5 Background Art
An assay is an investigative procedure for qualitatively or quantitatively measuring the
presence, amount or activity of an analyte in an assay sample. Often, the assay sample is
a cell or a cell culture and the analyte is a protein or gene sequence of that cell or cell
culture, but analyses may more generally include metabolites, peptides, proteins, nucleic
10 acids, extracellular vesicles, organelles, cells, or tissues.
Often, the assay sample is exposed to a reagent prior to a measuring step. In fields such
as biology and pharmacology, it is common for assay samples to be exposed to a large
number of different reagents. For example, if the analyte is a protein or a gene sequence of
a cell culture serving as a disease model, different samples of the cell culture may be
15 exposed to a large number of different drug candidates. The interaction between the assay
sample and the reagent typically occurs in a well plate. The measuring step may take place
in the well plate or elsewhere. In some cases, the measuring step can take place in flow
conditions, for example, in a flow cytometer.
Some biological assay samples, such as certain types of cells, may be suspended in an
20 aqueous solution. However, there are many biological assay samples which cannot easily
be suspended in solution and which are most viable when attached to a surface, such as
adherent cells. Assays of such samples, including the step of measuring, typically have to
be performed while the assay sample is attached to the bottom of a well plate. In such
assays, the assay process may be disadvantageously limited by the fact that an assay
25 sample attached to the bottom of a well plate cannot easily be transferred to another
container without stripping it from the well plate. This may damage the sample.
Measuring an assay sample attached to a well plate is inefficient. The assay sample is
typically attached or seeded across the entire well plate but only one or two percent of the
assay sample is actually imaged or measured. This is a particular problem when the assay
30 sample is a scarce resource, for example cells that are particularly difficult to culture or
primary cells such as cells collected during a biopsy of a tumour.
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Adherent cells are an example of an assay sample that is most viable when attached to a
surface. There is a strong interest in assaying adherent cells under flow conditions using,
for example, a flow cytometer. However, this requires stripping of the adherent cells from
their attached state prior to the assay. This may bias the assay being performed because
5 the adherent cells may be damaged or because adherent cells forced into suspension may
not accurately represent the normal biological state of the adherent cell. The significant
extent of this problem is illustrated by the fact that in conventional assays, after stripping an
adherent cell from a substrate, it is usual to wait for 12 to 48 hours for the cell to return to a
normal or undisturbed state.
10 Another problem of performing an assay on an assay sample that is most viable when
attached to a surface, or which tends to agglomerate on surfaces, is that such assay
samples may have a tendency to clog channels in flow cytometry apparatus.
Summary of Invention
The invention provides a carrier system for an assay, a method of manufacturing a carrier
15 system for an assay and a method of using a carrier system for an assay, as defined in the
appended independent claims to which reference should now be made. Preferred or
advantageous features of the invention are set out in the dependent claims.
A first aspect of the invention may thus advantageously provide a carrier system for an
assay, comprising a carrier or particle for carrying an assay sample. The carrier is secured
20 to a substrate by a release layer. The carrier is advantageously suitable for receiving an
assay sample, and the release layer configured in use to release the carrier from the
substrate in the presence of a biocompatible aqueous solution. For example, the release
layer may be activated, to release the carrier, by the biocompatible aqueous solution. The
release layer, when activated, may thus release the carrier from the substrate.
25 An assay sample for introduction into an array system may commonly be suspended in a
biocompatible aqueous solution. When a user of the carrier system wants to perform an
assay, contact between the biocompatible aqueous solution carrying the assay sample
may then automatically release the carrier into the solution.
Preferably, the carrier system may thus be contacted with the biocompatible aqueous
30 solution carrying the assay sample so that the assay sample (for example an adherent cell)
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may attach to the carrier, before the carrier is released into solution, carrying the assay
sample.
In a preferred embodiment, the assay sample may be an adherent cell and the carrier, or
particle, may be of a suitable size for the cell to adhere to the carrier. For example, as
5 described in more detail below, the carrier may have a planar upper surface of lateral
dimension in the range of 5 to 200 micrometres, suitable for an adherent cell to attach to.
The assay sample may then be carried through the assay on the carrier. In a particularly
preferred embodiment, the carrier may be magnetic so that it, and the assay sample that it
carries, may be directed through the assay by the application of an external magnetic field.
10 Aspects of the invention are described herein with reference to a carrier or particle secured
to a substrate, but in a typical application the carrier may be one of many carriers or
particles secured to the same substrate and used for example to perform a multi-channel
assay. In a typical embodiment of the invention, therefore, a substrate may carry many
carriers, such as more than 10, 100, or 500 carriers, up to as many as 1000, 5000 or
15 10,000 or more. As described below, each carrier may be individually identifiable, or
subgroups of the carriers may be identifiable, by way of markings such as barcodes carried
by the carriers for use in a multi-channel assay. Multiple carriers, or assay-sample carriers,
secured to a substrate may be termed a carrier array or particle array.
For the sake of simplicity, however, embodiments of the invention described herein will
20 usually be described with reference to only one carrier.
In a preferred embodiment, an assay sample may be introduced to the carrier system so
that the assay sample may be received by, or onto, the carrier. The assay sample may be
a biochemical substance or a cell of an organism or an organic sample. Preferably, the
assay sample may be an adherent cell. The assay sample received on the carrier may
25 attach, adhere, seed onto or bind to a surface of the carrier, preferably before the carrier is
released from the substrate. An assay may then be performed on the assay sample
received on, and carried by, the released carrier. The assay may involve measuring the
presence, amount or activity of an analyte of an assay sample. When the assay sample is
a cell, the analyte may, for example, be a protein or gene sequence of that cell.
30 An assay sample received on a carrier may be convenient to manipulate and transport.
This may be advantageous when performing an assay, for example in order to move the
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assay sample to a well plate, to expose the assay sample to a reagent or to position the
assay sample with respect to a detector in a measuring step.
In a further embodiment of the invention, the receiving of an assay sample onto a carrier
may be used to conveniently store assay samples for use in a future assay.
5 A conventional, prior-art, method of storing cells for an assay may involve:
10
15
Stripping the adherent cells (e.g. chemically) to make a single cell suspension in
culture medium.
Concentrating the cells to about 1M cells per ml (done with a centrifuge)
Complementing the culture medium with 5-20% DMSO (dimethyl sulfoxide) or
equivalent.
This mixture is then added to freezing tubes (i.e. cryogenic storage vials)
These are then slowly cooled down to -70C at 1C per minute (typically this is done
by immersing the vials in isopropanol and placing that in a -70C freezer)
After that, the vials are stored at -70C (either in a freezer or in liquid nitrogen)
When thawing, the frozen vial is placed in a 37C warm water bath until thawed.
Fresh medium is then added, cells are spun down, and fresh medium is added
again.
Cells are then seeded onto a flask or plate or dish for an assay to be performed.
Using an embodiment of the present invention, this method may be significantly improved
20 as follows:
25
30
Attach the adherent cells to carriers attached to a substrate, as described herein.
Optionally, release the carriers from the substrate into liquid as described herein.
Alternately the carriers may be retained on the substrate.
Cells would then either be cultured for 1-2 days or frozen directly (the former is
preferred to maintain healthy cells).
The substrate carrying the carriers, or the free carriers, loaded with or carrying the
cells, are in a medium which is refreshed with medium containing 5-20% DMSO,
making sure there is about 1M cells per ml of liquid. Either a centrifuge or gravity
may be used to concentrate the carriers as the medium is refreshed. Alternatively,
if magnetic carriers are used, as described herein, the carriers may be magnetically
pulled down or held down as the medium is refreshed.
5
10
15
20
25
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Then the protocol would go as in conventional cell freezing, except that the cells are
received on carriers ready for use in an assay.
This embodiment of the invention provides numerous benefits:
Cells do not need to be stripped prior to freezing
Cells do not need to be seeded or attached to any substrate after freezing
It is possible to store live, adhered cells.
This is useful if researchers would want to do an experiment (which needs adherent
cells), but would like to store some of the assayed samples for later analysis or for
additional experiments.
This is useful when people culture a large batch of cells on which they want to do
multiple experiments over a long time. They may then attach the whole batch, or a
plurality of the cells, to the carriers, which are then frozen. They can then thaw a
portion or all of the cells when they want to do an experiment. This ensures minimal
inter-experimental variability and bias. What people need to do conventionally is
continuously grow cells (over multiple passages, i.e. cycles of stripping and reseeding
when cells are overgrown in culture), but mutations and changes
accumulate over these different passaging steps. The other known solution is to
always thaw a fresh batch prior to an experiment. This is however not only labour
intensive (because you need to thaw, grow for 2 days, strip and re-seed), but also
introduces variability and potentially bias.
The embodiment of the invention is useful when people want to do a quick assay.
This approach makes sure that people do not need to do the "thaw, grow for 2 days,
strip and re-seed" step prior to an assay. In this situation, cells are thawed and the
experiment/assay can either proceed directly or after 1-2 days of culturing (without
the strip and re-seed step).
Conventionally, assay-ready cells are available, but these cells are still suspended in liquid
and need to be attached to a substrate prior to assaying. The embodiment of the invention
ensures that these assay-ready cells are all frozen in the same "state" (e.g. how many
times they have been passaged, what cell cycle step they are in, making sure that they
30 have no artefacts such as mutations or metabolic issues). Thus, in an embodiment of the
invention assay-ready cells on carriers may be sold, for example in a frozen state, ready for
use. Further, as described herein, the assay-ready cells may be on carriers which are
individually identified by, for example, barcodes or other readable markings on each carrier.
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In a preferred embodiment of the carrier system of the invention, the release layer may be
activatable in the presence of an assay-sample solution or assay-sample medium for
containing or carrying an assay sample. The assay-sample solution may for example
comprise a biocompatible aqueous solution and an assay sample. Therefore, the single act
5 of introducing the assay-sample solution to the carrier system may result in the assay
sample being received by the carrier and the carrier being released from the substrate.
Many assay samples must be maintained and transported in a biocompatible aqueous
solution in order to maintain viability. Therefore the provision of carriers, for receiving an
assay sample, secured to a substrate by a release layer configured to release the carriers
10 in the presence of a biocompatible aqueous solution is particularly convenient. The
biocompatible aqueous solution may for example be non-cytotoxic.
In a preferred embodiment, the release layer may be configured such that, following
activation of the release layer, the biocompatible aqueous solution remains biocompatible.
The release layer may preferably not release non-biologically-compatible or toxic
15 components into the biocompatible aqueous solution. Such components might otherwise
render the biocompatible aqueous solution toxic to the assay sample. More generally, the
release layer may preferably not release into the biocompatible aqueous solution any
materials or components that may modify or change the assay sample in any way. Such a
release layer may advantageously not impact the results of an assay performed using the
20 carrier system. The release layer may for example be formed of a biocompatible material.
In a preferred embodiment, the assay sample is one that is at its most viable when
received on a surface. An example of such an assay sample may be an adherent cell.
Once such an assay sample has been received by the carrier, or onto a surface of the
carrier, and the carrier has been released from the substrate, the assay sample may
25 remain on the carrier rather than moving onto another surface. This may mean that such
assay samples, received on and carried by released carriers, are convenient to manipulate
and transport. Furthermore, such assay samples may remain in a representative biological
state while the assay is being performed. This may reduce any bias in an assay of such
assay samples. In particular, there may be no need to force such assay samples into
30 suspension via mechanical or chemical means, as would be required in prior art methods.
The agglomeration of such assay samples may also advantageously be reduced.
Assay samples received on carriers embodying the invention may advantageously be
measured for example using a flow cytometer or fluorescent microscopy.
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In a preferred embodiment, the release layer may comprise a material that is wateractivatable,
in other words a material that releases the carrier or particle in the presence of
water, such as water in a biocompatible aqueous solution. The water-activatable material
may be a water-soluble material. The water-soluble material may dissolve in the presence
5 of water to release the carrier. The release layer may or may not completely dissolve. The
release layer may only dissolve to such an extent that the bond between the release layer
and the carrier is weakened and the carrier is released from the substrate.
Alternatively, or additionally, the release layer may comprise a material having other
properties that change in the presence of water or an aqueous solution. For example, the
10 adhesion of the material of the release layer may be reduced in the presence of water or an
aqueous solution. As with water-soluble release layers, these release layers may have the
advantage that the carrier may be released from the substrate in the presence of water, but
preferably without adding any components of the release layer into the solution where they
may affect the assay sample.
15 In a preferred embodiment as described in more detail later in this document, the
manufacture of a carrier system may comprise a lithographic process. The release layer
may then be formed of a material suitable for or compatible with the required lithographic
process. The release layer may be applied to the substrate prior to at least some of the
steps of the lithographic process, and the carriers then formed on top of the release layer.
20 In order to be suitable for lithographic process, the release layer may advantageously not
be affected by the processes and chemicals used in the lithographic process.
In a preferred embodiment, the release layer may comprise a material that is not
activatable in a non-aqueous solvent such as ethanol. As described in more detail later in
this document, the method of manufacturing the carrier system may comprise applying to
25 the carrier a coating adapted for receiving an assay sample. This process may
advantageously be carried out while the carrier is secured to the substrate by the release
layer. The coating may for example be applied by immersing the carrier system in a
solution for forming or depositing the coating, which may be a polymer, for a period of time
such as between 10 and 120 minutes. Such solutions commonly comprise non-aqueous
30 solvents such as ethanol and so advantageously the release layer may be unaffected, or
may not be activated, by such solvents so that the release layer may retain the carrier
secured to the substrate while the carrier system is exposed to the non-aqueous solvent. In
particular, the release layer advantageously may not release the carrier when exposed to
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the non-aqueous solvent for the length of time, and under the conditions required, during
manufacture of the carrier.
Alternatively or additionally, the carrier system may undergo a sterilization process while
the carrier is secured to the substrate by the release layer. The sterilization may be part of
5 the manufacturing process or in preparation of the carrier system for performing an assay.
Sterilization advantageously eliminates unwanted forms of life or biological agents that
might otherwise adversely interfere with assays performed using the carrier system.
Sterilization may comprise immersing the carrier system in a non-aqueous sterilization
liquid such as ethanol. The sterilization liquid may comprise pure ethanol. The carrier
10 system may be immersed in the sterilization liquid for up to 10, 15, 20 or 25 minutes,
typically up to a maximum of about 30 minutes or 1 hour. If the release layer comprises a
material that is non-soluble in a non-aqueous solvent such as ethanol, the release layer
may advantageously retain the carrier secured to the substrate while the carrier system is
immersed in non-aqueous solvent. In particular, the release layer advantageously may not
15 release the carrier when exposed to the non-aqueous solvent for the length of time, and
under the conditions required, for sterilization of the carrier.
The release layer may comprise a sugar. Sugars are examples of materials that may be
biocompatible. A release layer comprising a sugar may release the carrier in the presence
of a biocompatible aqueous solution such as an aqueous solution. Sugars may be water-
20 soluble. Preferably, the release layer may comprise a sugar such as a dextran. A release
layer comprising a dextran may be biocompatible. In the manufacture of the carrier system,
the release layer may be applied to the substrate using a spin-coating technique. A release
layer comprising a sugar and, particularly, a dextran, may advantageously be suitable for
spin-coating.
25 Dextrans may comprise polymer molecules of a range of lengths, typically from about 3 to
2000 kDa or more. A solution of any dextran may be spin coated to form the release layer.
A dextran of 70 kDa has been found to provide a release layer activation time of between 1
and 10 minutes. Larger dextran molecules, in the range 5000 to 40000 kDa are less
soluble in the biocompatible aqueous solution and may be used to provide longer release
30 times. Therefore, smaller dextran molecules in the range of 3 kDa or 50 kDa to 500 kDa or
2000 kDa may be used to provide shorter release times, and larger dextrans such as in the
range of 2000 kDa or 3000 kDa or 5000 kDa to 6000 kDa or 1 0000 kDa or 40000 kDa may
be used to provide longer release times.
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A release layer comprising a sugar, such as a dextran, may be suitable for lithographic
processes, and may not be activatable in non-aqueous solvents such as solvents
containing ethanol, or in pure ethanol.
The parameters of the release layer, such as its material(s), structure and thickness, may
5 be selected or designed depending on the desired time taken for the carrier to be released
following the introduction of a biocompatible aqueous solution to the carrier system. In a
preferred embodiment, the parameters of the release layer may be selected to release the
carrier at a time after which the assay sample is likely to have been received on the carrier.
Receiving the assay sample on the carrier while the carrier is in contact with the substrate
10 may ensure that the assay sample is received only by an exposed surface of the carrier. As
only one surface of the carrier may typically be measured during an assay, this may
advantageously ensure that a greater proportion of the assay sample is measured and
reduces or eliminates wastage of the assay sample. This is particularly advantageous
when the assay sample is a scarce resource, for example cells that are particularly difficult
15 to culture or primary cells such as cells collected during a biopsy.
A release layer comprising a sugar such as a dextran may release the carrier relatively
quickly in the presence of a biocompatible aqueous solution, such as in 5 or 10 seconds or
less. Some assay samples may typically take much longer than five seconds to be received
on the carrier following the introduction of the assay sample to the carrier system. For
20 example, it may take an adherent cell three hours or more to be received on the carrier.
Therefore, the release layer may comprise materials having a longer release time.
The release layer may comprise a polyvinyl alcohol (PVA). The release time of a release
layer comprising polyvinyl alcohol may be much longer than the release time of a release
layer comprising dextran. A release layer comprising polyvinyl alcohol may be configured to
25 release the carrier about 1, 2, 3, 6 or 9 hours, up to as long as 12 hours or more, after the
introduction of a biocompatible aqueous solution and an assay sample to the carrier
system. A release layer comprising polyvinyl alcohol may therefore be designed to release
the carrier a suitable time after the introduction of biocompatible aqueous solution such that
an assay sample, such as an adherent cell, has been received on the carrier.
30 Like sugars, polyvinyl alcohols are materials that may be considered to be biocompatible. A
release layer comprising a polyvinyl alcohol may be water-soluble, suitable for spin-coating
and compatible with lithographic processes. A release layer comprising polyvinyl alcohol
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may not be activatable in non-aqueous solvents such as solvents containing ethanol, or in
pure ethanol.
Other materials may be used to form the release layer, such as PLGA (PLG or poly(lacticco-
glycolic acid)) or a poly( acrylic acid) (PAA). The structure of the release layer may also
5 be formed from multiple materials, such as layer-by-layer deposited polymers and/or
sugars.As described above, manufacturing a carrier system embodying the invention may
include a step in which a coating adapted for receiving an assay sample is applied to the
carrier(s). Alternatively or additionally, the carrier system may undergo sterilisation. As
described above, each of these processes may be performed using a non-aqueous solvent
10 such as ethanol and the carrier may advantageously remain secured to the substrate in the
presence of the non-aqueous solvent.
Alternatively, in some embodiments, the materials, structure and thickness of the release
layer may be selected to allow for the use of an aqueous solution during manufacture, for
example in the application of a coating or during a sterilization process. If the parameters of
15 the release layer are selected so that the carrier is only released after a period that is
greater than the cumulative time that the carrier system is immersed in aqueous solutions
for either or both of the above processes, then these processes may be carried out and the
carrier may remain secured to the substrate. It should also be borne in mind that the
release layer should still provide time, when an assay is to be performed, for an assay
20 sample to be received on the carrier while the carrier is secured by the release layer to the
substrate.
In general, the materials and other properties, such as the thickness and structure, of the
release layer may be varied or predetermined, in order to achieve a desired release time.
In a preferred embodiment, the carrier may comprise a magnetic material. This may have a
25 number of advantages during an assay. When an external magnetic field is applied, the
carrier may have a sufficient magnetic moment for the external field to apply a desired
force to the carrier.
For example, the force may be applied to retain the carrier in contact with the substrate
even after the release layer has otherwise released the carrier. As described above, it may
30 be advantageous for the assay sample to be received by the carrier while the carrier is in
contact with the substrate. By applying an external field to retain the carrier in contact with
the substrate even after the release layer has released the carrier, the carrier can be held
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in place on the substrate for any length of time. This may, for example, remove the need to
match the release time of the release layer with the typical time taken for a particular assay
sample to be received by the carrier, or it may allow a carrier which is retained by a release
layer designed to have a particular release time to receive assay samples which require
5 any length of time longer than that release time to be received on the carrier. In other
words, a release layer having an activation time that is less than the typical time taken for
the particular assay sample to be received by the carrier may be used.
Similarly, the force applied by the external field is advantageously sufficient to move or
drive the carrier through a solution during an assay, while carrying the assay sample. If the
10 sample is a cell, for example, the force should be sufficient to move the carrier and the cell.
The carrier may be lithographically defined or fabricated. A lithographically defined carrier
may advantageously have a high aspect ratio. Such a carrier may advantageously have a
large surface area for receiving an assay sample relative to the volume of the carrier. In a
preferred embodiment, the carriers may conveniently be lithographically defined or
15 fabricated on or over the release layer of the carrier system.
The carrier may comprise a photoresist layer which may be an artefact of a lithographic
process used to define or fabricate the carrier. The photoresist layer may advantageously
comprise a material or materials that are not soluble in water.
During manufacture of the carrier, a photoresist layer may be patterned by exposing it to
20 radiation and washing away or dissolving regions of the photoresist layer in a solvent. In
lithographic processes, photoresists are typically water-soluble and the solvent is an
aqueous solvent. In embodiments of the invention, a photoresist that is not soluble in water
is preferred. The step of washing away the photoresist may then advantageously involve a
solvent other than a water-based solvent. Therefore, the release layer may advantageously
25 be unaffected by the washing process.
The photoresist layer may comprise an SU-8 photoresist. An SU-8 photoresist may be
soluble in a solvent comprising, for example, propylene glycol methyl ether acetate,
gamma-butyrolactone or cyclopentanone. The SU-8 photoresist may advantageously not
be soluble in a biocompatible aqueous solution. An SU-8 photoresist may therefore be
30 biocompatible, if present in an assay.
In a preferred embodiment, a surface of the carrier may be adapted or modified for
receiving an assay sample. Such an adaption may advantageously improve the efficiency
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or rate at which an assay sample is received by that surface and so may reduce the time
for an assay sample to be received on the carrier and increase the strength of bonding or
attraction between the assay sample and the carrier so that the assay sample is
advantageously securely bonded to the carrier. This may also reduce any tendency for the
5 assay sample to migrate or move away from the carrier during an assay. The surface of the
carrier may be adapted for receiving adherent cells. The adapted surface may be a surface
of the carrier that is exposed when the carrier is secured to the substrate.
The surface of the carrier may comprise a coating adapted for receiving an assay sample.
Carriers comprising such a coating may be referred to as bio-functionalized. The coating
10 may be adapted for receiving adherent cells as an assay sample. The coating may be a
charged coating. This may be particularly preferable when the assay sample is inherently
charged. For example, cells typically have a membrane potential of between negative 40
and negative 80 millivolts. Providing a coating defining a surface having a positive charge
may encourage attachment of the assay sample onto the charged surface of the carrier. It
15 has been found that providing a charged surface, and preferably a positively charged
surface, may advantageously lead to more effective cell adhesion. The coating may
comprise a charged polymer.
In some embodiments, the coating is applied on a gold layer of the carrier in the form of a
gold cap.
20 The gold cap of the carrier may only be on one side or surface of the carrier. In particular,
the gold cap may only be on a surface of the carrier that is exposed while the carrier is
secured to the substrate. This ensures that only one surface of the carrier is biofunctionalized
and so only one surface of the carrier tends to receive the assay sample.
The advantages of only receiving the assay sample on one surface of the carrier are
25 described herein. In some embodiments, the carrier may have been fabricated using
lithographic processes. Lithographic processes may be particularly suitable for fabricating a
carrier with only a specific surface comprising a gold cap.
The polymer (charged polymer) may be covalently bound to the gold cap. The surface
adapted for receiving the assay sample may comprise a gold cap layer on to which a
30 polymer is covalently bonded by a thiol group. This advantageously ensures that each
polymer adsorbed onto the surface has the same orientation. Alternatively, the polymer
may be adsorbed onto the gold cap of the carrier by Vander Waals forces.
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The charged polymer may be a polymer comprising a positive-charge-carrying group. The
polymer may be a polyornithine or a poly-d-lysine. The polymer may be a polyelectrolyte.
Alternatively or additionally, the coating may comprise a plurality of ligands. The plurality of
ligands may comprise antibodies. When the assay sample of the assay is a cell, the
5 plurality of ligands may comprise antibodies that specifically bind to cell receptors such as
integrins.
When the assay sample is a cell, the coating may alternatively or additionally comprise an
extracellular matrix protein. The extracellular matrix protein may be a protein selected to
increase or enhance cell adhesion. The protein may be a collagen, laminin or Matigel, a
10 protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.
A surface of the carrier may comprise a physical structure adapted for receiving adherent
cells. The physical structure or topography of a surface can have an effect on the degree to
which adherent cells adhere or seed onto that surface. Cell adhesion may advantageously
be increased by increasing the surface area of the surface of the carrier. The surface area
15 may be increased by increasing the roughness of the surface. For example, the surface
may have been etched using an ion beam.
The hardness of a surface may also have an effect on the rate or efficiency to which
adherent cells are received on that surface of the carrier. Cell adhesion is closely related to
the Young's modulus of the surface. Therefore, the hardness, or Young's modulus, of the
20 surface of the carrier may be tuned to optimize the adhesion of the cell that is the assay
sample.
To summarise, the steps in an assay according to a typical embodiment of the invention, in
an example where the assay sample comprises adherent cells, may include the following;
1. Carrier array placed in a well plate, flask, or any other cell culture vessel.
25 2. Cells that have been stripped (single cells) are mixed with culture medium and
loaded onto the carrier array.
3. The cells will settle down and attach to the carriers (typically minimum 2-3h)
4. The aqueous medium slowly dissolves the sacrificial (release) layer.
5. The carriers with attached cells are released into liquid.
30 6. Carriers with cells can be moved, assayed, measured, stored, etc.
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As described above, the carrier may comprise a magnetic material. When an external field
is applied, the carrier may have a sufficient magnetic moment for the external field to apply
a desired force to the carrier. The desired force may depend on the application of the
carrier. In practical implementations, the applied external field is typically less than 2T, or
5 1T or 0.5T, and may typically be greater than 0.05T, or 0.1T or 0.25T. A carrier comprising
a magnetic material may be steerable by the application of an external magnetic field. For
example, the carrier may be steerable within a fluid medium. The fluid medium may be the
biocompatible aqueous solution introduced to the carrier system to release the carrier. The
magnetic moment may be due to magnetization of the carrier itself, or it may be induced in
10 the carrier by the external field. It is important, however, for the carrier to contain sufficient
magnetic material to enable the desired force to be generated. The magnetic moment that
can be generated by an external field applied to a carrier may depend on the total volume
V of magnetic material in the carrier multiplied by that material's magnetization Ms. Thus,
the value of V.Ms for a carrier embodying the invention is preferably greater than a
15 predetermined value, such as 10-18 J/T or 5x10-18 J/T or 10-17J/T.
The magnetic material may comprise a material selected for example from metals or
metallic alloys such as Fe, Co, Ni, CoFe, CoFeB, FePt, CoNi and NiFe.
The external field may be applied to orientate the carrier, and the assay sample received
on the carrier, with respect to a detector. This may advantageously improve the
20 consistency and quality of results from an assay performed with such carriers as the carrier
may be placed in the optimum position and angle with respect to the detector. The ability to
orientate the assay sample received on the carrier is particularly advantageous when the
carriers are in suspension, for example, in an aqueous solution and may be particularly
beneficial in microfluidic or flow cytometry applications.
25
The structure of the magnetic carriers enables a number of advantages in an assay, such
as enabling the changing of the liquid in which the carriers and assay samples are
supported, sorting specific carriers (e.g. after measurement for collection), the settling of
carriers to the bottom of a well or flask with the face of the carriers carrying the assay
30 samples face-up (so that the assay samples are not pressed between carrier and well).
A preferred embodiment of an imaging assay using the magnetic carriers, for an assay
sample comprising cells, may then comprise steps such as the following:
5
10
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Assay sample attached to carriers, while using magnetic field to hold carriers on
substrate after release layer activated
Assay sample (on carriers) moved to a fresh container or well (to remove
unattached cells (i.e. any cells not attached to carriers) and refresh the culture
medium)
Magnetically pull down the carriers with cells to the bottom of the container with
cells facing up (for incubation or cell growth).
Add assay reagents (e.g. a drug)
Remove liquid (using magnetic field to keep carriers in place)
Add various assaying liquids sequentially (e.g. wash buffers, labelled antibodies)
(using magnetic field to hold carriers in place as liquids are changed)
Flip over the carriers (using magnetic field) for microscope imaging through the
bottom of the well.
15 In an alternative embodiment, using a flow-based assay (again described in the context of
an assay sample comprising cells) the use of carriers embodying the invention may provide
further advantages, such as not aggregating (clogging), allowing the imaging of groups of
cells or single cells on carriers that pass detectors, and allowing measurement using either
fluorescent intensity (spectrometry) or by taking full images of cells on carriers.
20
In an embodiment of the invention, the carrier or particle may comprise a layered structure
between a top surface of the carrier and an opposed bottom surface of the carrier, the
layers including one or more magnetized layers; in which the ratio of a lateral dimension of
the one or more magnetized layers to a thickness or aggregate thickness of the
25 magnetized layer or layers is preferably greater than 500. Carriers having such a structure
may have a low stray field at the surfaces of the carrier. This may advantageously mean
that when a plurality of carriers having the same structure are provided the carriers may not
interact with another. In particular, the carriers may not aggregate or clump. This may be
the case whether or not the carriers are magnetically remanent.
30 The carrier may further comprise a non-magnetic layer, which may advantageously provide
mechanical support to the magnetic layer, and may determine physical characteristics of
the carrier such as its mechanical properties and its density. The non-magnetic layer may
provide a suitable substrate for the magnetized layer. The non-magnetic layer may thus
advantageously comprise a material selected from AI, Ta, Pt, Pd, Ru, Au, Cu, W, MgO, Cr,
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Ti, Si, lr, Si02, SiO, Sn, Ag, polymers and plastics, alloys of these materials, and
composites or mixtures comprising these materials.
In a preferred embodiment, the carrier may comprise readable information, such as a
5 readable code selected from a barcode or a 20 code. This may allow the carrier to be
identified remotely by reading the information, for example with a camera and suitable
software. A multi-channel assay may be performed by providing a plurality of carriers in
which each carrier carries readable information.
In a preferred embodiment, the readable information may be used to identify the assay
10 sample received on the carrier. In a multi-channel assay, more than one type of assay
sample may be analysed at once. Where the assay sample is a cell, more than one
different type of cell may be analysed at once. The readable code may indicate the assay
sample type on a particular carrier. The assay may be performed using a plurality of
differently coded carriers, with the differently coded carriers carrying different types of
15 assay sample. Following the assay samples being received on the carriers, the carriers
having different types of assay sample may be pooled. For example, a plurality of carriers,
each comprising (carrying) different types of cells, may be pooled or mixed in a well plate
containing a particular drug in order to expose the various cells to that drug. The
identification of carriers using readable information in this way may advantageously provide
20 a multiplexed platform in which the carriers, and therefore the assay samples received on
the carriers, can be accurately distinguished from each other.
It may be that assay samples of a particular type are received by carriers having identical
readable information. In that case, the readable information need only distinguish between
carriers carrying different assay sample types. In other words, the number of channels, or
25 the plex, of the assay equals the number of individual assay sample types used in the
assay. However, it may be advantageous to provide each carrier with unique readable
information. A subset of the carriers may comprise unique readable information relating to
a particular assay sample type. The unique readable information of the carriers in the
subset may also be used to identify the test conditions experienced by each assay sample
30 in the subset during the assay, for example, the particular drug that the assay sample is
exposed to during the course of the assay. In this case, the readable information needs to
be capable of distinguishing between every carrier of the plurality of carriers of the carrier
system. In other words, the plex of the assay may equal the number of carriers used in the
assay.
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The use of barcodes, or 20 codes may provide a significantly more robust process for
identifying different carriers than in existing multiplexed assay platforms, with minimum
crosstalk between plex channels. In addition, the use of readable information in this way
may enable the use of very much larger numbers of multiplex channels than is currently
5 possible. For example, barcoding or 20 codes may enable 1000 plex, or 10,000 plex, or
more if desired (i.e. an assay may involve 1000, or 10,000 or more multiplex channels).
Use of barcodes or 20 codes may be particularly advantageous when each carrier is
required to comprise unique readable information.
In a preferred embodiment, the (or each) carrier may comprise a magnetic material and a
10 suitable external magnetic field may be applied to steer or move or drive the carrier through
a fluid medium to a predetermined location for reading the code or information. The fluid
medium may comprise or consist of the aqueous solution introduced to the carrier system
to release the carrier. For example, a carrier having a high-aspect-ratio shape with a large
top or bottom surface for displaying a code or information, may be directed so that it is in
15 contact with a substrate or other supporting surface for convenient reading of the code or
information. A magnetic field may be applied to steer the carrier to a reading position, and
an assay result obtained by reading the readable code and the assay sample received on
the carrier.
In a preferred embodiment, the carrier system may comprise a plurality of carriers for
20 receiving an assay sample secured to the substrate by the release layer. An assay sample
may be received on each of the carriers. The same type of assay sample may be received
on each carrier. Alternatively, different types of assay sample may be received on some or
each of the carriers. By providing a plurality of carriers, a multiplexed assay may
advantageously be performed using the carrier system.
25 A second aspect of the invention may provide a method for manufacturing a carrier system
for an assay, the method comprising the steps of: providing a substrate; forming a release
layer on the substrate; and depositing or forming a carrier on the release layer such that
the carrier is secured to the substrate, wherein the release layer is configured in use to
release the carrier from the substrate in the presence of a biocompatible aqueous solution.
30 In a preferred embodiment, the step of forming the release layer on the substrate may
comprise spin-coating the release layer. The release layer may comprise a material that is
particularly suitable for spin-coating. For example, the release layer may comprise a sugar
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such as a dextran or a polyvinyl alcohol (PVA) or a poly(lactic-co-glycolic acid)) (PLGA) or
a poly( acrylic acid) (PAA).
In a preferred embodiment, the step of depositing the carrier on the release layer may
comprise fabricating the carrier on the release layer. In other words, the method of
5 manufacturing the carrier system may comprise the step of fabricating the carrier. This
conveniently may allow the carrier to be manufactured in the same process as the
manufacture of the carrier system. Alternatively, the step of depositing the carrier may
comprise depositing an already fabricated carrier on the release layer. For example, the
carrier may have been manufactured by a third party.
10 A plurality of carriers may be deposited or fabricated on the release layer.
The method of manufacturing the carrier system may comprise a lithographic process. In
particular, the carrier may be lithographically defined. This may advantageously provide a
carrier having a high aspect ratio. The fabrication of the carrier may comprise a
lithographic process. The lithographic process may comprise a physical vapour deposition
15 process which advantageously enables sub-nanometer control in the deposition of the
various layers used to fabricate the carriers. While the thickness of carriers manufactured
using the lithographic process may be of the order of nanometers, the minimum size of the
carriers in a lateral dimension may be between 5 and 200 microns, or between 20 and 200
microns; the carriers (in the lateral dimension) may be of any convenient shape, such as
20 square or rectangular or circular. A carrier having a minimum lateral dimension of 5 or 10 or
20 microns may be suitable for receiving single cells and so advantageous in assays where
single cells are of interest. A larger carrier, for example a carrier having a minimum lateral
dimension of closer to 200 microns, may be suitable for receiving a plurality of cells. In a
preferred embodiment, the carrier may have a minimum lateral dimension of 100 microns.
25 The fabrication of the carrier may comprise forming a photoresist layer on or over the
release layer.
The photoresist layer may be spin coated over the release layer. As discussed above, the
photoresist layer may advantageously comprise a material or materials that are not soluble
in water, so that portions of the photoresist layer may be washed away, or dissolved, using
30 a non-aqueous solvent that does not affect the release layer.
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If the photoresist layer is not soluble in an aqueous solvent, then it may be considered
biocompatible. For example, it may comprise a biocompatible polymer.
A suitable photoresist layer may be an SU-8 photoresist layer. An SU-8 photoresist layer
may not be soluble in water. The lithographic process may comprise applying a photomask
5 to the photoresist and exposing the photoresist layer to ultraviolet radiation, before washing
away or dissolving unwanted regions of the photoresist layer.
Once the photoresist layer has been patterned to define the areas of the carriers on the
substrate, the release layer in between the carriers may be removed, if desired. This may
advantageously reduce the volume of the release layer which is exposed to the
10 biocompatible solution during subsequent release of the carriers, reducing any
concentration of the release layer that dissolves in the solution and minimising any
influence that concentration may have on assay results.
During fabrication of carriers, subsequent layers of materials may be deposited on the
carriers as described herein. These materials may also be deposited on the substrate or
15 release layer between carriers. It is important that if any such materials are deposited
between the carriers, they do not impede access of the biocompatible solution to the edges
of the release layer beneath the carriers when an assay is performed. If required, materials
between the carriers can be removed during fabrication to ensure that edges of the release
layer beneath the carriers are exposed, so that the release layer can be activated by the
20 biocompatible solution to release the carriers.
The method for manufacturing the carrier system may further comprise the step of
sterilizing the carrier system by immersing it in ethanol, following the step of depositing or
forming or fabricating the carrier on the release layer. Sterilization may advantageously
eliminate unwanted forms of life or biological agents that might otherwise adversely
25 interfere with assays performed using the carrier system. The carrier system may typically
be immersed in the sterilization liquid for up to 10, 15, 20 or 25 minutes. Sterilization may
comprise immersing the carrier system in a non-aqueous sterilization liquid such as
ethanol. The sterilization liquid may comprise pure ethanol. This may be advantageous
when the release layer comprises a material, such as dextran or polyvinyl alcohol, which is
30 not activatable in a non-aqueous solvent, because the carrier will remain secured to the
substrate while the carrier is immersed in the sterilization liquid. A similar effect may be
achieved even if the sterilization liquid does affect the release layer, provided the release
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time of the release layer is greater than the period for which the carrier system is immersed
in the sterilization liquid.
The step of sterilizing the carrier system may also be conducted using dry heat, steam, or
gaseous sterilization methods. Dry heat sterilization may be the best suited for this as the
5 release layer is least likely to be affected by this sterilization method.
The step of depositing or fabricating the carrier may comprise forming a magnetic structure
or layer. In particular, when the method comprises a lithographic process, the method may
comprise depositing a magnetic material on top of the photoresist layer. Additional
materials such as gold may also be deposited on top of the photoresist layer or on top of
10 the magnetic material of the carrier. A gold cap may provide biocompatibility. Furthermore,
barcodes, QR codes or other readable information may be lithographically added to the
carriers.
The method may further comprise the step of depositing a layer of gold on the carrier to
form a cap. The gold cap may advantageously be biocompatible. The gold may be
15 deposited only on a surface of the carrier that is exposed when the carrier is in contact with
the substrate.
The method may further comprise adapting a surface of the carrier for receiving an assay
sample. In a preferred embodiment, the surface of the carrier may be adapted for receiving
adherent cells.
20 The step of adapting a surface of the carrier for receiving an assay sample may be
performed before the step of depositing a carrier on the release layer. Alternatively, the
step of adapting a surface of the carrier for receiving an assay sample may be performed
after the step of depositing a carrier on the release layer. When the step of depositing the
carrier on the release layer comprises fabricating the carrier on the release layer, the step
25 of adapting the surface of the carrier for receiving the assay sample may be a step of that
fabrication process.
The step of adapting a surface of the carrier for receiving an assay sample may comprise
applying a coating to the surface of carrier, as has been described above.
The coating may be a charged coating. The coating may comprise a charged polymer. The
30 charged polymer may be a polymer comprising a positive charge carrying group. The
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polymer may be a polyelectrolyte. The charged polymer may be a polymer comprising a
positive charge carrying group. The polymer may be a polyornithine or a poly-d-lysine.
The polymer may be applied by immersing the carrier system in a solution comprising the
polymer for a predetermined period of time, such as between 20 and 120 minutes. The
5 solution may comprise the polymer in a concentration of between about 1nM to 10mM,
depending on the polymer used. The solution may comprise a non-aqueous solvent such
as ethanol. The solution may consist of the non-aqueous solvent and polymer. The use of a
non-aqueous solvent may be advantageous when the release layer comprises a material,
such as dextran or polyvinyl alcohol, that may not be activatable in a non-aqueous solvent.
10 This may be because the carrier will remain secured to the substrate while the carrier is
immersed in the solution comprising the polymer.
The polymer may comprise a thiol group and it may be the thiol group that covalently bonds
with the gold cap of the carrier. This advantageously ensures that each polymer bonded
onto the surface has the same orientation. Alternatively, the polymer may be adsorbed onto
15 the gold cap of the carrier via Van der Waals forces.
Alternatively or additionally, the coating may comprise a plurality of ligands. The plurality of
ligands may comprise antibodies. The plurality of ligands may comprise antibodies that
specifically bind to cell receptors such as integrins. Alternatively or additionally the coating
may comprise an extracellular matrix protein. The extracellular matrix protein may be a
20 protein selected to increase or enhance cell adhesion. The protein may be a collagen,
laminin or Matrigel, a protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells.
The step of adapting a surface of the carrier for receiving an assay sample may comprise
modifying the surface topology of the carrier for example, by increasing the surface area of
25 the carrier. The surface area may be increased by increasing roughness of the surface. For
example, the surface may have been etched using an ion beam. Alternatively or
additionally, dots, pits, protrusions or grooves may be provided on the carrier surface.
The step of adapting a surface of the carrier for receiving an assay sample may comprise
modifying the hardness of a surface of the carrier. This may be achieved by the application
30 of a coating comprising a dense layer of proteins or polymers.
A third aspect of the invention may provide a method of performing an assay using the
carrier system as in the first aspect of the invention or manufactured as in the second
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aspect of the invention described above. The method comprises a step of introducing a
biocompatible aqueous solution to the carrier system to release the carrier. The method
may further comprise the step of introducing an assay sample to the carrier such that the
assay sample is received by the carrier. The assay sample may advantageously be
5 received by the carrier while the carrier is in contact with the substrate. An assay may be
performed on a carrier having an assay sample received thereon. The advantages of the
assay sample being received on the carrier while the carrier is in contact with the substrate
have been described in relation to the first aspect.
In a preferred embodiment, the carrier may comprise a magnetic material and the method
10 may further comprise applying a magnetic field to the carrier, the magnetic field acting to
retain the carrier in contact with the substrate even after the release layer has released the
carrier from the substrate. This may be particularly advantageous when the release layer
releases the carrier in a time that is less than the typical time for an assay sample to be
received by the carrier. For example, if the release layer comprises a sugar such as a
15 dextran, the release layer, in the presence of a biocompatible aqueous solution, may
release the carrier in five seconds or less. However, the typical time for an assay sample to
be received by the carrier may be longer than five seconds. The assay sample may then
advantageously be received by the carrier while the magnetic field retains the carrier in
contact with the substrate (by magnetically applying a force urging the the carrier against
20 the substrate) for the time required for the assay sample to be received by the carrier. This
may remove the need to match the release time of the release layer with the typical time
taken for a particular assay sample to be received by the carrier, while maintaining the
advantages of receiving the assay sample on the carrier while the carrier is contact with the
substrate.
25 The magnetic field may be applied to retain the carrier in contact with the substrate for at
least five seconds. In other words, the magnetic field may be applied to retain the carrier in
contact with the substrate for longer than the typical time for activating a release layer
comprising a sugar such as a dextran in the presence of water. The magnetic field may be
applied for much longer than five seconds. For example, the magnetic field may be applied
30 for at least 1, 5 or 10 minutes, or at least 1, 3 or 12 hours. In a preferred embodiment, the
magnetic field may be applied for 2 to 4 hours, or for about 3 hours, where the assay
sample is an adherent cell. This is a typical time for an adherent cell to be received on the
carrier. The magnetic field may typically be applied for less than 10 minutes, or less than
1, 3 or 12 hours, but this time will depend on the assay application.
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It is clear that features described in relation to one aspect of the invention may be applied
to other aspects of the invention.
A carrier system comprising a carrier or particle for receiving an assay sample has been
described above. Particularly preferable features for the carrier have also been described
5 above. However, it should be understood that the carrier system may comprise any carrier
suitable for receiving and carrying a sample which may be secured to a substrate by a
release layer and that such a carrier system may have the advantages described above.
Further specific preferred embodiments of the invention may include carriers on a wafer or
substrate with or without pre-functionalization. A wafer may carry anywhere from 1000-
10 10 million carriers, or more, depending on customer needs. Alternatively, carriers on
wafers or substrates may be provided with the wafers each in a well of a well plate. In this
case there may typically be 100-10,000 carriers per wafer depending on the well size. An
example of this embodiment may be a well plate containing the carriers and substrates
already installed in one or more wells. In a further embodiment, carriers on a wafer or
15 substrate may be provided with an assay sample, such as cells, in a frozen state already
attached and ready to assay. Such a wafer may typically carry 1000-100,000 carriers.
Carrier systems embodying the invention may be provided to users in a variety of forms. In
one embodiment, a carrier system comprising carriers on a substrate may be sterilised and
packaged, for example in dry, sterile packaging or wrapping, for supply to users wishing to
20 carry out assays. Such a carrier system may also be supplied in a sterile medium, which
may be a liquid or gas. Alternatively, as described above carrier systems may be provided
pre-loaded with assay samples, such as cells, usually frozen ready for use in an assay, In
such carrier systems, the carriers may be suitably marked for identification, such as for
identifying individual carriers, or groups of carriers.
25 PCT/GB2019/053188, a co-pending application filed by the applicant, describes the
fabrication of lithographically defined carriers for multichannel assays. Many features
described in PCT/GB2019/053188 are directly applicable to the present invention although
in the context of the present invention the processes described in PCT/GB2019/053188
need to be modified to avoid using a water-soluble photoresist. For example, an SU-8
30 photoresist could be used. PCT/GB2019/053188 is incorporated herein in its entirety by
reference, and is reproduced below in an Annex.
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It may be noted that in the prior art, different types of magnetic particles for use in assays
have been described. For example, patent publication W02009/029859 describes a
method for forming magnetic nanodiscs, of 1 nm to 200nm width, by electrodeposition or
chemical vapour deposition. The nanodiscs are formed either on a layer of sodium chloride
5 or potassium chloride, which is then dissolved in water, or on a layer of copper, silver or
aluminium, which is then dissolved in an acid/metal etchant. The product of this method is
free nanodiscs in solution which are provided to users for use in DNA arrays. (The solution
may be the solution which was used to remove the nanodiscs from the salt layer, or the
nanodiscs may be washed and dispersed again into a suitable solution for use in DNA
10 arrays.) The nanodiscs are formed with suitable molecules on their surfaces so that when
they are in solution, they can bind to specific desired targets, i.e. particular structures on
the surfaces of cells, so that those targets can be identified by use of a magnetic field
sensor to sense the magnetic field of the nanodisc. The nanodiscs are too small to carry or
support a cell, and are not usable in an assay on a substrate because they are much too
15 small to support a cell. The nanodiscs may individually be influenced by an external
magnetic field, but the volume of magnetic material in such a nanodisc is insufficient to
direct an assay sample such as a cell within an assay, even after a nanodisc has bound to
the assay sample.
Patent publication US2013/0052343 describes a another type of magnetic particle for
20 targeting assay samples in solution. These particles are of 2 or 3 micrometres up to 100
micrometres or 500 micrometres diameter, and are formed by deposition in moulds defined
in a layer of a photosensitive resin deposited over a layer of polymethyl methacrylate
(PMMA). The photosensitive resin is selectively removed in the areas where particles are
to be formed, the particles are deposited, and then the PMMA is dissolved in a solvent
25 such as acetone to free the particles. The particles are then washed and suspended in a
suitable solution for use. They are supplied in this form for users to target and enable
recognition of desired molecular or cellular species.
Specific Description
Specific embodiments of the invention will now be described by way of example, with
30 reference to the accompanying drawings in which:
Figure 1 is a schematic view of a carrier system comprising a plurality of carriers or
particles secured to a substrate by a release layer, according to a first embodiment of the
invention;
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Figure 2 illustrates a process for manufacturing the carrier system of Figure 1;
Figure 3 shows a number of different surface adaptations of the carrier to improve the rate
or efficiency with which adherent cells are received on the carrier; and
Figure 4 shows a series of schematic views of one of the plurality of carriers of Figure 1
5 during the process of introducing a biocompatible aqueous solution comprising an assay
sample; Figure 4a shows the carrier immediately prior to the introduction of the
biocompatible aqueous solution comprising the assay sample, Figure 4b shows a carrier
that, following the introduction of the biocompatible aqueous solution, is no longer secured
to the substrate by the release layer but that is retained in contact with the substrate by an
10 external field, and Figure 4c shows an assay sample received on a released carrier.
Sheets 5 to 13 of the accompanying drawings reproduce the drawings of co-pending
application PCT/GB2019/053188 incorporated herein in its entirety by reference, and
reproduced in the Annex below.
A carrier system 100 according to a first embodiment of the invention is shown in Figure 1.
15 The carrier system 100 comprises a substrate 10 in the form of a silicon chip. Four carriers,
or particles, 12 are secured to the substrate 10 by a release layer 14 comprising a dextran.
The carrier system can be used in an assay of adherent cells. Each of the carriers 12 is
suitable for receiving an adherent cell or a plurality of adherent cells. Each carrier is in the
shape of a flat, or high-aspect-ratio, cuboid with relatively large, square upper and lower
20 surfaces. The upper surface 16 of each carrier, opposite to a lower side of the carrier
contacting the release layer, is adapted for the cells to adhere to. The carriers have a
lateral dimension of 100 microns. Each carrier comprises a layer of magnetic material, not
shown in Figure 1.
Each carrier comprises a barcode 18 such as a quick response (QR) code or 20 data
25 matrix code. Predetermined readable codes are assigned to each carrier to identify the
carriers. For example, the predetermined barcode 18 can refer to a particular adherent cell
received on the carrier 12 or a particular adherent cell and particular reagent that the
adherent cell is exposed to as part of an assay. In other words, the barcodes 18 allow for a
multichannel assay to be performed by the carriers 12.
30 As shown in Figure 1, the release layer 14 is discontinuous, so that the substrate is
exposed 15 between each of the carriers 12. However, during manufacture the release
layer 14 was originally formed by spin-coating a continuous layer of dextran covering the
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entire surface of the substrate 10, and the portions of the release layer between the
carriers were removed after formation of the carriers. Therefore, the release layer 14
beneath the carriers may be considered a single layer.
While Figure 1 shows four carriers 12 secured to the substrate 10, any number of carriers
5 12 can be secured to the substrate. A single carrier 12 can be secured to the substrate.
A lithographic process for manufacturing the carrier system 100 according to an
embodiment of the invention is illustrated in Figure 2.
Figure 2a shows a release layer 14 formed on a silicon substrate 10 by spin-coating onto
the substrate an aqueous solution comprising Dextran 70 in an amount 20 % by weight.
10 Following the spin-coating of the release layer, the silicon substrate 10 is baked. In the
case of a Dextran 70 release layer, the baking is at 150 degrees Celsius for 2 minutes. A
photoresist layer 20 is formed on top of the release layer 14 by spin-coating an SU-8
photoresist on top of the release layer 14. The SU-8 photoresist is then baked.
In an alternative embodiment, the release layer 14 can be formed of polyvinyl alcohol
15 (PVA) rather than Dextran 70, by spin-coating an aqueous solution comprising a polyvinyl
alcohol onto the substrate.
Figure 2b shows a lithographic patterning step being performed on the SU-8 photoresist
layer 20, by exposing the photoresist to ultraviolet radiation 24 through a patterned
photomask 22 and baking the exposed resist at 95C for 2 minutes. This exposes specific
20 regions of the photoresist layer 40 to the ultraviolet radiation 24 causing cross-linking in
those regions. The remainder of the photoresist layer 24 remains soluble and can be
washed away in a suitable non-aqueous solvent. This process allows for discontinuities to
be formed in the photoresist layer to define the base of each carrier, without affecting the
underlying release layer.
25 The barcodes are also added during the lithographic patterning of the SU-8 photoresist
layer by making an array or pattern of holes in the SU-8. These holes can be read in
transmission mode in a microscope and act as a barcode that can used to identify the
carrier, or in the identification of the assay sample received on that carrier.
Figure 2c shows the photoresist layer 20 following exposure to a suitable solvent. The
30 primary solvents for SU-8 photoresists are PGMEA, gamma-butyrolactone or
cyclopentanone. The soluble, non-cross-linked regions of the photoresist are washed away
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by the solvent to create a discontinuous SU-8 photoresist layer. Because gammabutyrolactone
or cyclopentanone are non-aqueous solvents, washing away the non-crosslinked
regions of the photoresist layer does not dissolve the release layer 14. The release
layer 14 is not soluble in non-aqueous solvents such as gamma-butyrolactone or
5 cyclopentanone. Following washing, the remaining SU-8 forms the base 26 of each carrier.
An oxygen plasma etch is then applied, using the photoresist as a mask, to remove the
release layer 14 from the substrate 10 in the regions between carriers. Figure 2d shows the
release layer and the overlying portions of the photoresist layer which form the bases 26 of
the carriers, following this etching process.
10 Figure 2e shows the carrier system after further layers 28 comprising magnetic materials
have been deposited over the photoresist bases 26 of the carriers. These layers 28 include
a plurality of layers of magnetic material interspersed with layers of non-magnetic material
and are added by magnetron sputtering. In order to ensure that the carrier has a low stray
field to avoid aggregation of the carrier, a layered structure comprising 11 layers is used,
15 the layers being as follows (thickness in nm):
Au(20.0)/Ta(2)/Pt(4)/CoFeB(0.6)/Pt(1.2)/CoFeB(0.6)/Pt(1.2)/CoFeB(0.6)/Pt(1.2)/
CoFeB(0.6)/Pt(5.0). It may be noted that the SU-8 photoresist layer of the carrier is about
1.5 to 2 microns in thickness, and advantageously provides mechanical stability to the
carrier. Therefore, the magnetic layer is mechanically supported.
20 A cap of gold is then formed on the carrier using a top-down lithographic process. The gold
cap is deposited on a surface of the carrier that is exposed while the carrier is secured to
the substrate. The gold cap provides biocompatibility, and further coatings or surface
adaptions can be applied to the gold cap depending on the desired assay application of the
carriers.
25 Figure 2f shows the carrier system after the gold cap layer has been adapted to be
particularly suitable for receiving adherent cells by adding a coating 29 comprising a
polymer.
The polymer comprises a thiol group and is applied by immersing the carrier system in a
solution comprising the polymer for between 20 and 120 minutes. The solution comprises
30 the polymer in a concentration of between about 101JM to 10mM. The polymer covalently
bonds to the gold cap of the carrier by the thiol group. The solution comprises a nonaqueous
solvent such as ethanol. The release layer is non-soluble in such a non-aqueous
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solvent and so the carrier will remain secured to the substrate while the carrier system is
immersed in the solution comprising the polymer. Figure 3a shows a schematic of a carrier
12 comprising a coating of charged polymers 32 comprising a thiol group covalently
bonded to the carrier 12.
5 Alternative coatings can be applied to carrier. Instead of a charged polymer, the coating 29
can comprise a plurality of ligands comprising antibodies that specifically bind to cell
receptors such as integrins or an extracellular matrix protein such as collagen, or Matigel, a
protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Figure
3b shows a schematic of a carrier 12 where a coating has been applied comprising a
10 plurality of ligands 34.
Alternatively to applying a coating, the method can comprise modifying the physical surface
of the carriers to be particularly suitable for receiving adherent cells, for example by
modifying the gold cap. Figure 3c shows an embodiment where the physical structure of
the carrier, and particularly, the gold cap, has been adapted for receiving adherent cells. In
15 this embodiment, the surface area has been increased by providing grooves 30 on the top
surface of the carrier. The grooves 30 are formed by an extra lithography step or by etching
the carrier (for example by plasma etching) during the manufacture process, before the
deposition of the gold. The physical structure or topography of a surface can have an effect
on the degree to which adherent cells adhere or seed onto that surface. Cell adhesion can
20 be increased by increasing the surface area of the surface of the carrier.
The final step of the method of manufacturing the carrier system is sterilization, not shown
in the figures. In order to sterilize the carrier system, it is immersed in pure ethanol for 20
minutes. This eliminates unwanted forms of life or biological agents that might otherwise
adversely interfere with assays performed using the carrier system. Again, as dextran or
25 polyvinyl alcohol are not soluble in such a non-aqueous solvent, the carrier remains
secured to the substrate while the carrier system is sterilized.
Figure 4 illustrates the carrier system in use in an assay of adherent cells 42, including a
process of introducing a biocompatible aqueous solution 40 and adherent cells 42 to the
carrier system 100. The adherent cells 42 to be received by the carrier 12 are transported
30 in the biocompatible aqueous solution in order to maintain their viability. Once an adherent
cell has been received by the carrier, the cell reaches a desired or natural adhered
morphology. The coating 29 on the carrier, described above, improves the efficiency or rate
at which the cells are received by carrier and so reduces the time required for the cells to
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be received on the carrier and increases the strength of bonding or attraction between the
assay sample and the carrier. The assay sample is thus advantageously securely bonded
to the carrier.
In practice, the carrier system 100 would be placed in a container and immersed in the
5 biocompatible aqueous solution. This is schematically represented in Figure 4a by the test
tube 44 containing a quantity of biocompatible aqueous solution comprising the adherent
cells 42 and the drop of biocompatible aqueous solution 40. The biocompatible aqueous
solution 40 and the adherent cells 42 are not drawn to scale.
When the biocompatible aqueous solution 40 comes into contact with the release layer 14
10 comprising dextran, the release layer 14 dissolves. This releases the carrier 12. Because
dextran is biocompatible and non-toxic, dissolving the dextran release layer 14 in the
aqueous solution 40 has a minimal effect on the adherent cells 42 and so a minimal effect
on the results of the assay to be performed on the adherent cells 42. Figure 4b shows the
release layer 14 as completely dissolving to release the carrier 12. However, in other
15 embodiments, using different materials for the release layer, the release layer 14 may not
dissolve or may not dissolve completely. In such cases, it is only required that the bond
between the release layer 14 and the carrier 12 is weakened such that the carrier 12 is
released.
In the presence of a biocompatible aqueous solution, a dextran release layer 14 will
20 typically release the carrier in five seconds or less. This is considerably shorter than the
typical time taken for the adherent cell to be received by the carrier. It may be necessary to
allow three hours or more for the adherent cell to be received by the carrier 12. However, it
is preferred for the adherent cell to be received on the carrier while the carrier is in contact
with the substrate. This ensures that the outer, or top, surface 16 of the carrier 12 remains
25 the main exposed surface of the carrier, preventing adherent cells 42 from being received,
for example, on a bottom surface of the carrier 12.
As illustrated in Figure 4b, in order to retain the carrier in contact with the substrate even
after the release layer has released the carrier, an external magnetic field is applied. As
described above, the carriers 12 comprise layers 28 that include magnetic material. The
30 external field is arranged to apply a force to the carrier 12 that urges the carrier against the
substrate and holds it in contact with the substrate 10 even after the release layer 14 has
released the carrier 12. The external field is represented by the dotted arrows in Figure 4b.
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In an alternative embodiment, the release layer can be designed with a release time that
matches or exceeds the typical time for the adherent cell to be received on the carrier. In
that case, the step of applying an external field, as shown in Figure 4b, may not be
necessary. A release layer comprising a polyvinyl alcohol is biocompatible and can be
5 made to have a longer release time, such as twelve hours or longer. This can
advantageously be achieved through a combination of formulation of the PVA layer
(hydrolysis degree in the range 85-99%, or a ratio of two PVAs with different hydrolysis
degrees) and baking, typically at about 115C. If the baking step is omitted, PVA layers with
much shorter release times, down to a few minutes or less, can be fabricated. In this way
10 the release time of the release layer can be designed as required for specific assay
applications.
Therefore, when a carrier system 100 comprising a suitably-fabricated polyvinyl alcohol
release layer 14 is used with adherent cells that typically take three hours to be received by
the carrier 12, there may be no need to apply an external field to retain the carrier in
15 contact with the substrate, as described above.
Once the carrier has been released from substrate, the released carrier 12 is free to move
through the aqueous solution 20 surrounding the carrier system. This is shown in Figure
4c. External magnetic fields can be used to apply forces to the carrier in order to
manipulate and move the carrier as desired to perform the assay. The biocompatible
20 aqueous solution 20 also provides a suitable environment for the adherent cells 22 to
maintain their viability. It is more convenient to manipulate and transport adherent cells
received on carriers than trying to manipulate and transport the adherent cells on their own,
particularly because adherent cells are most viable when received on a surface. Receiving
the cells on the carriers 12 allows each adherent cell to be transported as if it is in
25 suspension while remaining in a biologically representative, adhered state.
Once the carriers having adherent cells received thereon are released from the substrate,
an assay can be performed.

Claims
1. A carrier system for an assay comprising a carrier secured to a substrate by a
release layer, the carrier being suitable for receiving an assay sample, and the release
5 layer being configured in use to release the carrier from the substrate in the presence of a
biocompatible aqueous solution.
2. A carrier system according to claim 1, adapted to receive the assay sample onto the
carrier while the carrier is in contact with the substrate.
3. A carrier system according to claim 1 or 2, in which the assay sample is suspended
10 in the biocompatible aqueous solution, and in which when the carrier system is contacted
with the biocompatible solution, the assay sample is received onto the carrier and the
carrier is released from the substrate.
4. A carrier system according to any preceding claim, wherein the release layer is
configured such that, following activation of the release layer, the biocompatible aqueous
15 solution remains biocompatible.
5. A carrier system according to any preceding claim, wherein the release layer
comprises a material that is water-activatable, such as a material that is water-soluble.
6. A carrier system according to any preceding claim, wherein the release layer is not
activatable in a non-aqueous solvent such as ethanol.
20 7. A carrier system according to any preceding claim, wherein the release layer
comprises at least one of a sugar, such as a dextran, or a polyvinyl alcohol or a poly( acrylic
acid) or a poly(lactic-co-glycolic acid)).
8. A carrier system according to any preceding claim, wherein the carrier comprises a
magnetic material.
25 9. A carrier system according to claim 8, wherein the carrier comprises a layered
structure between a top surface of the carrier and an opposed bottom surface of the carrier,
the layers including one or more magnetized layers.
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10. A carrier system according to claim 9, in which the ratio of a lateral dimension of the
one or more magnetized layers to a thickness or aggregate thickness of the magnetized
layer or layers is greater than 500.
11. A carrier system according to any preceding claim, in which a minimum lateral
5 dimension of the carrier is between 5 micrometres and 200 micrometres.
12. A carrier system according to any preceding claim, wherein the carrier is
lithographically defined.
13. A carrier system according to any preceding claim, wherein the carrier comprises a
photoresist layer, such as an SU-8 photoresist.
10 14. A carrier system according to any preceding claim, wherein a surface of the carrier
is adapted for receiving the assay sample, for example wherein the surface comprises a
gold cap layer on to which a polymer is covalently bonded by thiol group.
15. A carrier system according to any preceding claim, wherein the carrier comprises
readable information, such as a readable code selected from a barcode or a 20 code.
15 16. A carrier system according to any preceding claim, comprising a plurality of carriers,
wherein each of the carriers is secured to the substrate by the release layer.
17. A carrier system according to any preceding claim, comprising sterile packaging
from which the carrier secured to the substrate is removable for use.
18. A method of manufacturing a carrier system for an assay, the method comprising
20 the steps of:
providing a substrate;
forming a release layer on the substrate; and
depositing a carrier for receiving an assay sample on the release layer such that the carrier
is secured to the substrate,
25 wherein the release layer is configured in use to release the carrier from the substrate in
the presence of a biocompatible aqueous solution.
19. A method according to claim 18, wherein the step of forming the release layer on
the substrate comprises spin-coating the release layer.
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20. A method according to claim 18 or 19, wherein the step of depositing the carrier on
the release layer comprises fabricating the carrier on the release layer, for example by a
lithographic process.
21. A method according to any of claims 18 to 20, wherein the release layer is adapted
5 to release the carrier from the substrate, in use, in the presence of the biocompatible
aqueous solution within a time of between 1 hour to 72 hours.
22. A carrier system according to any of claims 18 to 21, in which the assay sample is
receivable by the carrier while the carrier is in contact with the substrate.
23. A method according to any of claims 18 to 22, further comprising the step of
10 adapting a surface of the carrier such that the surface is suitable for receiving an assay
sample.
24. A method according to any of claims 18 to 23, further comprising the step of
sterilizing the carrier system by immersing the carrier system in ethanol, following the step
of depositing the carrier on the release layer.
15 25. A method according to any of claims 18 to 24, further comprising the step of
packaging the carrier system within sterile packaging, with the carrier secured to the
substrate.
26. A method according to any of claims 18 to 25, wherein the step of depositing the
carrier comprises forming a magnetic structure.
20 27. A method of performing an assay using the carrier system as defined in any of
claims 1 to 17, the method comprising the step of introducing a biocompatible aqueous
solution to the carrier system to release the carrier.
28. A method of performing an assay according to claim 27, further comprising the step
of introducing a sample for an assay to the carrier such that the sample is received by the
25 carrier.
29. A method of performing an assay according to claim 28, wherein the sample is
received by the carrier while the carrier is in contact with the substrate.
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30. A method of performing an assay according to any of claims 27 to 29, in which the
assay sample is suspended in the biocompatible aqueous solution.
31. A method of performing an assay according to any of claims 27 to 30, wherein the
carrier comprises a magnetic material and the method further comprises applying a
5 magnetic field to the carrier, the magnetic field acting to retain the carrier in contact with the
substrate even after the release layer has released the carrier from the substrate.
32. A method of performing an assay according to claim 31, wherein the magnetic field
is applied to retain the carrier in contact with the substrate for at least five seconds, or at
least one minute, or at least 5 minutes or at least 30 minutes.
10 33. A method of using the carrier system as defined in any of claims 1 to 17, comprising
the steps of introducing a sample for an assay to a carrier such that the sample is received
by the carrier while the carrier is in contact with the substrate, and storing the sample
received on the carrier.
34. A method according to claim 33, including the step of freezing the sample received
15 on the carrier for storage.
35. A method as defined in claim 33 or 34, further comprising the step of releasing the
carrier from the substrate before storing the sample received on the carrier.
36. A method for carrying out an assay, comprising the steps of;
providing a carrier secured to a substrate by a release layer which is configured to release
20 the carrier from the substrate in the presence of a biocompatible aqueous solution;
contacting the carrier secured to the substrate with a biocompatible solution in which an the
assay sample is suspended;
receiving the assay sample onto the carrier while the carrier is in contact with the substrate;
and releasing the carrier from the substrate by activation of the release layer by the
25 biocompatible solution.
37. A carrier system or a method according to any preceding claim, in which the assay
sample comprises a cell, preferably an adherent cell.