not be construed as limiting
the scope. The drawings described are only schematic and are
non-limiting. In the drawings, the size of some of the elements may
be exaggerated and not drawn on scale for illustrative
purposes.
[0039] Where the term "comprising" is used in the present
description and claims, it does not exclude other elements or
steps. Where an indefinite or definite article is used when
referring to a singular noun e.g. "a" or "an", "the", this includes
a plural of that noun unless something else is specifically
stated.
[0040] Furthermore, the terms first, second, third and the like in
the description and in the claims, are used for distinguishing
between similar elements and not necessarily for describing a
sequential or chronological order. It is to be understood that the
terms so used are interchangeable under appropriate circumstances
and that the embodiments of the invention described herein are
capable of operation in other sequences than described or
illustrated herein.
[0041] The terms or definitions used herein are provided solely to
aid in the understanding of the invention.
DEFINITIONS
[0042] The term "analyte" as used herein refer to a compound in a
sample of which the detection of presence and/or concentration is
desired.
[0043] The term "analyte-specific probe", as used herein, refers to
a compound which can bind with the analyte.
[0044] The term "reaction chamber" as used herein refers to a
region within a device or a cartridge, where different reagents
taking part in a reaction are contacted with each other.
[0045] The term "sensor surface", as used herein, refers to the
part of the reaction chamber, suitable for binding probes, such as
analyte-specific probes. Generally, it is also the area where the
most important sensitive detection takes place.
[0046] The term "region outside the sensor surface" as used herein,
refers to a region within a reaction chamber of a device or
cartridge, located next to or around the sensor surface, and in the
same plane as the sensor surface.
[0047] The term "essentially parallel" as used herein refers to
movement of particles or to magnetic fields or magnetic field
gradients. As the movement of magnetic particles is determined by
magnetic field gradients there is often a relationship between the
flow and the magnetic filed gradient orientation. With respect to
flow, the term refers to orientations corresponding to an angle of
less than 45.degree., or less than 20, less than 10 or less than
5.degree.. With respect to magnetic fields or magnetic filed
gradients the term generally only relates to a component of the
field or gradient which has an orientation corresponding to an
angle of less than 45.degree., or less than 20, less than 10 or
less than 5.degree. with the sensor surface. Notwithstanding the
above, the magnetic vector, e.g. magnetic field gradient, may make
other angles with the substrate or the sensor surface, e.g. higher
than 45.degree. such as 85.degree. or higher.
[0048] Typically, in detection and/or quantification methods and
systems making use of magnetic particles, the magnetic particles
are provided with the analyte (or sample comprising analyte) or a
probe, and detection is based on the reaction with a probe or the
analyte, respectively, which can be bound to a surface. The present
invention provides methods and systems wherein magnetic particles
are moved through a reaction chamber, based on the application of
one or more magnetic fields, of which the movement is essentially
parallel to a sensor surface provided within the reaction
chamber.
[0049] The force exerted on a magnetic particle by a magnetic field
is given by the gradient of the potential energy of the particle in
the magnetic field. The magnetic potential energy can be calculated
by taking the integral of the magnetization and the field (see for
example J. D. Jackson, Classical Electrodynamics, John Wiley &
Sons, Inc., 1999). As a result, the force on a magnetic particle
relates to the gradient of the magnetic field. In other words, a
magnetic particle has a tendency to move from a region of lower to
a region of higher magnitude of the magnetic field. Providing a
magnetic field with a gradient which is essentially parallel to the
sensor surface or has at least a component parallel to the sensor
surface allows the movement of the magnetic particles, from an area
next to or within the sensor surface, across the sensor surface, so
as to allow the molecules present on the magnetic particles to bind
with the molecules present on the sensor surface. The lateral
movement of particles is generally obtained by a magnetic field
with its largest gradient component parallel to the sensor surface.
However, any magnetic field gradient differing from perpendicular
to the sensor surface can be used as long as it has a component
parallel to the sensor surface. In addition, the same or another
magnetic field gradient which is essentially parallel to the sensor
surface or has at least a component parallel to the sensor surface,
ensures removal of the particles not bound, or aspecifically bound
to the sensor surface. In this way, the one or more magnetic fields
ensure and speed up both the contacting and the washing step and
minimizing reagent use.
[0050] Alternatively, a field can be generated that also has a
gradient component perpendicular to the sensor surface and wherein
said component is directed from the reaction chamber (2) to the
sensor surface (5). This component keeps the magnetic particles
close to the sensor surface and enhances the exposure of the sensor
surface to the magnetic particles. Meanwhile, aspecific sticking of
particles to the sensor surface can be avoided, e.g. by optimizing
the properties of the coating of the sensor surface, the coating of
the particles, the fluid composition, and by limiting the size of
the perpendicular magnetic force. In addition or as an alternative
to this perpendicular magnetic force, other means can be provided
to maintain or bring the magnetic particles to the sensor surface,
such as a decreased size of the reaction chamber at the sensor
surface, or other forces such as a fluid flow or an acoustic or
ultrasonic fluid excitation. In a first aspect, the invention
provides a system comprising a reaction chamber having a sensor
surface and at least one region outside the sensor surface and
further comprising one or more means for applying one or more
magnetic fields, which is/are placed such that the flow of
particles induced by the magnetic field is essentially parallel to
the sensor surface and spans the sensor surface and the one or more
regions outside the sensor surface.
[0051] According to the present invention, the system uses one or
more magnetic fields, each of which is generated by one or more
magnetic field generating means. Different types of magnetic field
generating means are envisaged in the context of the present
invention, such as permanent magnets, electromagnets, coils and/or
wires. The strength of the magnetic force on the particles should
be such that the induced travel distance is larger then the
distance traveled without magnetic fields, i.e. the magnetic forces
should be dominant over translational Brownian motion. Most
particularly, the magnetic field generated by the magnetic field
generating means ensure that the travel distance of the magnetic
particles (upon activation) of the magnetic field generating means
is larger than the size of the sensor surface.
[0052] According to the present invention, the magnetic field(s)
generated by the one or more magnetic field generating means can be
constant, pulsating, or can vary in strength. Moreover, where more
than one magnetic field is generated, their exact orientation may
be fixed or may vary, provided that the field gradient is
essentially parallel to the detection surface or has at least a
component parallel to the sensor surface.
[0053] According to one embodiment the system of the present
invention contains one single means for generating a magnetic
field. An example hereof is shown in FIG. 1. In order to allow
removal of the magnetic particles from the sensor surface, the
magnetic field generating device is placed such that the magnetic
field gradient spans across the sensor surface as well as spanning
at least one region outside the sensor surface. According to an
alternative embodiment, the magnetic field generating means is
placed below the axis of the sensor field, typically just below the
plane of the reaction chamber comprising the sensor surface. In
these embodiments, the generated magnetic field gradient has a
component parallel with the sensor surface as well as perpendicular
to the sensor surface. The parallel component drags the magnetic
particles over the sensor surface. The magnetic field generating
means can be either fixed, spanning the sensor surface and the
region outside the sensor surface within its field simultaneously
or moveable, sequentially moving the magnetic field over the sensor
surface and the region outside the sensor surface. The same effect
can be achieved by fixing the magnet and moving the reaction
chamber in relation thereto.
[0054] According to a particular embodiment, the magnetic field
generating means is an electromagnet. This makes it possible to
avoid mechanical moving of parts in the device. Alternatively
permanent magnets may be arranged to move to and from the reaction
chamber.
[0055] According to a particular embodiment, the magnetic field
generating means is placed below the sensor surface and generates
an essentially out-of-plane field with smaller in-plane
components.
[0056] According to another embodiment the system of the present
invention makes use of more than one magnetic field generating
means for generating a magnetic field. A non-limiting embodiment of
a system with two magnetic field generating means (3) and (3') is
illustrated in FIG. 2. In this illustrated embodiment, the device
comprises a reaction chamber with a sensor surface and a first (6)
and a second (6') region outside the sensor surface. The
alternating activation of both magnets allows the movement of
magnetic particles back and forth over the sensor surface from a
first region outside the sensor surface (6) to a second region
outside the sensor surface (6'). Alternatively, the magnetic field
of the magnetic field generating devices spans the sensor surface
and one region outside the sensor surface, and the magnetic
particles can be moved back and forth between the sensor surface
and the two magnetic field generating means. In an alternative
embodiment, a magnetic field generating means placed below the
sensor surface generating an out-of-plane field is combined with a
magnet placed in the plane of the sensor surface for generating an
in-plane field.
[0057] Yet a further aspect of the present invention provides a
system comprising a reaction chamber having a sensor surface and at
least one region outside the sensor surface, and the magnetic field
generating means are placed in such a way that they allow the
generation of magnetic fields of different strength and/or
orientation, depending on the assay step. One particular embodiment
of this aspect of the invention provides for a system wherein the
magnetic field generating means ensure a first magnetic field for
the contacting step (movement of the particles towards and/or
across the sensor surface) and a second, stronger magnetic field,
for the washing step (removal of unbound particles from the sensor
surface). According to one embodiment, the device of the invention
comprises a first and a second region outside the sensor surface
and one magnetic field generating device placed so as to generate a
magnetic field between the first region outside the sensor surface
and the sensor surface, and a second magnetic field generating
device such that the magnetic field between the sensor surface and
the second region outside the sensor surface. In another embodiment
both magnetic field generating devices are placed so that their
respective magnetic field gradients are oriented in opposite
directions between a region outside the sensor surface and the
sensor surface and alternating activity of the magnetic field
generating devices allows movement back and forth to the sensor
surface (as described above).
[0058] In accordance with a specific embodiment, the second
magnetic field can also be used for magnetically-controlled reagent
release. For example, magnetic particles are concentrated at a
location in the reaction chamber by a concentrating magnetic field,
either before or after exposure to the sample fluid. At a
controlled time, the concentrating magnetic field is switched off
and the magnetic field that generates lateral transport of magnetic
particles over the sensor surface is switched on.
[0059] According to the present invention, systems are provided
which ensure the movement of magnetic particles essentially
parallel to the sensor surface in a magnetic field gradient that
has at least a component parallel to the sensor surface. It is
nevertheless envisaged that the magnetic fields of the present
invention can be combined with other forces for moving/immobilizing
magnetic particles. Examples of other forces envisaged in this
context are other (non parallel) magnetic fields, electrical
fields, acoustic forces, hydrodynamic forces, gravitational forces
. . . etc. Thus, according to one embodiment, the devices of the
present invention comprise magnetic field generating devices which,
in addition to the magnetic field gradient which is essentially
parallel to the sensor surface, allowing a lateral movement of the
particles over the sensor surface, generate magnetic fields that
cause movements of the magnetic particles perpendicular to the
sensor surface, which can be alternated with the lateral movement.
This can be of interest in the context of stirring, to remove
unbound or aspecific bound magnetic particles or to improve binding
to the sensor surface. Examples of the use of magnetic fields in
the context of stirring are described in U.S. Pat. No. 5,445,971,
U.S. Pat. No. 6,548,311, U.S. Pat. No. 6,180,418, Perrin et al.
(1999), J. Immun. Meth. 224, 77-87; Luxton et al. (2004) Anal.
Chem. 76, 1715-1719; and Ferreira et al. (2005) Appl. Phys Lett.
87, 013901. Typically, in the detection and/or quantification
devices of the present invention, the sensor surface is placed on
the horizontal bottom plane of the reaction chamber. Thus, the
magnetic field gradient(s) essentially parallel to the sensor
surface are also horizontal. It is however also envisaged that the
bottom of the reaction chamber and the sensor surface are inclined,
or that the sensor surface in the system is present on a vertical
wall of the reaction chamber. In such a configuration one or more
magnetic field generating devices can be used in combination with
the gravitational force on the magnetic field. According to a
particular embodiment, a system is provided comprising a sensor
surface on a vertical wall of a reaction chamber and one magnetic
field generating device which can be switched on and off. When the
magnetic field generating device is switched on, magnetic particles
are pulled upwards by the magnetic field generating device, over
the sensor surface, allowing specific binding of the magnetic
particles to the sensor surface. By switching the magnet off,
specifically bound magnetic particles will remain bound to the
vertical surface and aspecifically bound particles are pulled
downward by gravity. A similar effect can be achieved where the
wall of the reaction chamber comprising the sensor surface is
inclined. The combination with the gravitational force on the
magnetic particles (in the absence of a magnetic field) makes it
possible to make one of the steps (such as the contacting step)
less stringent. For instance, where the sensor surface is inclined
and the magnetic particles can be applied at the top of the
inclination, the particles will roll or slide by gravity over the
sensor surface. The particles can then be pulled further down or
back up the inclination using a magnetic field generating device.
This will allow removal of the non-specifically bound particles. As
an alternative to gravitational forces which are small, the
particles can also be manipulated by a fluid flow or an acoustic or
ultrasonic fluid excitation, generating a hydrodynamic shear force
to the particles and removing non-specifically bound particles.
[0060] Using the magnetic field generating devices, e.g. as known
in the art, different methods are available to achieve a maximal
interaction of the magnetic particles with probes on the sensor
surface. According to one embodiment, the magnetic field(s) is/are
applied in such a way that the magnetic particles slide or roll
over the magnetic surface. Additionally or alternatively, magnetic
field generating devices are used which can be turned on and off.
In particular embodiments, one or more magnetic field generating
means are provided which can be reversed one or more times. By
making use of one or more of these features, the sensitivity of the
assays performed with the devices of the present invention can be
increased. For instance, by manipulating the magnetic field
generating means it is made possible to ensure that the particles
can pass one or more times forwards and backwards over the sensor
surface, which increases the chance of a magnetic particle to bind
to the sensor surface. It is also possible to decrease the size of
the reaction chamber near the sensor surface to improve binding of
magnetic particles to the sensor surface.
[0061] According to the present invention, systems are provided
which allow quicker movement of magnetic particles to and from the
sensor surface. The sensor surface is typically a specially
derivatized surface to which molecules, more particularly probes
can be bound. Examples of suitable surfaces include, glass, metal,
plastic, an organic crystal or an inorganic crystal (e.g. silicon),
an amorphous organic or an amorphous inorganic material (e.g.
silicon nitride, silicon oxide, silicon oxinitride, aluminum
oxide). Suitable surface materials and linking chemistries are
known to the person skilled in the art, and are described for
instance in "Diagnostic Biosensor Polymers", by A. M. Usmani and N.
Akmal, American Chemical Society, 1994 Symposium Book Series 556,
Washington D.C., USA, 1994, in "Protein Architecture, Interfacing
Molecular Assemblies and Immobilization Biotechnology", edited by
Y. Lvov and H. Mhwald (Marcel Dekker, New York, 2000), in "The
Immunoassay Handbook" by David Wild (Nature Publishing Group,
London, 2001, ISBN 1-56159-270-6) or "Handbook of Biosensors and
Electronic Noses. Medicine, Food and the Environment" by
Kress-Rogers (ISBN 0-8493-8905-4). Supports for coupling proteins
to coated and uncoated plastic and glass supports are disclosed in
Angenendt et al. (2002) Anal Biochem. 309, 253-260. Dufva (2005) in
Biomol Eng 22, 173-184, review the methodology to attach
oligonucleotides and factors influencing this process.
[0062] The invention is generally performed on planar sensor
surfaces (e.g. planar glass biochip), but also can be performed in
a flow-through system (e.g. flow-through sensors made of porous
aluminum oxide, porous silicon, or a porous column containing
microparticles).
[0063] Typically, where the sensor surface is modified for the
binding of probes or analytes thereto, the region outside the
sensor surface is a surface which is not so modified.
Alternatively, the sensor surface differs from the region outside
the sensor surface only in that no molecules have been bound to
that area of the reaction chamber surface.
[0064] Further aspects of the system of the present invention are
mentioned hereafter. The system of the invention will usually
comprise one or more inlet means for introducing sample, magnetic
particles or reagents into the reaction chamber, and optionally an
outlet means for removing reagents, reaction waste, and optionally,
magnetic particles, from the reaction chamber. These can optionally
be coupled to sources comprising each of the reagents. According to
one embodiment, an inlet means for sample and/or magnetic particles
directs the sample and/or magnetic particles to a region outside
the sensor surface or directly to the sensor surface. Additionally
or alternatively, one or more inlet and outlet means are provided
so as to ensure the direct delivery of the sample and/or magnetic
particles and/or other reagents or buffers in the reaction chamber.
The different inlet and/or outlet means can be connected to
connection means such as valves and tubing, which can be driven by
pumps.
[0065] As detailed above, the devices of the invention comprise,
within a reaction chamber, one or more regions outside the sensor
surface. This/these regions can be used to collect the particles at
the start of the assay, or to collect the non-bound particles when
the `washing` step is being performed. For instance, the magnetic
field generated in the device of the invention may span, besides
the sensor surface, one or more regions outside the sensor surface.
The magnetic field can be such that the unbound particles are
pulled back out of the sensor surface and into a region outside the
sensor surface. In some embodiments the physical separation between
the bound and the unbound magnetic particles allows the detection
of both fractions, providing a further control on the method.
[0066] As indicated above, the devices of the present invention are
envisaged to contain one or more regions outside the sensor
surface. In one embodiment, the reaction chamber comprises at least
one region outside the sensor surface which is adapted to the
collection of the magnetic particles prior to the assay, e.g. by
the presence of an indentation or small crevices, or by the
presence of a controllable magnetic field generating means. Such
dedicated regions can also be provided within the sensor
surface.
[0067] According to one embodiment, the reaction chamber comprises
a first region outside the sensor surface where the particles
reside before the assay and a second region outside the sensor
surface, to which the unbound particles are moved (these regions
being e.g. on opposite sites of the reaction chamber as depicted in
FIG. 1). In other embodiments, such as depicted in FIG. 2, the
magnetic particles are moved back and forth from a region outside
the sensor surface to the sensor surface. Optionally, the magnetic
particles can be moved back and forth several times prior to the
detection step.
[0068] The system of the present invention either further comprises
or is used in combination with a detection means, capable of
detecting the binding of magnetic particles to the sensor surface.
Detection of the bound magnetic particles on the sensor surface can
be done by various means, either based on the properties of the
magnetic particles themselves or using a label. The label can be
attached to the magnetic particles, or can be bound to or
incorporated into the analyte.
[0069] Accordingly, the detection means present in or used in
combination with the systems of the present invention are detection
means capable of detecting the relevant signal such as, but not
limited, to a magnetic signal, magnetoresistance, a Hall effect, an
optic signal (reflection, absorption, scattering, fluorescence,
chemiluminescence, RAMAN, etc.), an acoustical signal (quartz
crystal microbalance (QCM), surface acoustic waves (SAW), Bulk
Acoustic Wave (BAW) etc.). These may be generated by liposomes,
micelles, bubbles, microbubbles, microspheres, lipid-, or polymer
coated bubbles, microbubbles and/or microspheres, microballoons,
aerogels, clathrate bound vesicles, and the like. Such vesicles may
be filled with a liquid, a gas, a gaseous precursor, and/or a solid
or solute material.
[0070] Typical labels useful in the context of the present
invention are those labels which are classically used in in vitro
assays such as, but not limited to, chromophoric groups,
radioactive labels, electroluminescent, chemiluminescent,
phosphorescent, fluorescent or reflecting labels.
[0071] Magnetic particles used in the present invention can be
completely inorganic or can be a mixture of an inorganic and an
organic material (e.g. a polymer). Accordingly, labels can be
attached via the inorganic or via the organic component at the
outside or can be incorporated into the particle.
[0072] Magnetic particles are widely used in biological analysis,
e.g. in high-throughput clinical immunoassay instruments, sample
purification, cell extraction, etc. Several diagnostic companies
(Roche, Bayer, Johnson & Johnson, Abbott, BioMerieux, etc.)
fabricate and sell reagents with magnetic particles, e.g. for
immunoassays, nucleic-acid extraction, and sample purification.
Magnetic particles are commercially available in various sizes,
ranging from nanometers to micrometers. For attachment or binding
of the particles of the invention to the bioactive molecules, the
particles may carry functional groups such as hydroxyl, carboxyl,
aldehyde or amino groups. These may in general be provided, for
example, by treating uncoated monodisperse, superparamagnetic
particles, to provide a surface coating of a polymer carrying one
of such functional groups, e.g. polyurethane together with a
polyglycol to provide hydroxyl groups, or a cellulose derivative to
provide hydroxyl groups, a polymer or copolymer of acrylic acid or
methacrylic acid to provide carboxyl groups or an aminoalkylated
polymer to provide amino groups. U.S. Pat. No. 4,654,267 describes
the introduction of many such surface coatings. Other coated
particles may be prepared by modification of the particles
according to the U.S. Pat. Nos. 4,336,173, 4,459,378 and 4,654,267.
For example, macroreticular porous polymer particles, prepared from
styrene-divinylbenzene and with a diameter of 3.15 um were treated
with HNO.sub.3 to introduce-NO.sub.2 groups at the surface of the
pores. Then the particles were dispersed in an aqueous solution of
Fe. The Fe.sup.2+ is oxidized by the NO.sub.2 groups which leads to
precipitation of insoluble iron oxy-hydroxy compounds inside the
pores. After heating the iron exists as finely divided grains of
magnetic iron oxides throughout the volume of the porous particles
The NO.sub.2 groups are reduced by the reaction with Fe to NH.sub.2
groups. To fill up the pores and to introduce the desired
functional groups at the surfaces, different monomers are caused to
polymerize in the pores and at the surface. In the case of a
preferred type of particle, the surface carries-OH groups connected
to the polymeric backbone through (CH.sub.2CH.sub.2O) 8-10
linkages. Other preferred carry --COOH groups obtained through
polymerization of methacrylic acid. For example, the NH.sub.2
groups initially present in the particles may be reacted with a
diepoxide as described in U.S. Pat. No. 4,654,267 followed by
reaction with methacrylic acid to provide a terminal vinyl
grouping. Solution copolymerization with methacrylic acid yields a
polymeric coating carrying terminal carboxyl groups. Similarly,
amino groups can be introduced by reacting a diamine with the above
product of the reaction with a diepoxide, while reaction with a
hydroxylamine such as aminoglycerol introduces hydroxy groups.
[0073] The coupling of a bioactive molecule to a particle can be
irreversible but can also be reversible by the use of a linker
molecule for the crosslinking between particle and bioactive
molecule. Examples of such linkers include peptides with a certain
proteolytic recognition site, oligonucleotide sequences with a
recognition site for a certain restriction enzyme, or chemical
reversible crosslinking groups as those comprising a reducible
disulfide group. A variety of reversible crosslinking groups can be
obtained from Pierce Biotechnology Inc. (Rockford, Ill., USA).
[0074] According to a particular embodiment, the sensor of the
detection unit is integrated into the reaction chamber (e.g.
magnetoresistive sensor is integrated), which can be provided as a
disposable cartridge. Alternatively, the sensor is provided as
separate part from the reaction chamber (e.g. optical unit). In
this embodiment, the reaction chamber optionally comprises a
detection window, which allows the detection of the signal of the
magnetic particles and/or labels bound to the sensor surface. The
location of the detection window is of course determined by the
location of the sensor surface and the detection means. Most
particularly, the detection window is opposite to the sensor
surface. Alternatively, sensor surface of the reaction chamber is
provided on the detection window. Where the detection is based on
magnetic field or optical methods, the material of the reaction
chamber may render the provision of a specific detection window
superfluous. Additionally or alternatively, the systems of the
present invention provide for the detection of magnetic particles
in a region outside the sensor surface. This can be of interest in
the context of controls or validation measurements (see below), or
where the detection method is based on a competitive binding at the
detection surface. Where appropriate, the systems of the present
invention are thus provided with an additional detection window
and/or an additional detection means or a means which can
simultaneously or sequentially detect a signal both on the sensor
surface and in a region of the reaction chamber outside the sensor
surface.
[0075] Typically the system of the present invention is a
single-chamber (bio)sensor, with low reagent use and small required
sample volume. A (bio)sensor in accordance with the present
invention comprises can be operated with a minimum of equipment,
washing steps and buffers.
[0076] Another aspect of the present invention provides an
optionally disposable cartridge comprising a reaction chamber
having a sensor surface and at least one region outside the sensor
surface. The disposable cartridge can further comprise magnetic
particles integrated therein or these can be provided separately.
The material of the cartridge is such that magnetic fields can be
generated therein. For example, the cartridge is made of glass or a
synthetic material, such as plexiglass [poly(methy)methacrylate] or
clear PVC (polyvinyl chloride) or PC (polycarbonate) or COP (e.g.
Zeonex) or PS (polystyrene).
[0077] The cartridge can further comprise at least one magnetic
field generating device or a part thereof, which can be used in the
generation of a magnetic field gradient essentially parallel to the
detection surface within the reaction chamber or having at least a
component parallel to the detection surface.
[0078] According to yet another aspect the invention provides
methods whereby the systems and/or cartridges of the present
invention are used for the detection and/or quantification of
analytes. The methods of the present invention thus generally
comprise providing within a reaction chamber having a detection
surface and a region outside the detection surface, an
analyte-specific probe, magnetic particles and a sample believed or
known to comprise the analyte and applying at least one magnetic
field gradient to the particles, which is essentially parallel to
the sensor surface or has at least a component parallel to the
sensor surface. The methods performed in accordance with the
present invention are rapid, sensitive, and robust.
[0079] The methods of the present invention are applicable in the
detection of any molecule, more particularly biomolecules such as
DNA, RNA, proteins, carbohydrates, lipids and organic anabolites or
metabolites. The nature of the sample comprising the analyte to be
detected is not critical and can be for instance any sample of a
living or dead organism (body fluid such as, but not limited to
blood or urine, hair, stool, etc.), an environmental sample (water,
soil, plant material), food or feed products or products used in
the manufacturing thereof, a sample of a chemical reaction process
etc. . . . . The detection can be performed on a sample which is
the result of a pre-processing step such as a semi-purification,
purification, semi-purification and/or amplification of the
analyte. According to a particular embodiment, the analyte is a
single stranded nucleotide sequence, which has been amplified using
PCR.
[0080] The methods of the present invention provide for a detection
of an analyte based on the reaction/binding of the analyte with an
analyte-specific probe. Typical specific interactions include
DNA/DNA or DNA/RNA binding, protein/protein, protein/DNA and
protein/carbohydrate interactions antibody/antigen interactions,
receptor/ligand binding. Also synthetic molecules can be used to
detect an analyte (e.g. enzyme inhibitors, pharmaceutical
compounds, lead compounds isolated from library screenings).
Accordingly, examples of analyte-specific probes include but are
not limited to oligonucleotides, antibodies, enzyme substrates,
receptor ligands, lectins etc. . . . . Examples of envisaged sensor
surface and analyte interactions are a sensor surface comprising
antibody probes to detect antigens in a sample or antigens attached
as probe to the sensor surface for the detection of antibodies in a
sample.
[0081] According to a particular embodiment of the invention, the
analyte-specific probe is an analyte-specific oligonucleotide, i.e.
an oligonucleotide comprising a sequence which is complementary to
a sequence specific for the analyte.
[0082] Different assay principles are envisaged in the detection
methods of the invention. In one embodiment, the detection and/or
quantification method used is a direct detection method. For
example, the analyte present in the sample is bound to magnetic
particles, which are moved across a sensor surface comprising an
analyte-specific probe and the signal generated by the particles
bound to the sensor surface is directly proportionate to the number
of magnetic particles bound to the sensor surface, and thus to the
presence (and/or amount) of analyte present in the sample.
Alternatively the detection can be based on competitive binding of
the analytes to the sensor surface. For example, the sensor surface
is first provided with magnetic particles bound to the
analyte-specific probe on the sensor surface through an
analyte-like compound (weak binding to the sensor surface) and upon
contacting with magnetic particles to which the analyte is linked,
displacement of the analyte-like compound takes place. Where the
magnetic particles comprising the analyte-like compound are
labeled, the signal is inversely proportionate to the amount of
analyte in the sample. Alternatively, the signal of the magnetic
particles not bound to the sensor surface can be measured in a
region outside the sensor surface. In other embodiments, the
detection of the analyte is envisaged to require the addition of
further reagents such as secondary antibodies, labels, substrates
etc. . . . . More specifically for specific analytes (large
molecules, e.g. proteins, possessing at least two epitopes)
sandwich assays can be envisaged. In a sandwich assay, molecules of
interest (proteins) from an applied sample fluid are trapped
(`sandwiched`) between a probe (a first antibody) on a biologically
active sensor surface and a biologically active molecule (a second
antibody) with a label (a magnetic particle). Although the molecule
of interest is the protein sandwiched between the two antibodies,
the antibody on the sensor surface and the protein act as the probe
and the labeled second antibody as an analyte according to the
terminology of the present invention. In another embodiment of a
sandwich assay, the antibody on the sensor surface acts as the
probe and the labeled second antibody and the protein act as an
analyte according to the terminology of the present invention.
[0083] Thus, as indicated above in a particular embodiment of the
methods of the invention, one or more analyte-specific probes are
bound to the sensor surface and the analyte is provided on the
magnetic particle. In this embodiment, the analyte is bound to the
magnetic particle either directly (non-specific adsorption of the
sample or a pre-processed fraction thereof to the magnetic
particle) or specifically (through the use of an analyte-specific
probe, attached to the surface of the molecule).
[0084] The magnetic particles suitable for use in the methods and
devices of the present invention are known to the skilled person.
Magnetic particles of different size (10 nm to 5 .mu.m, typically
between 50 nm and 1 .mu.m), shape (spheres, spheroids, rods),
composition (see before in this application), and magnetic
properties (magnetic, paramagnetic, superparamagnetic,
ferromagnetic, i.e. any form of magnetism which has a magnetic
dipole in a magnetic field, either permanently or temporarily) have
been described. It is envisaged that different types of magnetic
particles, e.g. with different magnetic and/or optical properties
can be used simultaneously within one reaction chamber (magnetic
particle multiplexing).
[0085] Attachment of the analytes or probes to the surface of
magnetic particles can be performed by methods described in the
art, as described above in this application.
[0086] According to one embodiment, the invention provides methods
for the detection and/or quantification of an analyte, comprising
the step of contacting a sample comprising an analyte with magnetic
particles and subjecting the magnetic particles to a magnetic field
in a reaction chamber (2) with a sensor surface (5) comprising an
analyte-specific probe (8). The step of subjecting the magnetic
particles to a magnetic field is performed such that the magnetic
particles are moved laterally over the sensor surface (5), whereby
the movement of magnetic particles (7) spans between at least one
region outside the sensor surface (6) and the sensor surface, and
detecting the signal generated by the binding of the magnetic
particles to the sensor surface. Depending on whether or not the
magnetic particles are present within the reaction chamber, the
method can further comprise the step of introducing magnetic
particles within the reaction chamber. Typically, the magnetic
particles are applied to first a region outside the sensor surface
and, the one or more magnetic fields ensure the movement of the
particles from the first region outside the sensor surface to the
sensor surface and laterally over at least part of the sensor
surface (5). Alternatively, the magnetic particles are introduced
into the reaction chamber in a random fashion (e.g. magnetic
particles, to which analyte has been bound are applied to a
reaction chamber and than shaken) and magnetic forces can be used
to attract the magnetic particles to the sensor surface. In yet
another alternative embodiment the magnetic particles are
introduced into the reaction chamber directly on the sensor
surface.
[0087] It is envisaged that the methods of the present invention
are performed using reaction chambers comprising an
analyte-specific probe bound thereto. Alternatively however, the
methods of the present invention further encompass the step of
providing an analyte-specific probe on the sensor surface.
Depending on the nature of the material of the sensor surface, the
binding of the analyte-specific probe to the sensor surface will
require a specific chemical reaction and/or a blocking step.
[0088] In the methods of the present invention the binding of the
analyte to the magnetic particles (7) is either performed within or
outside the reaction chamber. Thus in a particular embodiment, the
reaction chamber comprises magnetic particles in a region outside
the sensor surface and the methods further comprise the step of
introducing the sample into the reaction chamber in the same region
outside the sensor surface thereby contacting the sample with the
magnetic particles. Alternatively, the methods of the invention
comprise the step of contacting the sample with the magnetic
particles outside the reaction chamber and introducing the magnetic
particles to which the sample/analyte has bound in the reaction
chamber.
[0089] According to a particular embodiment, the one or more
magnetic fields further ensure the movement of the particles not
bound to the sensor surface to a second region outside the sensor
surface (6). Optionally the first and the second region outside the
sensor surface are the same (i.e. movement is back and forth).
[0090] As detailed above, the movement of the magnetic particles
can be ensured using one magnetic field. Alternatively, the methods
of the invention comprise the generation of different magnetic
field gradients essentially parallel to the detection surface or
having at least a component parallel to the detection surface for
the contacting of the analyte with the sensor surface (movement of
particles towards sensor surface) and the removal of the unbound
magnetic particles (movement of particles away from sensor
surface). Moreover the essentially parallel magnetic field
gradients or having at least a parallel component can be alternated
or combined with other movements of the particles generated by e.g.
magnetic, electrical, hydrodynamic, sonic, or gravitational forces
in the context of washing steps, introduction of the magnetic
particles into the reaction chamber and/or removal of the particles
from the reaction chamber (as described for the systems of the
invention above).
[0091] The methods of the present invention involve the contacting
of sample with magnetic particles and the movement of particles
within a reaction chamber. Magnetic particles can be part of a
fluid reagent or of a dry reagent. Besides magnetic particles, the
reagent can for example contain buffer salts, detergents,
biomolecules that assist in the biological interactions, etc. These
steps can be performed in a liquid, which is any liquid compatible
with the reagents used (i.e. analyte, analyte-specific probe,
label), such as standard buffers, or minimally pre-treated or even
pure sample (e.g. blood or saliva). Liquid can be introduced in the
reaction chamber for rinsing purposes. A liquid current can also be
applied to provide a flow contrary to the movement of the magnetic
particles. Such a flow can be applied with pumps but also without
pumps using an electric field to generate electro-osmosis.
[0092] Alternatively, the methods are performed with a minimum
amount of liquid at the detection surface and optionally the region
outside the detection surface. To avoid spreading of irrelevant
sample components which can interfere with binding or detection,
the sample can be applied in a solution or matrix which avoids such
movement, such as glycerol or in a gel which disintegrates in the
reaction chamber.
[0093] Upon application of a magnetic field only the magnetic
particles will move while buffer, and other components of the
sample which can interfere with binding or detection (e.g. traces
of a body fluid such as blood or urine) remain at the site of
application.
[0094] The presence of the liquid will influence the strength of
the magnetic field required to move the particles For example, the
flow resistance is lower in a clean buffer than in a sample with
high viscosity, e.g. saliva.
[0095] When the magnetic particles are supplied in a small volume
at a distance removed from the sensor surface, no or minimal
interchange by diffusion will occur between the liquid wherein the
magnetic particles are supplied and the liquid which is present at
the sensor surface.
[0096] The methods of the present invention further comprise a
detection step, which allows the detection and/or quantification of
the magnetic particles to the sensor surface. The detection step is
ensured using one or more detection means as described for the
systems of the invention above.
[0097] According to the present invention, the binding between,
e.g. an analyte present on the surface of a magnetic particle and
an analyte-specific probe on the sensor surface of the reaction
chamber ensures that these magnetic particles are immobilized on
the sensor surface. According to one embodiment the magnetic field
of the magnetic particles bound to the sensor surface through the
analyte can be used to detect the presence of the particle on the
sensor surface and consequently of the analyte attached to the
magnetic particle. In addition or alternatively, the detection of a
magnetic particle is performed visually by the presence of the
particle itself or by labels such as chromophoric groups in or on
the particle.
[0098] According to a particular embodiment, the sensor surface
comprises one analyte-specific probe. Alternative embodiments are
also envisaged whereby a variety of analyte-specific probes are
arrayed on the sensor surface to allow simultaneous detection of
different compounds in a sample. (sensor multiplexing) (a sensor
array with different capture molecules). Another modification is
chamber multiplexing. Herein a system has multiple reaction
chambers. In one embodiment, the sample is spread over different
chambers to run different assays in parallel. Alternatively,
magnetic particles which passed over a first sensor surface in a
first reaction chamber are further transported to a next reaction
chamber wherein they can react with probes on a next surface sensor
(system with several reaction chambers).
[0099] As detailed above, the methods of the present invention
involve the use of a reaction chamber comprising a sensor surface
and at least one region outside the sensor surface.
[0100] In the methods and devices of the present invention,
magnetic particles are concentrated near the sensor surface,
thereby reducing the time needed to allow binding between probe an
analyte and increasing the chance of the binding of a rare analyte
to a probe. During the flow-over transportation, the magnetic
particles have a dynamic movement (driven by a magnetic field
gradient, magnetophoresis), combined with translational and
rotational diffusion of the particles.
[0101] Non-homogeneous magnetic particle solutions, e.g. due to a
large size distribution of particles or the presence of clusters of
sticking particles, can cause large signal variations and signal
offsets in standard assays with magnetic particles. The methods
performed in accordance with the present invention are to a large
degree not influenced by the presence of a fraction of large
magnetic particles or magnetic particle clustering, due to the
lateral magnetic force on the particles, which is higher for
particles with higher magnetization. This stronger magnetophoretic
force gives the larger particles a higher velocity, which reduces
the residence time near to the sensor surface. In addition, larger
particles have a higher average travel height above the sensor
surface due to the strong hydrodynamic friction near to the
surface, which further reduces the number of collisions with the
sensor surface. As a result, larger magnetic units will have a
reduced interaction with the sensor surface compared to smaller
particles. In the methods of the present invention, the binding of
large magnetic particles or large clusters to the sensor surface is
inherently suppressed, which makes the detection more reliable.
Magnetic particles can also temporarily form multi-particle
structures in a magnetic field, due to dipole-dipole interactions.
For example, chains of particles can form, oriented along the lines
of the magnetic field. Such structures can change or dissemble when
the field is removed and/or when the chain is excited by other
forces. In the method of the present invention, the lateral
movement of particles and multi-particle structures provides a
source of excitation. The movement along the surface causes a
dynamic process of continuous assembly/disassembly, refreshment,
and reformation of multi-particle structures. As a result, there is
good contact between the particles and the sensor surface.
[0102] The system and methods described in the present invention
can be used as rapid, robust, and easy to use point-of-care
biosensors for small sample volumes. The reaction chamber can be a
disposable item to be used with a compact reader, containing the
one or more magnetic field generating means and one or more
detection means.
[0103] Also, the systems and methods of the present invention can
be used in automated high-throughput testing for centralized
laboratories. In this case, the reaction chamber is e.g. a well
plate or cuvette, fitting into an automated instrument. When
applied in e.g. immunoassays, a minimum number of fluid
manipulation steps are needed, incubation occurs at high speed and
washing steps are reduced to a minimum with a minimal fluid
waste.
[0104] Other arrangements for embodying the invention will be
obvious for those skilled in the art.
[0105] It is to be understood that although preferred embodiments,
specific constructions and configurations, as well as materials,
have been discussed herein for devices and methods according to the
present invention, various changes or modifications in form and
detail may be made without departing from the scope and spirit of
this invention. The invention is illustrated by the Examples
provided below which are to be considered for illustrative purposes
only and the invention is not limited to the specific embodiments
described therein.
EXAMPLES
Example 1
Magnetic Binding Assay
[0106] A region of a polystyrene reaction chamber is coated with
morphine coupled to BSA (grey zone in FIG. 3). Afterwards the
remainder of the reaction chamber is coated with BSA to avoid
aspecific binding to polystyrene. A solution containing magnetic
particles coated with anti-morphine Ab (200 nm Protein G coated
magnetic particles) (black circles in FIG. 3) and magnetic
particles without antibody (white circles in FIG. 3) is applied to
a region outside the sensor surface (top part of FIG. 3A). The
amount of morphine on the sensor surface is sufficient to bind all
antibody coated magnetic particles.
[0107] To avoid any movement of the magnetic particles on the
reaction chamber, they are applied to the reaction chamber in a
solution of low melting agarose. After application the reaction
chamber is filled with buffer. An electromagnet (M in FIG. 3) is
placed under the reaction chamber at the opposite end from the
place where the magnetic particles were applied. The configuration
of the assay before a magnetic field is applied is shown in FIG.
3A.
[0108] The reaction chamber is heated to about 40.degree. C. which
releases the magnetic particles from the agarose gel. The
electromagnet is switched on and generates a magnetic field, which
draws the magnetic particles over the sensor surface. Particles
with antibodies bind to the morphine, while the remainder travels
further towards the electromagnet (FIG. 3B). The magnetic particles
which are collected at the electromagnet are assayed for binding to
morphine. This control shows the efficacy of the assay.
Example 2
Competition Assay
[0109] A pilot experiment was performed using morphine as a
detection agent. Morphine is a small molecule, with only one
epitope, so a competitive assay has to be performed to indicate the
amount of morphine in a sample. On a polystyrene surface (96 wells
titre plate), 1 .mu.l of BSA-morphine (1 mg/ml in phosphate
buffered saline (PBS)) was spotted in a corner of each well and
dried [morphine-3-glucoronide was coupled in excess BSA via its
lysine residues]. After coating, the wells were blocked with 10
mg/ml BSA+0.65% Tween-20 in PBS for 1 hour. Then, the blocking
solution was discarded and 10 mg/ml BSA+0.65% Tween-20 in PBS
containing anti-morphine Ab coated magnetic particles (200 nm
Protein G coated magnetic particles) were applied to the wells
(1:10 dilution of magnetic particles, total amount of solution was
50 .mu.l. The titer plate was placed on a magnet and after 1 minute
pictures were taken (FIG. 4) The magnetic particles are detected by
optical reflection. During this period magnetic particles move over
the place where the morphine is spotted. The magnetic particles
which are specifically bound via the antibodies to the morphine,
remain located at the place where the morphine was applied (arrow
1) (FIG. 4 top panel), while the remaining magnetic particles
(having no antibodies or having antibodies which did not react with
morphine) moved towards the magnet (arrow 2).
[0110] When sufficient morphine (5 or 100 ng/ml in a microtiter
well) was added to the antibody-coated magnetic particles prior to
the application to the titre plate, antibodies were saturated with
morphine and could only bind in a non specific way to the wells of
the plate. The middle and bottom panel of FIG. 4 show that such
interactions are not sufficient to elicit a visible spot of
bounding magnetic particles.
[0111] FIG. 2 shows that morphine in a sample, already at 5 ng/ml,
prevents the binding of the magnetic particle coupled antibodies to
the spotted morphine. There are not enough magnetic particles bound
to elicit a visible spot of bound magnetic particles.
* * * * *