Technical Field of the Invention

The present invention relates to a device and an associated method for accurate measurement of magnetic particles in assay apparatus, especially immunoassay strips.

Background to the Invention

WO2011/128696 describes an immunoassay apparatus in the form of an assay strip. The strip has a reservoir for receiving a fluid, especially a biological fluid, the reservoir containing magnetic particles (or "beads"). The magnetic particles are bonded to the reservoir, and contain probes for a substance of interest, such that when a fluid containing the substance of interest is introduced into the reservoir, the magnetic particles bond preferentially to the substance and flow with the fluid through a microfluidic channel. Traps are provided in a sensing area of the channel, and a magnetic field is applied to draw the particles in the fluid into the traps, where they become concentrated whilst excess fluid flows to an exit point.

WO2011/128696 proposes that the trapped magnetic particles may be quantified by any suitable means, including a hall effect sensor, a capacitive measurement circuit or a magnetoresistor; additionally, or alternatively, photovoltaic cells are proposed to detect the change in incident light falling in the sensing area. No detail is provided as to how the magnetic techniques could work and in practice, optical detection using photovoltaic methods are used. However, these can suffer from magnetic particles overlying one another, such that whilst a qualitative reading of the approximate amount of magnetic particles in the sensing area can be achieved, a quantitative measurement is not possible.

This invention seeks to provide a measurement device that can detect magnetic particles in sensing areas of assay apparatus, especially apparatus of the kind described above.

Summary of the Invention

In its broadest sense, a first aspect of the invention concerns a measurement device comprising a magnetic flux generating means defining at least one gap. At least one magnetic flux detector (for example a hall effect sensor) is arranged to detect changes in flux across the gap. The device comprises means for monitoring the output of the flux detector to detect differences in magnetic permeability of the at least one gap. The measurement device further comprises a holder arranged to hold an assay apparatus such that a sensing area of the assay apparatus is provided in the first gap, whereby the magnetic flux detector quantitatively measures the presence of magnetic particles in the first gap. The device further comprises balancing means to balance the input to, or output from the at least one magnetic flux detector when the holder is empty.

The balancing means may be in the form of magnetic flux generating means, arranged to balance the magnetic flux at the flux detector to zero when the holder is empty. To achieve this, the magnetic flux generating means may comprise body defining one or more loops. Two loops may be provided by two spaced apart limbs and an intermediate portion between the limbs, and a central member extending from the intermediate portion of the body to define a first gap and a second gap with the respective limbs of the body.

The at least one magnetic flux detector may be provided in the at least one gap, or may be provided in another gap in the one or more loops. In particular, the magnetic flux detector may be positioned between the central member and the intermediate portion of the body.

The magnetic flux generator that generates a flux across the gap may be one or more magnet. If two loops are provided at least two magnets may be provided, one associated with each limb and the polarities of the magnets being opposed so that magnetic fields flow across the first and second gaps; whereby the magnetic flux in the central member resulting from the magnet associated with one limb is in the opposite direction to the magnetic flux in the central member resulting from the magnet associated with the other limb.

As an alternative to the balancing means being in the form of magnetic flux generating means, which balances the input to the magnetic flux detector when the holder is empty, balancing means may be provided by electronic balancing means arranged to balance the output from the magnetic flux detector such that a zero reading is obtained by the means for monitoring the output of the flux detector when the holder is empty. Such electronic balancing means can be provided by an adder (digital/analogue) arranged to supply a counterbalancing voltage to the means for monitoring, so as to offset the voltage from the magnetic flux detector. Alternatively, a second magnetic flux detector could be provided in a position which will not be affected by the introduction of magnetic particles into the loop, and the output from the second magnetic flux detector may be subtracted from the output from the first magnetic flux detector to produce a zero reading when nothing is in the holder, which will increase when magnetic particles are detected by the first magnetic flux detector.

According to a preferred embodiment of the invention, there is provided a measurement device comprising a body defining two spaced apart limbs and an intermediate portion between the limbs, and a central member extending from the intermediate portion of the body to define a first gap and a second gap with the respective limbs of the body; a magnetic flux detector positioned between the central member and the intermediate portion of the body and at least two magnets, one associated with each limb; the polarities of the magnets being opposed so that magnetic fields flow across the first and second gaps; whereby the magnetic flux in the central member resulting from the magnet associated with one limb is in the opposite direction to the magnetic flux in the central member resulting from the magnet associated with the other limb; and means for monitoring the output of the flux detector to detect differences in magnetic permeability of the gaps; wherein, the measurement device comprises a holder arranged to hold an assay apparatus such that a sensing area of the assay apparatus is provided in the first gap, whereby the magnetic flux detector quantitatively measures the presence of magnetic particles in the first gap.

This quantitative measurement makes the assay extremely sensitive. The provision of two limbs with magnetic fields flowing in opposite directions, allows the magnetic flux to be balanced out in the region of the flux detector, when there is nothing in either gap. If there are imbalances, balancing means may be provided. Various techniques are known to balance magnetic bridges, some disclosed GB2207510. For example iron screws (or other ferromagnetic materials) can be provided in tapped holes in the body/magnets and inserted/retracted to balance the flux at the detector.

The intermediate portion and/or the central member may comprise a region that is thicker than the limbs to provide an easy path for the magnetic field.

The two magnets associated with the limbs may be permanent magnets or electromagnets (electromagnets can make the balancing easier by independently supplied the coils).

This balance means that a sensitive flux detector, such as a hall effect sensor, e.g. a high sensitivity hall effect sensor, with a sensitivity of over 300V/AT may be used. The measurement device may comprise an amplifier and the output can be highly amplified to detect small changes in magnetic permeability in the first gap caused by magnetic particles. The magnetic particles may for example be paramagnetic nanoparticles, each only producing a small amount of magnetism and only when in a larger magnetic field.

The measurement device may comprise a further magnetic field source arranged to concentrate magnetic particles in one or more traps in the sensing area of the assay apparatus. Such a magnetic field source may be arranged such that its magnetic field is orthogonal to the field of the magnetic flux across the first gap. This aids in drawing the magnetic particles into the sensing area.

The holder may comprise a slot, and/or a groove, arranged to receive the assay apparatus, which may be an assay strip, or a cuvette. A second aspect of the invention comprises a kit of parts comprising a measurement device as set out in the first aspect of the invention and at least one assay apparatus, the assay apparatus comprising a sample receiving area and a sensing area in fluid connection therewith, the measurement device and assay apparatus arranged such that when placed in the holder the sensing area of the assay apparatus is provided in the first gap.

The sensing area may comprise one or more traps to trap magnetic particles.

Magnetic particles comprising selective receptors may be bonded to the assay apparatus upstream of the sensing area (for example in the sample receiving area, or a channel between that area and the sensing area) so as to be selectively released by a sample of interest and flow to the sensing area. The magnetic particles bound to the surface may be released into the fluid by becoming attached to the substance of interest or by being displaced by the substance of interest. This may occur though a specific bonding substance such as an antibody. The magnetic particles may be magnetic nanoparticles. The magnetic particles may be tagged and/or coated with selective receptors. The selective receptors may selectively bind to specific markers, for example cardiac markers, such as Troponin.

The assay apparatus may comprise an outlet in fluid connection with the sensing area, to which fluid is arranged to flow, with magnetic particles remaining in the sensing area.

The assay apparatus may be an assay strip. The or each fluid connection may be provided by a microfluidic channel. The sensing area may have a volume of less than ΙΟμΙ., preferably less than 5μ The fluid described may be a liquid or gas, and may be a biological fluid such as a body fluid.

Substances of interest may include naturally occurring substances, substances that are the result of a chemical or biological reaction, such as drug by-products, and substances introduced into a fluid sample. The substance may be a compound, especially a molecule and could be, for example a protein, hormone or DNA section.

By "magnetic" particles is to be understood particles of non-zero magnetic susceptibility. The or each magnetic particle may be ferromagnetic, diamagnetic, paramagnetic or superparamagnetic. A homogeneous or heterogeneous mixture of such particles may be employed. In one embodiment the or each particle is formed from iron oxide. Particles of size in the range 5 nanometres to 100 micrometres may be used or in some embodiments particles of size in the range 5 nanometres to 50 micrometres may be used.

The or each particle may become attached to a substance of interest by means of a further substance, referred to as a bonding substance. The or each particle may be coated with the bonding substance. The bonding substance may be a protein, and in some embodiments it is an antibody or probe (ligand).

The or each magnetic particle may be coated with a material to facilitate adherence of a bonding substance to the particle. A suitable coating material is polystyrene.

By appropriate selection of the bonding substance it is possible to arrange for magnetic particles to attach to a variety of substances of interest or to be displaced by a variety of substances of interest. The or each magnetic particle may be arranged so that it can only become attached to or displaced by a single unit of a substance of interest, for example a single molecule. As such each particle may be provided with a single antibody or capture probe.

Advantageously there is no requirement for a secondary antibody capture site to "collect" the magnetic particles together for sensing. Additionally, there is no requirement for complex alignment systems between sensor and the sensing area to give accuracy and consistency. The microfluidics are designed to guide and focus the particles towards the sensing area where the combination of the microfluidic channel particle trap and the applied magnetic field act to concentrate all free magnetic particles in the sensing area. In a particular embodiment of the present invention, the entire fluidic chamber may be less than 500nm in depth in order to ensure a monolayer of magnetic particles in flow through the entire microfluidic chamber.

A third aspect of the invention provides a method of measuring the quantity of magnetic particles in the sensing area of an assay apparatus, the method comprising providing a loop and generating a magnetic flux through the loop, providing a magnetic flux detector in the loop and providing a gap in the loop; introducing an assay apparatus into the gap and measuring the change in magnetic flux in the region of the magnetic flux detector to determine the quantity of magnetic particles in the gap.

The method may comprise zeroing the output from the magnetic flux detector before introducing the assay apparatus.

The method may comprise generating a counterbalancing magnetic flux in the region of the magnetic flux detector to zero the output. The loop, the magnetic flux detector, the gap in the loop, and the counterbalancing magnetic flux in the region of the magnetic flux detector may be provided by providing a measurement device according to the first aspect of the invention (optionally including any optional features thereof), and/or, the assay apparatus that is introduced into the gap may be an assay apparatus as set out in the second aspect of the invention (optionally including any optional features thereof).

Alternatively, the output may be zeroed electronically, for example, a digital or analogue adder may generate a counterbalancing voltage to zero a voltage output by the magnetic flux detector, before the assay apparatus is introduced. As a further alternative, a counterbalancing output may be provided by a second magnetic flux detector arranged in the gap, but positioned such that introduction of the assay apparatus does not affect its output. That is to say, a first magnetic flux detector may be arranged in the gap such that introduction of an assay apparatus will result in magnetic particles in one or more traps in the assay apparatus disturbing the magnetic flux it reads, and a second magnetic flux detector may be arranged in the gap such that the traps in the assay apparatus are sufficiently far away that any magnetic particles therein do not affect the magnetic flux it reads.

The method may further comprise the steps of passing a fluid (optionally a biological fluid) over a surface of the assay apparatus having a quantity of magnetic particles bound thereto, the magnetic particles free to be released into solution either in response to the presence of a substance of interest in the fluid or in response to the absence of a substance of interest in the fluid; introducing the fluid and any magnetic particles released into the fluid into a microfluidic channel or chamber, having a sensing area (optionally comprising one or more particle traps) wherein the released magnetic particles thus become concentrated in sensing area (optionally in the one or more traps).

This could be done after introducing the assay apparatus into the gap, but is preferably done beforehand. Detailed Description of the Invention

In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 shows a schematic view of an exemplary assay apparatus for use in the method of the invention;

Figure 2 shows a side view of a measurement device according to the invention;

Figure 3 shows an exploded isometric view of the measurement device of figure

2;

Figure 4 shows the magnetic bridge of figures 2 and 3 with a further magnetic field source, in a first orientation; and

Figure 5 shows the magnetic bridge of figures 2 and 3 with a further magnetic field source, in a second orientation.

The assay of the present invention is operable to test a sample for the presence of a substance of interest. For the ease of explanation, the following description will detail an example wherein the present invention is applied to the determination of the presence of Troponin-I in a blood sample. The level of Troponin-I in the blood can indicate various heart disorders. In particular, Troponin is a highly specific marker for myocardial infarction or heart muscle cell death. It is of course obvious to the skilled man that the assay of the present invention could be suitably adapted to test for the presence of other substances of interest of or other types of sample.

Referring now to figure 1, the blood sample is added to an application area 1 on the assay strip 6. The sample flows along a microfluidic channel on the assay strip 6. The flow along the microfluidic channel is first over an area 2 containing pre-deposited Troponin receptor labelled with magnetic particles that contain a specific probe for the Troponin-I molecule. During the flow, by kinetics or preferential binding, the magnetic particles become bound to free Troponin-I in the sample to produce magnetic Troponin- I complexes which are released into the solution.

The flow along the microfluidic channel next passes a sensing area comprising either a single magnetic particle trap 7 as shown in figure 1 or multiple magnetic particle traps (not shown) as shown in figure 2. Each trap 7 comprises a deviation in the profile of the microfluidic channel geometry in the sensing area. In the particular examples shown each trap 7 comprises a convergence of straight edges (as in a V shape) which provides a particle dwell / suspension region in the flow path of the fluid.

A magnetic field source 5 (which may for example be created by coil 30, 31 mentioned in relation to the embodiments described with reference to figures 3 and 4 below) is arranged in the region of the trap 7, to draw magnetic particles in the solution into the trap. At the trap 7 the local magnetic field combines with the trap geometry to preferentially retain free moving magnetic particles within the traps 7. This results in the accumulation of the magnetic particles and thereby provides a quantifiable indication of the presence or absence of a substance of interest. After passing the traps 7 within the strip, the excess sample fluid is then routed by capillary action along the microfluidic channel 3 to the exit point 4 where a pad could be placed to act as a sink. In the event that a multiple traps were provided, they can be arranged such that one fills first, then when full, magnetic particles begin are trapped in subsequent traps (not shown).

As is well known, the assay strip 6 may be provided with a lid and base. Either or both of the lid and base may be formed from plastic material of a hydrophilic nature.

The quantity of magnetic particles in the trap (which is directly correlated to the quantity of Troponin-I in the sample) is measured using the measurement device 8 of the invention shown in figures 2 and 3.

The measurement device of the invention comprises a casing 9 formed in two halves joined together by fasteners 10 screwed through one half into the other.

A slot 11 is provide through the casing 9 to receive the assay apparatus 6, and a holder 12 is provided in the slot 11 to hold the assay apparatus. The holder 12 comprises a groove 13 sized to the width of the assay strip 6, and open at its ends.

Of course, those skilled in the art will appreciate that the holder 12 could be integral with the casing 9, rather than being a separate part fastened thereto (by fastener 14). The purpose of the holder 14 is to hold the assay apparatus, such that the sensing area is held in a defined position in the measurement device. Whilst the preferred arrangement involves detection in a single sensing area having one or more traps 7, in an alternative arrangement, as shown in figure 3, the assay strip 6 may have a plurality of sensing areas 15 (four in this case), each associated with a notch 16 in the side of the assay apparatus, and with a retainer 17 in the form of a sprung ball plunger arranged to locate the assay apparatus 6 such that a respective sensing area is arranged in the defined position in the measurement device.

Contained within the two halves of the casing 9 in a recess (not shown) is a magnetic bridge 18. The magnetic bridge 18 is comprised of a body defining two spaced apart limbs

19 and an intermediate portion 20 between the limbs, and a central portion 21 extending from the intermediate portion 20 of the body to define a first gap 22 and a second gap 23 with the respective limbs 19 of the body. The magnetic bridge is generally "8- shaped", made up of a first part in the form of a T-shaped central member 21, combined with a generally C-shaped or E-shaped body forming the two limbs 19 and the intermediate portion 20.

A third gap 24 is provided between the central member 21 and the intermediate portion 20, with the intermediate portion being provided with a projection 25 extending towards the gap 24. The projection is thicker than the remainder of the body, to provide an easy route for magnetic flux. In the region of all three gaps 22, 23, 24, the parts either side of the gap have wedge shaped tips.

A magnetic flux detector 26 is sandwiched in the gap 24 between the central member 21 and the projection 25 of the intermediate portion 20 of the body. This magnetic flux detector 26 is, in this embodiment, a hall sensor, provided on a PCB 27 attached to the casing 9, and extending out therefrom, such that the outputs (not shown) from the magnetic flux detector 26 may be connected to a suitable means for monitoring and amplifying the output (e.g. a computer terminal). Two magnets 28 are also provided, one associated with each limb 19. In this embodiment, each magnet is a simple permanent magnet, and the magnets are formed as integral parts of the limbs 19, with the remainder of the limbs (and the remainder of the entire body) formed from a highly magnetically permeably material (e.g. iron). The polarities of the magnets 28 are opposed so that magnetic fields flow across the first and second gaps 22, 23. For example, each magnet 28 may have its north pole extending towards the free end of its respective limb 19, or each magnet 28 may have its south pole extending towards the free end of its respective limb 19.

The permanent magnets can be replaced with electromagnets placed in the outer limbs of the bridge. The generally C-shaped or E-shaped body made up of the limbs 19, intermediate portion 20 and projection 25 can can all be made as one part.

Field lines 29 showing the magnetic flux are included in figure 5 to show the manner in which the magnetic fields from the magnets 28 flow through the magnetic bridge in loops in two opposite directions around the 8-shaped magnetic bridge. The magnetic flux in the central member 21, resulting from the magnet associated with one limb 19 is in the opposite direction to the magnetic flux in the central member 21 resulting from the magnet 28 associated with the other limb 19. In consequence in the third gap 24, where the magnetic flux detector 26 is sandwiched, when both of the first and second gaps 22, 23 are empty, the magnetic flux from one magnet 28 is equal and opposite to that from the other magnet 28, so they cancel each other out.

In practice, if magnets 28 of precisely the same strength are not available, or precise machining of the body is not possible, or for any other reason the magnetic flux at the magnetic flux detector is not exactly equal and opposite, various means for balancing the opposing magnetic fields are known, as discussed for example in GB2207510, the content of which is incorporated herein by reference. GB2207510 also proposes a number of alternative arrangements of magnetic bridges which could be substituted for that described above. With this arrangement, the means for monitoring the output of the flux detector

26 is able to detect differences in magnetic permeability of the gaps 22, 23. In practice the magnetic permeability of the second gap 23 will not change, as the casing 9 prevents anything being introduced into that gap, so the magnetic field through the loop that includes that gap is fixed. Therefore in the method of the invention, the magnetic flux detector 26 quantitatively measures the presence of magnetic particles in the sensing area of an assay apparatus 6, when that sensing area is in the first gap 22 and the particles disturb the magnetic field through the loop including that first gap 22.

In order to carry out the method of the invention, having introduced a sample into the sample-receiving application area 1 of the assay strip 6, allowed it to flow over the area containing the magnetic particles, and trapped the magnetic particles in the traps 7 in the sensing area, the strip is introduced into the holder 12, such that the sensing area is in the first gap 22.

A magnetic flux is generated through the loop in which the first gap 22 is provided, and a counterbalancing magnetic flux is generated through the loop in which the second gap 23 is provided. The perfectly balanced magnetic fields from the magnets 28 will create a zero field reading from the sensor. This allows a significant amplification of the sensor output. The balance is disturbed by the presence of magnetic particles in the gap 22, with each magnetic particle increasing the magnetic permeability of the gap 22. The change in magnetic flux caused by introduction of the assay strip 6 into the measurement device, i.e. the difference between the magnetic permeability of the gap before and after introduction, is determined by the means for monitoring the output of the magnetic flux detector 26. The output from the magnetic flux detector can be greatly amplified, to produce a highly accurate reading of the amount of magnetic particles present.

With suitable configuration and calibration of the means for monitoring the output of the magnetic flux detector 26, the output value can be correlated to the precise number of magnetic particles in the sensing area. Unlike in optical methods, overlapping particles will not affect each other's readings, so the measurement device quantitatively measures the presence of magnetic particles in the sensing area.

Figure 4 shows schematically key components of a modified embodiment of the apparatus of figures 2 and 3. In addition to all the components of the measurement device, such as the magnetic bridge 18 and other parts (not shown), a further electromagnet 30 is provided as the magnetic field source 5 to attract the magnetic particles in solution into the traps 7 in the assay strip. The electromagnet 30 is positioned outside the plane of the magnetic bridge, in which the loops of magnetic flux are generated and generates a magnetic field orthogonal to the field produced by the magnet 19 at the first gap 22. It is arranged to be switched off during measurement of the output from the magnetic flux detector 26. In consequence, it does not interfere with the measurement of the change in magnetic permeability in the gap; rather, prior to measuring that change, it draws the magnetic particles into the sensing area, so that they can be measured. This is advantageous over having a separate magnetic field source 5 used to draw the magnetic particles into the trap before putting the assay apparatus 6 into the measurement device, because it reduces the number of operations required, since the measurement device also carries out this function. It also avoids any possibility of particles escaping the traps 7 when the assay apparatus 6 is moved from the field source 5 that attracts the magnetic particles to the measurement device.

Yet another modified form of the apparatus is shown in figure 5, again schematically, with only the magnetic bridge 18 and the hall sensor 26 shown from the components in figures 2 and 3. Once again the modification concerns a further electromagnet 31 is provided as the magnetic field source 5 to attract the magnetic particles in solution into the traps 7 in the assay strip. The electromagnet 30 is positioned in the plane of the magnetic bridge 18, arranged within the loop of magnetic flux which is generated through the first gap 22. However, it is arranged to generate a magnetic field orthogonal to the field produced by the magnet 19 at the first gap 22. It is also arranged to be switched off during measurement of the output from the magnetic flux detector 26. In consequence, it does not interfere with the measurement of the change in magnetic permeability in the gap; rather, prior to measuring that change, it draws the magnetic particles into the sensing area, so that they can be measured.

The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention as defined in the appended claims. CLAIMS

1. A measurement device comprising a body defining two spaced apart limbs and an intermediate portion between the limbs, and a central member extending from the intermediate portion of the body to define a first gap and a second gap with the respective limbs of the body; a magnetic flux detector positioned between the central member and the intermediate portion of the body and at least two magnets, one associated with each limb; the polarities of the magnets being opposed so that magnetic fields flow across the gaps; whereby the magnetic flux in the central member resulting from the magnet associated with one limb is in the opposite direction to the magnetic flux in the central member resulting from the magnet associated with the other limb; and means for monitoring the output of the flux detector to detect differences in magnetic permeability of the gaps; wherein, the measurement device comprises a holder arranged to hold an assay apparatus such that a sensing area of the assay apparatus is provided in the first gap, whereby the magnetic flux detector quantitatively measures the presence of magnetic particles in the first gap.

2. A measurement device according to claim 1 further comprising balancing means to balance out magnetic flux in the region of the flux detector, when there is nothing in either gap. 3. A measurement device according to claim 2 or 3 wherein the intermediate portion and/or the central member comprises a region that is thicker than the limbs to provide an easy path for the magnetic field.