Note: Descriptions are shown in the official language in which they were submitted.
CA 02668839 2016-01-08
Method and Analysis Device Comprising a Substrate Zone
Technical field
The present invention concerns an assay device for the
analysis of a liquid sample.
Background
Quick, reliable, and cost effective analytical and diagnostic
devices for instance devices for use in point of care are
. 10 desirable.
In many assays, detection conjugate and possibly further
reagents are predispensed or integrated in the device,
setting aside the need for separate addition of reagents by
the user.
A common type of disposable assay device comprises a zone for
receiving the sample, a reaction zone, and optionally a
transport or incubation zone connecting the receiving and
reaction zone, respectively. These assay devices are known as
immunochromatography assay devices or simply referred to as
strip tests.
PCT/SE03/00919 relates to a micro fluidic system comprising a
substrate and provided on said substrate there is at least
one flow path comprising a plurality of micro posts
protruding upwards from said substrate, the spacing between
the micro posts being small enough to induce a capillary
action in a liquid sample applied, so as to force said liquid
to move.
PCT/SE2005/000429 shows a device and method for the
separation of a component in a liquid sample prior to the
detection of an analyte in said sample, wherein a sample is
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, .
added to a receiving zone on a substrate, said substrate
further optionally comprising a reaction zone, a transport or
incubation zone connecting the receiving and reaction zone,
respectively, forming a flow path on a substrate, wherein
said substrate is a non-porous substrate, and at least part
of said flow path consists of areas of projections
substantially vertical to the surface of said substrate, and
having a height, diameter and reciprocal spacing such, that
lateral capillary flow of said liquid sample in said zone is
achieved, and where means for separation are provided
adjacent to the zone for receiving the sample.
PCT/SE2005/000787 concerns a device for handling liquid
samples, comprising a flow path with at least one zone for
receiving the sample, and a transport or incubation zone,
said zones connected by or comprising a zone having
projections substantially vertical to its surface, said
device provided with a sink with a capacity of receiving said
liquid sample, said sink comprising a zone having projections
substantially vertical to its surface, and said sink being
adapted to respond to an external influence regulating its
capacity to receive said liquid sample.
PCT/SE2006/000745 relates to an absorbing zone for
establishing and/or maintaining fluid transport through or
along said at least one fluid passage is manufactured on the
basis of a non-porous substrate, having projections
substantially perpendicular to said surface, and said
projections having a height, diameter and a distance or
distances between the projections such, that lateral
capillary flow of said fluid in said zone is achieved.
Although the assay devices comprising projections according
to the prior art are working satisfactory there is still room
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for a further improvement regarding for instance the control
of the dissolution of a substance that is applied on the
device.
Summary of the invention
One object of the present invention is to provide a device
where the control of the dissolution of a predispensed
substance is further improved. There is made available an
analysis device for the analysis of a liquid sample, said
device comprising a substrate, said substrate at least partly
having projections substantially vertical to the surface of
said substrate, and having a height (H1), diameter (D1) and
center-to-center distance (xl, yl) such, that lateral
capillary flow of said liquid sample is achieved, wherein
said substrate comprises at least one substrate zone
comprising projections substantially vertical to the surface
of said substrate, and having a height (H2), diameter (D2)
and center-to-center distance (x2, y2), such, that lateral
capillary flow of said liquid sample is achieved and wherein
at least one substance is applied at least partly between the
projections in said at least one substrate zone.
Further aspects and embodiments of the present invention are
defined in the appended claims which are incorporated herein
by reference.
Short description of the drawings
The invention will be described in closer detail in the
following description, examples, and attached drawings, in
which
Fig. 1 shows an embodiment from above, where there is shown
one sample addition zone 1, one connecting zone 2, one
receiving zone 3 as well as a substrate zone 4 comprising
,
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projections with a diameter which is larger than the diameter
for the surrounding projections. There is also a reaction
zone 5. Between projections in the substrate zone 4 there is
an applied substance.
Fig. 2 shows an embodiment where there is a substrate zone
comprising projections with a diameter D2, which is larger
than the diameter D1 for the surrounding projections.
Fig. 3 shows an embodiment as in Fig 2 where a substance has
been applied in the substrate zone.
Fig. 4 shows an embodiment with a substrate zone comprising
projections with a larger diameter than the surrounding
projections. A substance has been applied between the
projections in the substrate zone. The arrows indicate the
direction of a flow of liquid.
Fig. 5 shows the same embodiment as in fig. 4 after a period
of time. The arrows indicate that a part of the substance has
dissolved in the liquid and is brought in the direction of
the flow.
Fig. 6 shows an embodiment with a substrate zone where a
substance has been applied between the projections in the
substrate zone. There is an area surrounding the substrate
zone where there are no projections.
Definitions
Before the present device and method is described, it is to
be understood that this invention is not limited to the
particular configurations, method steps, and materials
disclosed herein as such configurations, steps and materials
may vary somewhat. It is also to be understood that the
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,
terminology employed herein is used for the purpose of
describing particular embodiments only and is not intended to
be limiting since the scope of the present invention will be
limited only by the appended claims and equivalents thereof.
It must also be noted that, as used in this specification and
the appended claims, the singular forms "a", "an", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to a reaction mixture
containing "an antibody" includes a mixture of two or more
antibodies.
The term "about" when used in the context of numeric values
denotes an interval of accuracy, familiar and acceptable to a
person skilled in the art. Said interval can be + 10 % or
preferably + 5 %.
In describing and claiming the device and method, the
following terminology will be used in accordance with the
definitions set out herein.
As used throughout the claims and the description the term
"analysis" means the process in which at least one analyte is
determined.
As used throughout the claims and the description the term
"analysis device" means device by the aid of which an
analysis can be performed.
As used throughout the claims and the description the term
"analyte" means a substance or chemical or biological
constituent of which one or more properties are determined in
an analytical procedure. An analyte or a component itself can
often not be measured, but a measurable property of the
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analyte can. For instance, it is possible measure the
concentration of an analyte.
As used throughout the claims and the description the term
"capillary flow" means flow induced mainly by capillary
force.
As used throughout the claims and the description the term
"casing" means an element enclosing a part of or the entire
substrate.
As used throughout the claims and the description the term
"center-to-center distance" means the distance between
adjacent projections, measured from the center of a
projection to the center of an adjacent projection. For a
planar substrate the center-to-center distance is measured
both in the x direction and in the y direction, in an
orthogonal coordinate system in the substrate plane.
As used throughout the claims and the description the term
"center of a projection" means the center of gravity for an
infinitesimal thin slice of the projection taken on half of
the height of the projection in a plane parallel to the
surface of the substrate. For curved substrates the plane is
parallel to the surface of the substrate in a sufficiently
small surrounding around the projection.
As used throughout the claims and the description the term
"connecting zone" means a zone which establishes fluid
connection between at least two other zones.
As used throughout the claims and the description the term
"detectable group" means any arrangement of molecules or
atoms that can be detected when present on a substrate.
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As used throughout the claims and the description the term
"fluid connection" means a connection in which a fluid can be
transported.
As used throughout the claims and the description the term
"sample" means a mixture or a solution to be analyzed.
As used throughout the claims and the description the term
"substance" means any pure chemical or biological entity or
any mixture or solution comprising at least one chemical or
biological entity.
Detailed description
In a first aspect there is provided an analysis device for
the analysis of a liquid sample, said device comprising a
substrate, said substrate at least partly having projections
substantially vertical to the surface of said substrate, and
having a height, diameter and center-to-center distance such,
that lateral capillary flow of said liquid sample is
achieved, and said substrate comprises at least one substrate
zone comprising projections substantially vertical to the
surface of said substrate, and having a height (H2), diameter
(D2) and center-to-center distance (x2, y2), such, that
lateral capillary flow of said liquid sample is achieved and
wherein at least one substance is applied at least partly
between the projections in said at least one substrate zone.
When a liquid sample is added to the substrate a flow is
created by capillary forces due to the projections on the
surface, where the projections have a height (H1), diameter
(D1) and center-to-center distance (xi, yl). There is also at
least one zone which is covered with projections, where the
projections have a different height (H2), diameter (D2) or
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center-to-center distance (x2, y2), compared to the other
projections on the substrate with a height (H1), diameter
(D1) and center-to-center distance (xl, yl). In one
embodiment at least one of the properties height, diameter
and center-to-center distance is different between the zones.
In another embodiment the height, diameter and center-to-
center distance are different. In a further embodiment the
diameter and center-to-center distance are different. In one
embodiment H1 and H2 are different. In one embodiment D1 and
D2 are different. In one embodiment xl and x2 are different.
In one embodiment yl and y2 are different. In further
embodiments several of the parameters H, D, x and y are
different. In one embodiment all parameters H, D, x and y are
the same for at least two zones of the substance. In one
embodiment all parameters H, D, x and y are the same for the
entire device.
At least one substance is applied between projections on the
substrate. In one embodiment the substance is applied
entirely within the at least one substrate zone. In an
alternative embodiment at least one substance is applied both
in the at least one substrate zone and outside the at least
one substrate zone.
In one embodiment the projections in the substrate zone have
a different height (H2), diameter (D2) or center-to-center
distance (x2, y2) so that the substrate zone has an
appearance which is different from the surrounding. This is
an advantage when the device is manufactures since the zone
where a substance is to be applied can easily be identified
by a human eye or by an automated device.
In one embodiment the projections in the substrate zone have
a different height (H2), diameter (D2) or center-to-center
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distance (x2, y2) so that the substrate zone exerts a
different capillary force on a substance in solution compared
to the surrounding. This facilitates the addition of a
substance in a solution or a suspension. In one embodiment
the capillary force exerted by the substrate zone is higher
than for the surrounding and thus a substance in a solution
or a suspension can easier be applied to the substrate zone
only. The substance to be applied is in one embodiment
dissolved or suspended in a solvent and applied to the
substrate. The solvent is in one embodiment evaporated and
the substance is thus left on the substrate. This has the
advantage to create a well defined area in which the
substance is applied. The boundaries of the applied substance
are sharp.
In one embodiment the substance is applied in a volume
between the projections of the substrate zone. When the
substance is applied to the substrate the capillary force
exerted from the projections of the substrate zone in one
embodiment causes the substance in solution or suspension to
fill out essentially the entire volume between the
projections of the substrate zone.
In one embodiment the projections of the substrate zone(s)
have a height (H2), diameter (D2) and center-to-center
distance (x2, y2) such that the capillary force exerted by
the projections facilitate addition of said substance(s) to
the substrate zone(s).
In one embodiment the substance is an essentially pure
substance. In another embodiment the at least one substance
is a mixture of two or more substances.
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Examples of substances which can be applied on the substrate
include but are not limited to antibodies, DNA, RNA,
aptamers, antibodies directed to specific analytes in a
sample, fragmented antibodies, antibody fragments, synthetic
binders, chemical binders, receptors, ligands, affibodies,
cells, organelles, polypeptides, peptides, enzymes,
monoclonal antibodies, polyclonal antibodies, phage display
proteins, IgG immunoglobulins, chemical ligands, and
combinations thereof.
In one embodiment the substance to be applied to the
substrate comprises at least one further additive. Examples
of further additives include but are not limited to sugars,
polymers, detergents, surface active agents, cationic surface
active agents, non-ionic surface active agents, anionic
surface active agents, salts, and lipids or any combination
thereof.
In one embodiment there is added at least one further
substance to the substrate, where that substance is at least
one substance selected from a sugar, a polymer, a detergent,
a surface active agent, a cationic surface active agent, a
non-ionic surface active agent, an anionic surface active
agent, a salt, and a lipid.
In one embodiment the substrate further comprises in
sequence; at least one sample addition zone, at least one
connecting zone, and at least one receiving zone in fluid
connection, wherein said connecting zone comprises at least
one reaction zone, and wherein said at least one substrate
zone is between said sample addition zone and said reaction
zone. In one embodiment at least a part of the connecting
zone is covered by projections such that a lateral capillary
flow is achieved.
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In one embodiment at least one zone is surrounded by an area
where at least one of the height (H), diameter (D) and
center-to-center distance (x, y) of the projections is
different so that the surrounding projections exert a lower
capillary force on the sample liquid then the zone. In one
embodiment said zone is at least one zone selected from the
reaction zone and the substrate zone. In one embodiment said
zone is the substrate zone. In one embodiment the center-to-
center distance in an area surrounding a zone is larger
compared to within the zone.
In one embodiment there is an area without any projections
surrounding at least one of the substrate zone and the
reaction zone. In fig. 6 there is depicted one embodiment
where an area without projections is surrounding a substrate
zone. In one embodiment the surrounding area without
projections is from 15 to 100 pm wide. In another embodiment
the surrounding area without projections is from 20 to 40 pm
wide. In a further embodiment the surrounding area without
projections is from 25 to 35 pm wide. The width depends on
the viscosity of the substrate which is added to the
substrate zone and on the hydrophilicity of the surface
surrounding the substrate zone.
Advantages of a zone with a surrounding area with different
properties include that it is possible to apply a substance
to the zone without the substance flowing out of the zone.
Thus it is possible to apply a substance to a zone in a
reproducible and well defined manner.
In one embodiment the entire connecting zone is covered by
projections such that a lateral capillary flow is achieved.
In one embodiment at least a part of the sample addition
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=
zone, the connecting zone and the receiving zone are covered
by projections such that a lateral capillary flow is
achieved. In another embodiment the entire sample addition
zone, connecting zone and receiving zone are covered by
projections such that a lateral capillary flow is achieved.
In one embodiment the analysis device and the substrate is
such that it is possible to add a sample to the sample
addition zone. Due to capillary flow induced by the
projections a lateral flow is created and at least a part of
the sample reaches at least one substrate zone on the
substrate where there are projections with a height (H2),
diameter (D2) and center-to-center distance (x2, y2). In one
embodiment the substance applied in the at least one
substrate zone and/or near the at least one substrate zone is
gradually dissolved by the sample flowing by the at least one
substrate zone. The liquid sample thereafter reaches the
reaction zone. In one embodiment a measurement is made in the
reaction zone. Examples of measurement techniques include but
are not limited to detection of fluorescence, and
chemiluminescence. A person skilled in the art realizes that
also other detection principles can be used such as
absorption of light optionally at several different
wavelengths, and detection of emitted light. In one
embodiment the sample continues to a receiving zone, which
receives the liquid sample. The flow continues until there is
no more liquid sample or until the receiving zone is full of
sample liquid.
In one embodiment there is a surplus of sample liquid. In one
embodiment a part of the surplus of sample liquid is used so
that when all of the substance has been dissolved the
reaction zone and other parts of the device is washed so that
free or loosely bound substance is washed away.
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In one embodiment there are several receiving zones.
The at least one substance, which is applied to the at least
one substrate zone, is in one embodiment applied to the
substrate surface. In one embodiment the substance is applied
between the projections. In one embodiment the thickness of
the applied substance in a dried state is essentially
corresponding to the height of the projections. In an
alternative embodiment the thickness of the applied substance
in a dried state is lower than the height of the projections.
In one embodiment the thickness of the applied substance in a
dried state is higher than the projections.
In one embodiment the at least one substance is applied in a
volume between the projections in said at least one substrate
zone, so that the substance in a dried state fills a volume
up to a level at half of the height (H2) of the projections.
In an alternative embodiment the at least one substance is
applied in a volume between the projections in said at least
one substrate zone, so that the substance in a dried state
fills a volume up to essentially the same height (H2) as the
projections.
In a further embodiment the at least one substance is applied
in a volume between said projections in said at least one
substrate zone, so that the substance in a dried state fills
a volume up to a level so that there is essentially no liquid
flow on top of said at least one substance.
In one embodiment the volume between the projections of the
substrate zone is adjusted so that the amount of substance
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which is to be applied fills up to the desired level in a
dried state.
In one embodiment the substance is applied in a solution and
is dried so that a solvent is evaporated. In one embodiment
the solvent is water. When the solvent has evaporated the
remaining substance is called the substance in a dried state.
In embodiments where the there is no flow or essentially no
flow of liquid on top of the applied substance, the
dissolution of the applied substance occurs from the sides
and not from the top. This gives a controlled dissolution.
The dissolution occurs during a prolonged time and in a more
controlled manner compared to an embodiment where the liquid
sample also flows over the applied substance.
In one embodiment no liquid flows on top of the applied
substance. In an alternative embodiment only a minor part of
the liquid flows on top of the applied substance, examples of
such a part of the liquid include but are not limited to
0.1wt%, lwt%, 5wt96, and lOwt%. In one embodiment less than 1
wt% of the liquid flows on top of the applied substance. In
another embodiment less than 10 wt % of the liquid flows on
top of the applied substance.
By adjusting the diameter and center-to-center distance of
the projections for instance in an embodiment where
essentially no liquid flows over the applied substance, it is
possible to control the dissolution of the applied substance
in the liquid.
In one embodiment all components of the applied substance are
dissolved by the liquid sample. In an alternative embodiment
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not all applied substances are dissolved by the liquid
sample.
By adjusting the height, diameter and center-to-center
distance of the projections in the zone where a substance has
been applied and outside the zone where a substance has been
applied it is possible to control the lateral capillary flow
so that a desired dissolution of the applied substance occurs
at a desired rate.
In one embodiment the heights (H1, H2), diameters (D1, D2)
and center-to-center distances (xl, x2, yl, y2) are adapted
so that said at least one substance is gradually dissolved in
a flow of a liquid flowing by said substrate zone.
The invention is not limited to two different heights,
diameters and center-to-center distances of the projections.
There are provided embodiments with further zones, where each
zone has a height, diameter, and center-to-center distance,
where at least one of the parameters is different compared to
the other zones.
There is provided an embodiment where the analysis device
comprises at least one additional zone n, where each
additional zone n comprises projections substantially
vertical to the surface of said substrate, with a height
(Ho), diameter (Do) and center-to-center distance (xo, yn),
such that lateral capillary flow is created. There is
provided a substrate having n distinct zones, each zone
comprising projections having a height (Ha), diameter (Do)
and center-to-center distance (xn, yn), where n is a natural
number. n = 1, 2, 3, 4, 5, 6_
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In one embodiment there is at least one substance applied in
at least one of the n zones.
In one embodiment there is a substrate zone comprising
projections substantially vertical to the surface of said
substrate, and having a height, diameter and center-to-center
distance, such, that lateral capillary flow of said liquid
sample is achieved, where the height, diameter and center-to-
center distance in the substrate zone is different from
outside of the substrate zone. In one embodiment the height,
diameter and center-to-center distance of the projections
vary within the substrate zone.
In one embodiment there are at least two zones comprising an
applied substance, which zones have the shape of concentric
circles. In one embodiment the outer part comprises at least
one applied substance and the inner part comprises at least
one another applied substance. First the outer substance is
dissolved and then the inner substance is dissolved. Thereby
further possibilities to control the dissolution are
provided. There is also provided possibility to dissolve
several substance in sequence or together. In one embodiment
there is provided the possibility to dissolve more than one
substance in sequence. In other embodiments also other shapes
of the different zones with applied substances are provided.
In one embodiment there is at least one zone which first
comes into contact with the sample liquid and when a fraction
of or all of the substance in that zone has dissolved the
sample liquid comes into contact with another zone comprising
a different substance. Thereby it is possible to start the
dissolution of a substance in the sample liquid after a
certain period of time.
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Thus there is provided a method wherein the at least one zone
first comes into contact with liquid sample and when at least
a part of applied substance in said zone has dissolved, the
liquid sample comes into contact with another zone comprising
another applied substance.
In one embodiment there are two different zones, where each
zone comprises a distinct substance.
In one embodiment a part of the liquid sample passes the
zone(s) where a substance has been applied at such a distance
that essentially no substance is dissolved, and another part
passes near the zone where a substance has been applied so
that the substance dissolves in the liquid. Thus there is a
provided an embodiment where a part of the liquid stream
flowing through the device comprises a major part of
dissolved substance and another part comprises little or no
dissolved substance. Examples of a major part of the
dissolved substance include but are not limited to 75wt96,
90wt96, 95wt%, 99wt%, and 99.9wt%.
It is possible to control the height, diameter and center-to-
center distance of the projections inside and outside of the
zone where the substance to be dissolved is applied. In that
way it is possible to control the fraction of the liquid
sample that flows by the zone where the substance is
dissolved. By controlling the rate at which the substance
dissolves when the liquid sample flows by, it is possible to
control the concentration of the dissolved substance.
If a larger fraction of liquid sample passes outside the zone
where the substance is dissolved at a high flow, the result
is that downstream of the zone where the sample is applied;
there is a rather narrow concentrated flow of dissolved
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substance. On the other hand, if a smaller fraction of liquid
sample passes the zone where the substance is dissolved the
result is that downstream of the zone where the sample is
applied; there is a broader trace of dissolve substance. The
trace of dissolved substance is shown in Fig. 5 as a grey
area.
In one embodiment the result is detected in the middle of the
trace of dissolved substance downstream of the zone where the
substance is applied. In one embodiment the result of the
analysis is read in the reaction zone, downstream of the zone
where the substance is applied.
The shape of the zone where the substance is applied is in
one embodiment adapted to control the dissolution rate and/or
how the dissolved substance is distributed in the flow of
liquid sample.
Examples of shapes include but are not limited to a triangle,
a square, a rectangle, a parallelogram, a rhombus, a
trapezoid, a quadrilateral, a polygon, a circle, and an oval.
Also truncated shapes are encompassed, including but not
limited to a half circle, a half oval, a half polygon, and a
circle segment.
Further shapes include all possible combinations of shapes
including but not limited to a triangle and a square, a
triangle and a rectangle, a trapezoid and a rectangle, a half
circle and a rectangle and so on.
In one embodiment the shape of the zone where the substance
is applied is triangular with one of the corners pointing in
the direction from where the flow of sample liquid comes.
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In one embodiment the shape of the zone where the substance
is applied is an isosceles trapezoid.
In one embodiment the shape of the zone where the substance
is applied is an isosceles trapezoid combined with a
rectangle.
In one embodiment the shape of the zone where the substance
is applied is an isosceles trapezoid combined with a
rectangle, with the narrower zone pointing towards the
direction from where the flow of sample liquid comes.
In one embodiment the shape of the zone where is substance is
applied and the height, diameter and center-to-center
distance of the projections are adapted so that the
concentration of the dissolved substance is greater towards
the middle of the substrate.
In one embodiment the analysis device further comprises a
lid. Preferably the lid has at least one opening or an
aperture for the addition of a liquid sample. In one
embodiment there is an opening or a window allowing a
measurement result to be read from the analysis device. If a
lid is used the lid is not in capillary contact with the
projections on the substrate. The lid does not take part in
creating any capillary forces.
In one embodiment the analysis device comprises a casing. In
one embodiment the casing encloses the entire or a part of
the analysis substrate. Preferably the case has at least one
opening or an aperture for the addition of a liquid sample.
In one embodiment there is an opening or a window allowing a
measurement result to be read from the analysis device.
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In one embodiment the applied substance comprises a detection
conjugate. In one embodiment the detection conjugate
comprises at least one element selected from an antibody,
DNA, RNA, an aptamer, a fragmented antibody, an antibody
fragment, a synthetic binder, a chemical binder, a receptor,
a ligand, an affibody, a cell, an organelle, a polypeptide, a
peptide, an enzyme, a monoclonal antibody, a polyclonal
antibody, a phage display protein, an IgG immunoglobulin, a
chemical ligand. In one embodiment the detection conjugate
comprises more than one antibody. In one embodiment at least
one of the molecules in the applied substance comprises a
detectable group, which allows detection for instance in the
reaction zone. The detection conjugate facilitates detection
of an antigen bound to the conjugate. In one embodiment the
detection conjugate comprises a fluorescent molecule. In one
embodiment fluorescence from the detection conjugate is
measured.
In one embodiment at least one selected from an antibody,
DNA, RNA, an aptamer, a fragmented antibody, an antibody
fragment, a synthetic binder, a chemical binder, a receptor,
a ligand, an affibody, a cell, an organelle, a polypeptide, a
peptide, an enzyme, a monoclonal antibody, a polyclonal
antibody, a phage display protein, an IgG immunoglobulin, and
a chemical ligand, is bound to the substrate downstream of
the applied substance. Such an antibody or aptamer is able to
bind to the complex between an antibody and an antigen or to
a free antigen. In one embodiment an antibody or an aptamer
bound to the reaction zone is able to bind to the complex
between an antibody and an antigen or to a free antigen. In
one embodiment fluorescence from the detection conjugate in
the reaction zone is measured.
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In a second aspect there is provided a method for the
analysis of a sample comprising the steps of: a) addition of
a liquid sample on at least one spot on a substrate, and b)
performing at least one measurement on the substrate, wherein
an analysis device as described herein is used.
In one embodiment the substrate is first wetted by sample
liquid comprising essentially no dissolved substance and then
is brought into contact with liquid sample comprising
dissolved substance. The first wetting by the liquid sample
is achieved by a delay of the dissolution of the substance
applied to the substrate. The sample liquid that flows first
comprises essentially no dissolved substance, such as but not
limited to less than 0.001wt96, 0.01wt%, 0.1wt% or lwt96. When
the liquid sample comes into contact with the substance, the
dissolution process starts and the level of dissolved
substance gradually increases.
In one embodiment there are molecules bound to the substrate
which are first hydrated by the liquid sample, where the
liquid sample is essentially without any dissolved substance.
In one embodiment such molecules are antibodies. One
advantage of such a pre-hydration is that the antibodies or
molecules become more active. This first hydration is called
a pre wetting. In one embodiment a substantial part of the
substance(s) applied to the substrate is dissolved and
transported across the reaction zone after a steady lateral
flow has been established by the capillary force from the
receiving zone. A substantial part of the substance(s)
applied to the substrate is in this case more than 75wt%,
preferably more than 90 wt96, more preferably more than 95wt%
and most preferably more than 99wt%.
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In one embodiment the sample is added to a sample addition
zone, the sample flows through a connecting zone to a
receiving zone. The receiving zone has a capacity to receive
the sample liquid and has a large surplus of capillary force.
The capillary force of the receiving zone is in one
embodiment such that a steady and even lateral flow of sample
liquid is created. The capillary force of the receiving zone
acts like a pump and receives the sample liquid at a steady
rate. Before the added sample liquid has reached the
receiving zone the lateral capillary flow is not always even
and steady.
It is one advantage that a substantial part of the applied
substance is not dissolved until there is a steady lateral
flow, i.e. when the liquid sample has reached the receiving
zone. In that way the dissolution of the substance occurs at
a more controlled way due to the steady and even flow of
sample liquid.
In one embodiment the substrate comprises a receiving zone
with the capacity to receive liquid sample, and wherein more
than 95wt% of the applied substance is not dissolved until
any part of the liquid sample has reached the receiving zone.
In an alternative embodiment more than 90wt%, preferably
94wt%, more preferably 99wt% and most preferably 99.9wt% of
the applied substance is not dissolved until any part of the
liquid sample has reached the receiving zone. This implies
that at most lOwt%, preferably 6wt%, more preferably 1wt% and
most preferably 0.1wt% of the applied substance is dissolved
until any part of the liquid sample reaches the receiving
zone.
Further advantages include that there is provided a
possibility of increased control of the flow. There is also
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provided the possibility of dissolution during a steady
lateral flow. It is possible to control the start and stop of
dissolution of a substance. There is a possibility to provide
a wash of the reaction zone after the dissolved substance(s)
has passed. It is possible to obtain an even dissolution of
the reagent over time. There is the possibility to ensure
that the dissolved substance is spatially homogenously
distributed. There is also provided the possibility to
control the spatial distribution of the dissolved substance.
It is possible to ensure that all substance is dissolved in
the sample. A major portion of the sample is contacted with
the substance.
Examples
Example 1
A substrate with dimensions 25x75mm was made by injection
molding of a cyclo olefin polymer (COP). The substrate hade a
sample addition zone, a substrate zone, a connecting zone, a
reaction zone, and a receiving zone. On the substrate there
were projections with height 70 pm and diameter 50pm. The
distance between the projections were 15 pm in the sample
addition zone, the substrate zone, the connecting zone, and
the reaction zone. A distance between the projections of 15
pm and a diameter of 50 pm corresponds to a center-to-center
distance of 65 pm between the projections in both x- and y-
direction. The substance zone was surrounded by a gap where
the distance between the projections was 30 pm.
It was possible to apply detection conjugate to the substance
zone so that the detection conjugate did not move out of the
substrate zone. The application of the detection conjugate
was reproducible during several applications. The detection
conjugate was applied so that the height of the dried
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==
detection conjugate was at about the same level as the top of
the projections in the substance zone.
A detection conjugate comprising antibodies against N-
terminal pro-brain natriuretic peptide (NT-proBNP) was used.
Separated whole blood was added to the sample addition zone
and the detection conjugate dissolved gradually in the liquid
sample. The trace of dissolved detection conjugate was
continuous and the dissolution time could be controlled. The
experiment was repeated six times and the dissolution time of
the detection conjugate was 6 minutes and 8 seconds, whereby
the coefficient of variation was 3%.
The total assay time was 19 minutes and 19 seconds with a
coefficient of variation of 5% (six repeated experiments).
The result of the analysis was read by using an optical
reader.
Although the invention has been described with regard to its
preferred embodiments, which constitute the best mode
presently known to the inventors, it should be understood
that various changes and modifications as would be obvious to
one having the ordinary skill in this art may be made without
departing from the scope of the invention which is set forth
in the claims appended hereto.
