U.S. patent application number 13/054354 was filed with the patent office on 2011-12-15 for assay device and methods.
This patent application is currently assigned to L3 TECHNOLOGY LIMITED. Invention is credited to David Clarke, Laura Garcia, Mark Hudson, Gareth Jones, Anthony Nicholls.
Application Number | 20110306120 13/054354 |
Document ID | / |
Family ID | 41057998 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306120 |
Kind Code |
A1 |
Nicholls; Anthony ; et
al. |
December 15, 2011 |
ASSAY DEVICE AND METHODS
Abstract
The present invention relates to the use of amphipathic polymers
to enhance lateral flow and reagent mixing on assay devices. More
specifically, the invention relates to use of an amphipathic
polymer in assay methods including a device for determining the
concentration of lipids in blood serum or plasma.
Inventors: |
Nicholls; Anthony; (Ledbury,
GB) ; Garcia; Laura; (Ledbury, GB) ; Hudson;
Mark; (Ledbury, GB) ; Jones; Gareth; (Ledbury,
GB) ; Clarke; David; (Ledbury, GB) |
Assignee: |
L3 TECHNOLOGY LIMITED
Ledbury
GB
|
Family ID: |
41057998 |
Appl. No.: |
13/054354 |
Filed: |
July 15, 2009 |
PCT Filed: |
July 15, 2009 |
PCT NO: |
PCT/GB09/50861 |
371 Date: |
August 31, 2011 |
Current U.S.
Class: |
435/287.2 ;
422/69; 422/82.08; 435/288.7; 568/679 |
Current CPC
Class: |
B01L 2300/161 20130101;
B01L 2400/0406 20130101; G01N 33/92 20130101; G01N 21/64 20130101;
B01F 13/0084 20130101; Y02P 20/582 20151101; B01F 13/0071 20130101;
B01L 3/502769 20130101; B01L 2400/088 20130101 |
Class at
Publication: |
435/287.2 ;
435/288.7; 422/82.08; 422/69; 568/679 |
International
Class: |
C12M 1/34 20060101
C12M001/34; G01N 33/53 20060101 G01N033/53; C07C 43/10 20060101
C07C043/10; G01N 21/64 20060101 G01N021/64 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 15, 2008 |
GB |
0812907.4 |
May 28, 2009 |
GB |
0909130.7 |
Claims
1-47. (canceled)
48. An assay device for detecting the presence or quantity of an
analyte residing in an aqueous sample, the device comprising: (i)
at least one application area suitable for application of an
aqueous sample to the device; (ii) at least one probe, reporter or
reagent wherein, in use the at least one probe, reporter or reagent
is able to react with an analyte residing in the aqueous sample;
(iii) at least one test area wherein, in use the result and/or
progress of a reaction between the analyte and the at least one
probe, reporter or reagent may be determined; (iv) at least one
flow path being in fluid communication with the at least one
application area and the at least one test area; wherein the at
least one flow path comprises at least one amphipathic polymer that
is soluble in water and wherein, in use the passage of fluid along
the at least one flow path is greater than that expected by
capillary action alone.
49. The device of claim 48 wherein the flow path is coated with the
at least one amphipathic polymer.
50. The device of claim 49 wherein the amphipathic polymer is
printed and/or sprayed onto a surface of the device.
51. The device of claim 50 wherein the amphipathic polymer forms
one or more arrays of nano-, pico- or femto-litre droplets.
52. The device of claim 48 wherein the at least one flow path
comprises the at least one probe, reporter or reagent.
53. The device of claim 52 wherein the at least one probe, reporter
or reagent are combined with the amphipathic polymer.
54. The device of claim 52 wherein the at least one probe, reporter
or reagent are arranged above, below or beside the amphipathic
polymer.
55. The device of claim 48 wherein the result and/or progress of a
reaction is determined by means of optical measurement.
56. The device of claim 48 wherein the result and/or progress of a
reaction is determined by visual inspection.
57. The device of claim 55 wherein the result and/or progress of a
reaction is determined by means of fluorescence.
58. The device of claim 57 wherein the device comprises at least
two separable components.
59. The device of claim 58 wherein the at least two separable
components are a reader and a test cartridge.
60. The device of claim 59 wherein the reader comprises excitation
means and detection means.
61. The device of claim 60 wherein the excitation means are
operable to excite the sample so that it fluoresces, and the
detection means are operable to detect the fluorescence emitted by
the sample.
62. The device of claim 59 wherein the test cartridge comprises the
at least one flow path.
63. The device of claim 62 wherein the test cartridge is
disposable.
64. The device of claim 59 wherein the reader is re-usable.
65. The device of claim 59 wherein the reaction is an
immunoassay.
66. The device of claim 64 wherein the reaction is an ELISA.
67. The device of claim 57 wherein the reaction is a fluorometric
assay.
68. The device of claim 67 wherein the fluorometric assay is a
cholesterol assay.
69. The device of claim 67 wherein the fluorometric assay is a
lipoprotein assay.
70. The device of claim 67 wherein the fluorometric assay is a
triglyceride assay.
71. The device of claim 48 which comprises at least three test
areas and at least three flow paths wherein, a first flow path is
in fluid communication with the application area and a first test
area, a second flow path is in fluid communication with the
application area and a second test area and a third flow path is in
fluid communication with the application area and a third test
area.
72. The device of claim 48 wherein the at least one probe, reporter
or reagent is selected from the group consisting of Amplex Red,
K37, Nile Red, cholesterol esterase, cholesterol oxidase and horse
radish peroxidase.
73. The device of claim 72 wherein a first flow path comprises
Amplex Red, a second flow path comprises K37 and a third flow path
comprises Nile Red.
74. The device of claim 72 wherein a first test area comprises
Amplex Red, a second test area comprises K37 and a third test area
comprises Nile Red.
75. The device of claim 73 wherein the first flow path further
comprises cholesterol esterase, cholesterol oxidase and horse
radish peroxidise.
76. The device of claim 73 wherein the first test area further
comprises cholesterol esterase, cholesterol oxidase and horse
radish peroxidise.
77. The device of claim 71 wherein the application area comprises
the at least one probe, reporter or reagent selected from the group
consisting of Amplex Red, K37, Nile Red, cholesterol esterase,
cholesterol oxidase and horse radish peroxidise.
78. The device of claim 48 wherein the at least one probe, reporter
or reagent is in dry form.
79. The device of claim 78 wherein the at least one probe, reporter
or reagent further comprises a stabilizing agent.
80. The device of claim 79 wherein the stabilizing agent is a
copolymer of vinyl pyrrolidine and dimethylaminoethyl
methacrylate.
81. The device of claim 48 wherein the amphipathic polymer is
polyethylene glycol (PEG).
82. The device of claim 81 wherein the amphipathic polymer is
polyethylene glycol with a molecular weight of from 1000 Da to
20,000 Da.
83. The device of claim 82 wherein the amphipathic polymer is
polyethylene glycol with a molecular weight of from 1000 Da to 6000
Da.
84. A process for enhancing lateral fluid flow in a membrane-free
device for point of care use comprising coating a surface along
which a fluid may flow with an amphipathic polymer that is soluble
in water.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of amphipathic
polymers to enhance lateral flow and reagent mixing on assay
devices. More specifically, the invention relates to use of an
amphipathic polymer in assay methods including a device for
determining the concentration of lipids in blood serum or
plasma.
BACKGROUND TO THE INVENTION
[0002] Lateral flow assay devices and methods are known in the art.
Previously, such devices have been developed to test samples that
are easily available in large quantities. However, when the test
sample is blood or a component of blood, the collection of a large
sample is not always possible, particularly at a point of care such
as a doctor's surgery.
[0003] Generally these devices comprise a lateral flow matrix, for
example, nitrocellulose membranes and the like. A sample applied to
the matrix flows along the matrix, and one or more analytes within
the sample react with one or more reagents within the lateral flow
matrix. Typically, at least one of these reagents is immobilized
within the matrix allowing any reaction with the analytes to be
detected, for example, visually. Unfortunately, variations
associated with sample transfer and diffusion of the sample to the
membrane result in a flow that is largely uncontrolled and uneven
before reaching a test area. This is because such devices rely on
capillary action of the fluids alone. Such a reliance on capillary
action may have an adverse affect on the accuracy of the device
because the amount of analyte and/or label captured across a test
area is not consistent. The use of capillary action alone also
means that assays are slow because of unreliable fluid wicking. The
assays are also unsuitable for small fluid samples, such as in
nucleic acid detection, where the membrane may dry before the assay
is completed or there may be insufficient fluid to travel the
length of the test device.
[0004] As such, a need currently exists for a simple and efficient
technique for improving lateral flow assays, particularly to allow
faster testing whilst enabling low volume tests to be
performed.
[0005] One area where lateral flow point of care devices would be
useful is in the field of cholesterol and blood lipid testing.
[0006] It is well known that the concentration of various
lipoproteins in the blood is correlated with the risk of an
individual developing atherosclerosis. Atherosclerosis is a disease
that affects arterial blood vessels and is commonly referred to as
a "hardening" or "furring" of the arteries. It is caused by the
formation of multiple plaques on the blood vessel walls, in large
part due to the accumulation of macrophage white blood cells and
promoted by low density lipoproteins. Without adequate removal of
fats and cholesterol from the macrophages by high density
lipoproteins (HDL), a chronic inflammatory response develops in the
walls of the arteries.
[0007] Most of the circulating cholesterol in blood plasma is found
in three major classes of lipoproteins. Cholesterol and cholesterol
esters are water insoluble substances and are therefore carried by
these lipoproteins within the circulatory system for eventual
utilisation by the cells of the body.
[0008] Each of these lipoprotein classes carries varying amounts of
cholesterol. Total serum cholesterol is therefore a complex average
of the amount that each lipoprotein class contributes to the total
lipoprotein concentration of the serum.
[0009] Each class of lipoprotein plays a different role in
atherosclerosis. High density lipoproteins or HDL are generally
regarded as being `good cholesterol`, that is they are
anti-atherogenic. In contrast, Low density lipoproteins or LDL are
often referred to as `bad cholesterol` since they are known to be
highly atherogenic. Another class of lipoproteins, very low density
lipoproteins or VLDL are considered to be slightly atherogenic.
[0010] Levels of HDL in the blood have been extensively
investigated in view of the inverse relationship between HDL
cholesterol and the risk of atherosclerosis and, for example, heart
attack. Thus, if the levels of HDL cholesterol are determined to be
low, an individual may have an increased risk of developing
atherosclerosis. Therefore, this risk can be estimated by assaying
HDL cholesterol. From these assay results an approximate amount of
LDL cholesterol may be calculated using the following equation:
LDL Cholesterol=Total Cholesterol-1/5 Total Cholesterol-HDL
Cholesterol
[0011] To determine the cholesterol content of the various
cholesterol fractions, generally four methods have been used. These
include (1) ultracentrifugation, (2) fractional precipitation, (3)
calculation using the Friedewald equation and (4) electrophoretic
separation and precipitation.
[0012] Each of these methods suffers from a number of drawbacks.
For example, ultracentrifugation requires the use of specialised
laboratory equipment and may take several days to complete.
Fractional precipitation and electrophoretic separation are both
time-consuming and again require the use of specialised equipment.
The Friedewald equation is inaccurate because it estimates the
concentration of LDL cholesterol by subtracting the cholesterol
associated with other classes of lipoproteins. Thus, the equation
provides an indirect estimation based on three independent lipid
analyses each of which provides a potential source of error.
[0013] As a result of these drawbacks, the results of cholesterol
assays are not available for several hours or even days and cannot
be performed in smaller laboratories or by doctors in their
surgeries. Accordingly there is a need for a device and method of
performing a cholesterol assay that is both simple and inexpensive
to use. There is also a need for an assay that directly measures
the concentration of each class of lipoprotein without relying on
indirect estimates.
[0014] When solid hydrophobic molecules are added to an aqueous
solution or suspension they form an immediate precipitate and do
not enter the aqueous phase because the hydrophobic molecules
`stick` together rather than dissolve. Even with vigorous stirring
of the precipitate the number of direct encounters between the
hydrophobic molecules and molecules in solution is small. Such
interactions may also be thermodynamically unfavourable.
[0015] Methods of the prior art involve dissolving hydrophobic
molecules in suitable water-miscible organic solvents prior to
mixing with an aqueous solution or suspension. On entering the
aqueous solution, the hydrophobic molecules are mono-dispersed and
therefore have an increased probability of interacting with the
molecules already in solution. However, such methods suffer from
the drawback that the organic solvent is often toxic or can
interfere with enzymatic reactions or fluorescence measurement.
Such methods are generally also not suitable for point of care use
such as in a Doctor's surgery or clinic.
SUMMARY OF THE INVENTION
[0016] The Applicants have made the surprising discovery that by
use of amphipathic polymers in an assay system, considerable gains
in both the efficiency and/or speed of the assay can be achieved.
The Applicant's methods are also applicable to rapidly combining
hydrophobic reagents and compounds such as luminophores with
analytes, for example lipoproteins, in aqueous
solution(s)/suspension(s) without direct application of organic
solvents. This method enables the lipoprotein contents of blood,
foodstuffs, and the like to be easily quantified by measurement of
fluorescence. The use of such methods has also enabled the
development of a rapid and inexpensive point of care cholesterol
assay system.
[0017] In a first aspect of the invention, there is provided an
assay device for detecting the presence or quantity of an analyte
residing in an aqueous sample, the device comprising: at least one
flow path along which an aqueous sample can travel, characterised
in that the at least one flow path comprises at least one
amphipathic polymer wherein, in use the passage of fluid along the
flow path is greater than that expected by capillary action
alone.
[0018] An amphipathic polymer is a polymer possessing both
hydrophilic and hydrophobic properties. Such a compound may also be
called an amphiphilic compound or a non-ionic hydrophilic polymer.
In particular embodiments an amphipathic polymer is a substance
that is soluble both in water and a wide range of organic
solvents.
[0019] The present invention relates to the use of amphipathic
polymers such as polyethylene glycol for promoting and/or
controlling lateral fluid flow. The invention may also be utilised
to increase the interaction between an aqueous sample and at least
one reagent, preferably between hydrophobic luminophores and
lipoproteins.
[0020] Preferably the flow path is coated with the at least one
amphipathic polymer.
[0021] Surprisingly it has been found that the amphipathic polymers
may be used to coat the surface of, for example, plastics or glass
to enhance fluid flow to move and/or mix aqueous solutions, for
example with `dry` components combined within or as layers above or
below the coating of amphipathic polymers. The use of amphipathic
polymers also has the advantage that lateral flow of fluids is
improved, for example, over the traditional `wicking` with porous
materials such as is disclosed in U.S. Pat. No. 6,485,982. Wicking
methods of the prior art rely on the use of a support vehicle such
as paper or a membrane through which liquid is drawn by capillary
action. The use of amphipathic polymers removes the need for a
support vehicle which relies solely on capillary action and has the
surprising effect that liquids may travel greater distances or at
greater speeds, for example, along microtubes or surfaces than in
porous materials by capillary action alone.
[0022] As used herein, the term "lateral flow" refers to the
movement of a fluid on a surface wherein the fluid flows, for
example laterally, in a particular direction or along a particular
path. Preferably the fluid flows at a greater speed or for a
greater distance than that observed by capillary action across a
particular material alone. It should be noted that the term
"lateral flow" is meant to be descriptive and not limiting, as the
device could be configured in other ways with the same effect, for
example, radial or vertical flow can easily be envisaged employing
the same principles as the present invention, without departure
from the spirit of the invention. In particular embodiments the
fluid coincidentally interacts with, and analytes contained therein
may react with, various reagents as it flows or travels.
[0023] The device comprises at least one flow path along which an
aqueous sample can travel, and is characterised in that the at
least one flow path comprises at least one amphipathic polymer
wherein, in use the passage of fluid along the flow path is greater
than that expected by capillary action alone. In contrast to the
prior art, the device does not require a porous membrane for
lateral fluid flow, instead the fluid may travel along the flow
path comprising the at least one amphipathic polymer.
[0024] Surprisingly it has been discovered that fluids travel
faster and/or over greater distances along a flow path comprising
an amphipathic polymer than along flow paths comprising porous
materials, such as membranes.
[0025] In particular embodiments the flow path contains the at
least one amphipathic polymer.
[0026] Thus, an amphipathic polymer may be used to coat a surface
such as the inside of a tube, capillary, channel, well, membrane or
the like. It will be apparent that the amphipathic polymer may also
be used as a coating for membranes used in `standard` lateral flow
assays to speed up and/or provide greater control over the fluid
flow.
[0027] In other embodiments the flow path is formed by the at least
one amphipathic polymer.
[0028] The hydrophobic or amphipathic polymer may be printed and/or
sprayed onto a surface, such as a flat surface, for example forming
`tracks` and/or layers, by printing methods such as by inkjet or
bubble-jet printing, painting, spraying or other application
methods.
[0029] The amphipathic polymer may be in the form of a film.
[0030] In other embodiments, the film may be ground into
particulate material or may be formed into, for example, granules,
beads, pellets, microspheres or nanospheres or picospheres. The
amphipathic polymer itself may be in the form of granules, beads,
pellets, microspheres, nanospheres or picospheres.
[0031] In yet other embodiments the amphipathic polymer may be
printed as part of or forming one or more arrays of nano-, pico-,
or femto-litre droplets.
[0032] Preferably, the amphipathic polymer comprises at least one
probe, reporter or reagent. Preferably the reporter is a
hydrophobic compound or reagent such as a luminophore.
[0033] To combine a hydrophobic compound or reagent with the
polymer, said hydrophobic compound or reagent may first be
dissolved in a solvent, such as an organic solvent, that is
miscible with the amphipathic polymer. The amphipathic polymer is
also dissolved in a solvent such as water or a more volatile
solvent such as dimethyl formamide or chloroform although other
suitable solvents will be readily apparent to one skilled in the
art. The hydrophobic compound or reagent and amphipathic polymer
are then combined and then preferably dried into a thin film.
Surprisingly, the inventors have discovered that on drying, there
is no phase separation of the hydrophobic compound or reagent and
polymer.
[0034] The flow path may comprise at least one probe, reporter or
reagent. The flow path may contain the probe, reporter or
reagent.
[0035] Thus, the combination of at least one hydrophobic
luminophore and amphipathic polymer may be used to coat a surface
such as the inside of a tube, capillary, channel, well, membrane or
the like. In yet other embodiments, the combination may be printed
onto a surface, for example forming `tracks`, by printing methods
such as by inkjet or bubble-jet printing, painting, spraying or
other application methods. The probe, reporter or reagent may be
adjacent the at least one flow path. Alternatively, the probe,
reporter or reagent may be layered directly above or below the at
least one flow path. Thus, the reagents may be within the flow path
itself, for example combined with the amphipathic polymer or may be
arranged as discrete layers or printed as `dots` above, below or
beside the amphipathic polymer.
[0036] It will be apparent that the methods of the invention are
also applicable to reagents other than hydrophobic luminophores,
for example, such as enzymes, blocking reagents, chemicals and the
like.
[0037] Particularly the amphipathic polymer is polyethylene glycol
(PEG) with a molecular weight of from about 1000 to 20,000 Da, more
particularly between about 1000 to 6000 Da and yet more
particularly from about 1000 to 3000 Da. Particular PEGs include
PEG2000, PEG6000, PEG12000 and PEG20000.
[0038] Polyethylene glycol, also known as polyethylene oxide (PEO)
or polyoxyethylene (POE), is an oligomer or polymer of ethylene
oxide. PEGs are available over a wide range of molecular weights
from 300 g/mol to 10,000,000 g/mol. PEG has the following general
structure:
HO--(CH.sub.2--CH.sub.2--O--).sub.n--H
[0039] Numbers are frequently included in the names of PEGs to
indicate their average molecular weights. For example, a PEG with
n=80 would have an average molecular weight of approximately 3500
daltons and would be labeled PEG 3500.
[0040] Generally PEGs include molecules with a distribution of
molecular weights. Whilst PEGs having different molecular weights
find use in a variety of applications due to their differing
physical properties, such as viscosity, their chemical properties
are nearly identical. Different forms of PEG are also available
dependent on the initiator used for the polymerization process,
such as monofunctional methyl ether PEG (methoxypoly(ethylene
glycol)), abbreviated mPEG. PEGs are also available with different
geometries. Branched PEGs have 3 to 10 PEG chains emanating from a
central core group. Star PEGs have 10-100 PEG chains emanating from
a central core group. Comb PEGs have multiple PEG chains normally
grafted to a polymer backbone. PEGs may also be covalently coupled
to other molecules in a process known as PEGylation which may be
advantageous when using the fluid flow properties of PEG for
reagent mixing for example.
[0041] Further amphipathic polymers may include amphipathic
polypeptides, that is, a polypeptide that has a secondary structure
such that the polypeptide has both a hydrophilic and a hydrophobic
face. The design of amphipathic peptide structures (e.g.
alpha-helical polypeptides) is known in the art. For example, in:
Grell et al. (2001) J Pept Sci 7(3):146-51; Chen et al. (2002) J
Pept Res 59(1):18-33; Iwata et al. (1994) J boil Chem
269(7):4928-33; Cornut et al. (1994) FEBS Lett 340(1):29-33;
Negrete et al. (1998) Protein Sci 7(6):1368-79. Other amphipathic
or non-ionic polymers include polyvinyl alcohol (PVA) (Sigma
Aldrich: 360627-25G), Carboxymethyl cellulose (Sigma: C-5678);
O,O'-Bis(2-aminoethyl) PEG 2000 (polyoxyethylene bis(amine))
(Aldrich Chemistry: 14501) and PEG methyl ether 5000 (Aldrich
Chemistry: 81323-250G) and other ionic polymers.
[0042] Preferably the device further comprises at least one
application area for application of an aqueous sample.
[0043] An application area is the region on the apparatus wherein
the sample is applied. Preferably there is a single application
area from which a plurality but at least one, two, three or more
aliquots of the sample may be taken for performing assays according
to the method of the invention. Alternatively there may be separate
application areas for each or different aliquots of sample. It will
be appreciated that, in some embodiments, the device may be
designed such that the sample may be directly introduced into the
test area(s) obviating the need for an application area.
[0044] The term "aqueous sample" as used herein refers to any
liquid sample, preferably one from which an analyte may be
detected. Non-limiting examples of such samples include whole
blood, serum or plasma samples, urine, cerebrospinal fluid (CSF),
lymph, serous exudate or other biological fluids, animal tissue
homogenates, deproteinised tissue homogenates, milk, raw egg,
fermentation broths, animal feeds, and marine feeds. It should be
appreciated that a sample need only be in liquid or aqueous form
for at least an essential part of any assay. Thus, one skilled in
the art may envisage that such an assay may also be carried out at
temperatures above the melting point of a sample, such as a wax or
lipid for example, that is normally solid at room temperatures.
[0045] Preferably the device further comprises at least one test
area wherein the result and/or progress of a reaction between a
part of the aqueous sample and the probe, reporter or reagent is
determined.
[0046] The test area(s) are region(s) in which the result and/or
progress of a reaction between an analyte and probe, reporter or
reagent may be determined. The test area(s) may be brought into
contact with excitation means.
[0047] Preferably at least one flow path is in fluid communication
with at least one application area and at least one test area.
Preferably, the apparatus comprises a plurality of flow paths and
test areas. Preferably the flow path connects, for example by fluid
communication, an application area with at least one test area.
[0048] The flow path may be a channel, groove, capillary, track or
path and the like. Preferably the flow path comprises an
amphipathic polymer. The amphipathic polymer may be in the form of
a coating or film on the surface of the flow path or may be in the
form of a powder, pellets, microparticles, nanoparticles ,
picoparticles or filling within a cavity of the flow path. Where
the amphipathic polymer is a filling within a cavity it may fill
the cavity entirely or may be a partial filling with, for example,
gaps. Alternatively, the amphipathic polymer may be the flow path,
for example, provided as a track or path, for example, printed on a
surface along which fluid lateral flow occurs.
[0049] The flow path which comprises the amphipathic polymer may
further comprise other reagents required for the assay(s), for
example, at least one hydrophobic luminophore and/or at least one
enzyme. Different flow paths may comprise different reagents. A
flow path may also comprise different reagents along its length,
for example, allowing for sequential addition of reagents through
lateral flow of the aqueous sample. Alternatively the flow path
comprises the amphipathic polymer alone leading to a test area(s)
that comprises the amphipathic polymer in combination with other
reagents. The reagents may be within the flow path itself, for
example combined with the amphipathic polymer or may be arranged as
layers above, below or beside the amphipathic polymer.
[0050] Preferably the result and/or progress of a reaction is
determined by means of optical measurement. Alternatively the
result and/or progress of a reaction is determined by visual
inspection. More particularly the result and/or progress of a
reaction is determined by means of fluorescence.
[0051] Thus it is preferred that the test area is arranged so that
it may be brought into optical contact with excitation means. The
test area should be arranged such that fluorescence produced from
the assay may be detected by the detection means. There may be
separate test areas for different aspects of the same assay or for
different assays. "Excitation means" are means operable to excite
the sample so that at least one component of the sample--usually a
probe or reporter--fluoresces.
[0052] Use of the term `contact` above is not intended to imply or
limit to physical contact, in this context the term may simply mean
that the test area(s) is physically moved into the path of a beam
of light or that the test area is illuminated/excited by a beam of
light.
[0053] Detection means are means operable to detect fluorescence
emitted by the sample. In particular embodiments, measurement may
be performed simply by visual checking. In this instance,
excitation means and detection means may not be necessary.
[0054] In particular embodiments the device comprises at least two
separable components. More preferably the at least two separable
components are a reader and a test cartridge. Thus, the apparatus
may comprise two or more separate components, for example, a reader
and a cartridge adapted to be placed in functional communication
with the reader. Preferably, the cartridge may be inserted into,
placed on or attached to the reader. The reader may comprise
docking means in which the cartridge is inserted, placed or
attached. The docking means may be a slot. Hence, preferably, the
cartridge is removable from the reader. The cartridge may take a
variety of forms, for example, a card, chip or slide, and be
constructed from materials known in the art, for example,
cardboard, glass, silicon, plastics and the like. Preferably the
material comprises, or is, a hydrophobic material or has regions
that are hydrophobic.
[0055] Preferably the test cartridge comprises the at least one
flow path. Preferably the test cartridge is a disposable and may be
replaced with a new cartridge containing new assay reagents.
Preferably the reader is a re-usable component.
[0056] In preferred embodiments the device is an assay device for
detecting the presence or quantity of an analyte residing in an
aqueous sample comprising: [0057] (i) at least one application area
suitable for application of an aqueous sample to the device; [0058]
(ii) at least one probe, reporter or reagent wherein, in use the at
least one probe, reporter or reagent is able to react with an
analyte residing in the aqueous sample; [0059] (iii) at least one
test area wherein, in use the result and/or progress of a reaction
between the analyte and the at least one probe, reporter or reagent
may be determined; [0060] (iv) at least one flow path being in
fluid communication with the at least one application area and the
at least one test area, [0061] characterised in that the at least
one flow path comprises at least one amphipathic polymer and
wherein, in use the passage of fluid along the at least one flow
path is greater than that expected by capillary action alone.
[0062] In particular embodiments the apparatus comprises a single
application area and a plurality of flow paths, for example four,
leading to a plurality, for example at least three, test areas.
[0063] When the device comprises at least three test areas and at
least three flow paths preferably, a first flow path is in fluid
communication with the application area and a first test area, a
second flow path is in fluid communication with the application
area and a second test area and a third flow path is in fluid
communication with the application area and a third test area.
[0064] In one embodiment the first flow path and/or test area
comprises the amphipathic polymer in combination with a first
hydrophobic luminophore and optionally at least one enzyme. A
second flow path and/or test area comprise the amphipathic polymer
in combination with a second hydrophobic luminophore and optionally
at least one enzyme. The third flow path and/or test area comprises
the amphipathic polymer in combination with either the first,
second or a third hydrophobic luminophore and optionally at least
one enzyme. In use the sample (in aqueous form) applied to the
application area is urged along the at least one flow path into the
respective test area(s) by lateral flow effected by the amphipathic
polymer. As the sample flows along the at least one flow path it is
presented and mixed with the hydrophobic luminophore and/or other
reagents. Alternatively, the sample is only presented with the
hydrophobic luminophore and/or other reagents when it reaches the
test area. Preferably the sample moves by lateral flow not
requiring external forces, such as pumps, to move the aqueous
sample. Once the aqueous sample reaches the test area(s)
measurements may be taken.
[0065] Where the assay is a lipid profiling assay the probe,
reporter or reagent may be selected from the group consisting of
Amplex Red, K37, Nile Red, cholesterol esterase, cholesterol
oxidase or horseradish peroxidase.
[0066] Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine), available
from Invitrogen (catalogue numbers A12222 and A22177) amongst
others, reacts with Hydrogen peroxide (H.sub.2O.sub.2) with a 1:1
stoichiometry to produce highly fluorescent resorufin. K37
(4-dimethylamino-4'-difluoromethyl-sulphonyl-benzylidene-acetophone)
is disclosed by the inventors in their previous International
patent application PCT/GB2005/004757. Nile Red a lipophilic stain,
also known as Nile blue oxazone, is available from Invitrogen
(catalogue number N1142) or may be produced by boiling a solution
of Nile blue with sulphuric acid. Nile red stains intracellular
lipid droplets red and is also intensely fluorescent, with a strong
yellow-gold emission when in a lipid-rich environment.
[0067] Cholesterol esterase (Steryl-ester acylhydrolase, Registry
number: EC 3.1.1.13) is an enzyme that catalyses the hydrolysis of
cholesterol ester and some other sterol esters, to liberate
cholesterol plus a fatty acid anion. Cholesterol oxidase
(Cholesterol:oxygen oxidoreductase, Registry number: EC 1.1.3.6) is
an enzyme that catalyses the oxidation of cholesterol in the
presence of molecular oxygen to 4-cholesten-3-one and hydrogen
peroxide. Horseradish peroxidise (Sigma Aldrich, Registry number:
EC 1.11.1.7) is a Hydrogen peroxide oxidoreductase. Other
equivalent hydrogen peroxide oxidoreductases are known and may be
derived from, for example, soy bean.
[0068] For lipid profiling a first flow path comprises Amplex Red,
a second flow path comprises K37 and a third flow path comprises
Nile Red. Alternatively the first test area may comprise Amplex
Red, the second test area may comprise K37 and the third test area
may comprise Nile Red. Yet more preferably, when the first flow
path comprises Amplex Red, the first flow path further comprises
cholesterol esterase, cholesterol oxidase and horse radish
peroxidise. In alternative embodiments the first test area may
comprise cholesterol esterase, cholesterol oxidase and horse radish
peroxidise. In yet other embodiments the application area comprises
the at least one probe, reporter or reagent selected from the group
consisting of Amplex Red, K37, Nile Red, cholesterol esterase,
cholesterol oxidase or horse radish peroxidise.
[0069] Preferably the probe, reporter or reagent is in dry
form.
[0070] Advantageously, the apparatus may be used to carry out quick
and easy assays that can be conducted simultaneously, in parallel,
to determine the presence/absence or concentration of an analyte
from the biological fluid. For example, the use of an amphipathic
polymer reduces the time an assay takes from several hours or even
days to around as little as one minute.
[0071] In another aspect of the invention there is provided a
method of measuring an analyte in an aqueous biological sample
comprising: [0072] (i) contacting the aqueous biological sample
with a combination of at least one hydrophobic luminophore and at
least one amphipathic polymer wherein, the at least one hydrophobic
luminophore binds to at least one analyte in the aqueous biological
sample and when bound thereto fluoresces under appropriate
excitation; [0073] (ii) exciting the product(s) from step (i) at a
suitable excitation wavelength; [0074] (iii) measuring the
fluorescence emission following step (ii) at a suitable detection
wavelength.
[0075] It will be apparent to one skilled in the art that suitable
excitation and emission wavelengths will be dependent on the
particular dye or luminophore used. Selection of suitable
wavelengths may be determined using standard laboratory techniques
or available data relating to the dye or luminophore.
[0076] In one embodiment of the method of the invention, the
aqueous biological sample is contacted with the at least one
amphipathic polymer before contacting with at least one hydrophobic
luminophore.
[0077] In an alternative embodiment, the aqueous biological sample
is contacted with the at least one amphipathic polymer at
substantially the same time as it is contacted with at least one
hydrophobic luminophore.
[0078] By way of non-limiting example, the biological sample (in
aqueous form) is applied to an application area on, for example, a
test chip or card according to the first or second aspects of the
invention. The application area is connected to at least one,
preferably three, four or more test areas. The connection between
the areas may be by channels, capillaries, tracks and the like. The
channels, capillaries and the like are preferably coated with an
amphipathic polymer. Where tracks are used, they may be formed from
an amphipathic polymer, for example, printed or laid down onto a
surface. The amphipathic polymer `wicks` or draws the aqueous
sample from the application area, along the channels, capillaries,
tracks and the like, to the at least one test area. Where multiple
channels, capillaries, tracks and the like are used, the sample may
be divided or split into aliquots as the fluid travels along them.
In this manner the sample may be divided without manual processing
steps through the use of lateral flow effected by the amphipathic
polymer.
[0079] Each channel, capillary, track and the like may comprise at
least one hydrophobic luminophore in combination with the
amphipathic polymer. Where present, the hydrophobic luminophore in
each channel, capillary track and the like may be the same or a
different. Each different hydrophobic luminophore may bind to a
specific class or subclass of lipoproteins and when bound thereto,
modifies the fluorescence yield under appropriate excitation, which
is indicative of the concentration of the specific class or
sub-class of lipoproteins. Similarly, each channel, capillary or
track and the like may also comprise at least one enzyme or other
component or reagent. In this manner, as the sample travels or
flows (is drawn) along the channels, etc., it is presented and
mixes with other components or reagents without the need for manual
processing steps. It will be apparent to one skilled in the art
that, as a result, different quantities or combinations of reagents
or conditions may be applied to different aliquots of the sample
resulting in different assays being performed and measured in each
test area.
[0080] Preferably the at least one hydrophobic luminophore is a
dye, more particularly a fluorescent dye and yet more particularly
a dye that is uniquely fluorescent when it is bound to an analyte
such as a lipid component.
[0081] Human Serum Albumin (HSA) is a major component of blood
serum having a concentration of approximately 30-50 mg/ml. HSA is
known to have at least two types of binding site that are capable
of binding various ligands. A first type is referred to herein as
"a hydrophobic domain" whereas a second type of domain is referred
to herein as a "drug binding domain". These domains are known to
one skilled in the art and are distinguished from each other in a
paper in Nature Structural Biology (V5 p827 (1998)). This paper
identifies a hydrophobic domain as one to which fatty acids may
bind whereas the drug binding domain is capable of binding a number
of drugs that may be associated with HSA.
[0082] The inventors have established that hydrophobic lumiophores
may bind to hydrophobic binding sites/domains of HSA and may
fluoresce when bound to HSA. Therefore, the inventors believe that
this additional fluorescence may cause a substantial background
signal, potentially distorting measurements and leading to errors
in the determination of concentration of lipoproteins.
[0083] Thus, when the aqueous sample is a blood sample, the
hydrophobic binding sites of HSA at which hydrophobic luminophores
may bind, are blocked. Thus, prior to analysis of a sample a ligand
binding inhibitor is preferably added.
[0084] The ligand binding inhibitor may be hydrophobic. The
inhibitor may be amphipathic. The ligand binding inhibitor may
comprise a fatty acid or a functional derivative thereof, as well
as other hydrophobic molecules. Examples of suitable derivatives of
fatty acid, which may block the hydrophobic binding sites of HSA
may comprise a fatty acid, its esters, acyl halide, carboxylic
anhydride, or amide etc. A preferred fatty acid derivative is a
fatty acid ester.
[0085] The fatty acid or derivative thereof may comprise a C1-C20
fatty acid or derivative thereof. It is preferred that the fatty
acid or derivative thereof may comprise a C3-C18 fatty acid or
derivative thereof, more preferably, a C5-C14 fatty acid or
derivative thereof, and even more preferably, a C7-C9 fatty acid or
derivative thereof. It is especially preferred that the ligand
binding inhibitor comprises octanoic acid (C8) or a derivative
thereof, for example, octanoate: Preferably, the ligand binding
inhibitor is added as an alkali metal octanoate, preferably a Group
I alkali metal octanoate, for example, sodium or potassium
octanoate.
[0086] Preferably, between about 10-400 mM of the ligand binding
inhibitor is added to the sample prior to analysis, more
preferably, between about 20-200 mM, and even more preferably,
between about 30-80 mM is added. It is especially preferred that
about 50 mM of the inhibitor is added. Hence, in a preferred
embodiment of the method, about 50 mM of sodium octanoate may be
added to the sample before analysis.
[0087] Ligands for the drug binding domains of HSA include drug
molecules such as: thyroxine, ibuprofen, diazepam, steroid hormones
and their derivatives (drugs), haem, bilirubin, lipophilic
prodrugs, warfarin, coumarin based drugs, anaesthetics, diazepam,
ibuprofen and antidepressants (e.g. thioxanthine), benzoic acid or
a derivative thereof (e.g. trichlorobenzoic acid or triiodobenzoic
acid). Alternatively the HSA binding domains may be blocked or
removed by use of a HSA extraction means, for example, anti-HSA
antibodies.
[0088] In particular embodiments the aqueous sample is used `neat`,
that is, without dilution of the sample prior to use.
[0089] In other embodiments, the aqueous sample may be diluted
before use. For example, when the aqueous sample is derived from
blood, dilution of the sample may be desirable, for example using a
1 in 80 dilution, before the assay is performed. Preferably the
diluent is phosphate buffered saline (PBS) and comprises at least
one ligand binding inhibitor. In particular embodiments, the
diluent comprises two ligand binding inhibitors, one for the
hydrophobic binding site(s) and one for the drug binding site(s).
Preferably the ligand binding inhibitors comprise benzoic acid and
octanoic acid. Alternatively, the sample may be diluted with PBS
and the at least one ligand binding inhibitor is combined with the
amphipathic polymer. Thus, the ligand binding inhibitor is added to
and mixed with the aqueous sample as it travels by lateral flow,
for example, along the tube, capillary, channel or track.
[0090] In a preferred method a plurality of, and at least two or
three, fluorescence assays are carried out under similar conditions
in parallel.
[0091] According to a third aspect of the invention there is
provided a process for enhancing lateral fluid flow comprising
coating a surface along which a fluid may flow with an amphipathic
polymer.
[0092] Preferably in the process of the invention, the surface is
defined by a tube, capillary, channel, well, membrane or the
like.
[0093] Thus, an amphipathic polymer may be used to coat a surface
defined by the inside of a tube, capillary, channel, well, membrane
or the like, for example. It will be apparent that the amphipathic
polymer may also be used as a coating for membranes used in
`standard` lateral flow assays to speed up and provide greater
control over the fluid flow. The hydrophobic or amphipathic polymer
may be coated, printed and/or sprayed onto a surface for example
forming `tracks` and/or layers, by printing methods such as by
inkjet or bubble-jet printing or other application methods. In
particular embodiments of the process the amphipathic polymer is in
the form of a film. In other embodiments, the film may be ground
into particulate material or may be formed into, for example,
granules, beads, pellets, microspheres, nanospheres or picospheres.
The at least one amphipathic polymer may be in the form of
granules, beads, pellets, microspheres, nanospheres or
picospheres.
[0094] According to a fourth aspect of the present invention, there
is provided the use of Gafquat for stabilisation of a protein, in
particular an enzyme.
[0095] Surprisingly it has been discovered that co-polymers of
vinylpyrrolidone and dimethylaminoethyl methacrylate, sold by
International Speciality Products under the trade name
Gafquat.RTM., for example, are suitable enzyme stabilising
reagents. Gafquat (CAS Registry Number: 53633-54-8; 7732-18-5) is
the name for a range of water-soluble copolymers such as
Polyquaternium-11. It is the primary active ingredient in many hair
products such as mousses, gels, hairsprays and in special effects
makeup but has not been used before for the purposes of
stabilisation on an enzyme or protein. Preferred enzymes for
stabilisation with Gafquat include cholesterol esterase,
cholesterol oxidase and horseradish peroxidise although Gafquat may
be utilised for stabilisation of a wide range of enzymes.
[0096] According to a fifth aspect of the invention there is
provided use of the device, method or process of the invention in
an assay.
[0097] All of the features described herein (including any
accompanying claims, abstract and drawings), and/or all of the
steps of any method or process so disclosed, may be combined with
any of the above aspects in any combination, except combinations
where at least some of such features and/or steps are mutually
exclusive.
BRIEF DESCRIPTION OF DRAWINGS
[0098] The invention is described with the help of the following
figures, though not limited thereto. It should be understood, that
the figures are for illustrative purposes only.
[0099] FIG. 1 shows the components of an embodiment of a test
cartridge suitable for point of care use.
[0100] FIGS. 2 and 3 illustrate further embodiments of an assay
device
[0101] FIG. 4 illustrates embodiments of a capillary channel
defining an application area, fluid flow pathway, detection zone
and stop-flow junction.
[0102] FIG. 5 illustrates the effect of molecular weight of PEG on
vertical flow rate. Error bars indicate +/-1 standard deviation
(uncoated capillary tubes and their 1 order of standard deviation
are shown at zero molecular weight).
DETAILED DESCRIPTION OF THE INVENTION
[0103] Referring to FIG. 1 of the drawings, an assay device (1)
generally comprises a housing having a base (2a) and a cover (2b)
which protects the application area (3), flow paths (5) and test
areas (4) from contamination or damage.
[0104] The device is provided with a ridged handle portion (7)
enabling a doctor or clinician to hold the device. The housing over
the application area (3) comprises a well (3a), in this case,
having sloping sides to guide the sample onto the application area.
A window (4a) provides access to the test areas allowing the
results of the assay to be determined.
[0105] Within the housing (2a and 2b) is a strip of hydrophobic
plastic (6) which acts as a support for the PEG flow paths. The
flow paths (5) are printed onto the support leading from the
application area (3) to, in this instance, four test areas (4). A
filter (8) is glued onto the support to filter particulates from
the aqueous sample.
[0106] In use, a doctor or clinician applies an aqueous sample to
the application area (3). Fluid from the aqueous sample travels by
lateral flow along the flow path toward and through the filter (8).
The filter, which has been coated with PEG, blocks any cells in the
sample from travelling further along the flow paths. Once the fluid
approaches the test areas, junctions formed in the flow path split
the sample into four aliquots.
[0107] When utilised in the preparation of a lipid profile, as the
first aliquot travels along flow path 5(a), it is presented with
and mixes with K37 dye combined with the PEG in the flow path. The
second aliquot travels along flow path 5(b) and is mixed with a
second dye, Nile Red. The third aliquot travels along flow path
5(c) and as it does so, it mixes with three dried enzymes,
cholesterol esterase, cholesterol oxidase and horse radish
peroxidise and a dye, Amplex Red. Each of the three aliquots of
sample, mixed with the respective dyes flows into and collects in
the test areas (4). The fourth test area (5d) is used as a control
to measure background fluorescence.
[0108] The device is loaded into a reader which excites each of the
test areas in turn and measures any subsequent fluorescence
emission. The processing chip within the reader calculates the
concentration of analytes (such as total cholesterol,
triglycerides, HDL and other lipid components) within the sample
and displays the results on an LCD screen.
[0109] FIGS. 2 and 3 illustrate further embodiments of an assay
device according to the invention which may be used, for example,
in the determination of lipoprotein levels in blood.
[0110] In this embodiment, the assay device comprises a first,
support surface (1). The support surface generally comprises or
consists of an opaque material, for example a plastics material
incorporating an amount of a pigment such as carbon black. In this
case, a support surface is formed from a medical grade polymer
compatible with fluorescent, luminescent or photometric
measurement. Cyclic olefin polymers generally have excellent
mechanical properties, low autofluorescence and high UV
transmission. Suitable polymers include cyclic olefin copolymers
such as Topas.RTM. COC (CAS number 26007-43-2), Zeonor.RTM. COP,
Zeonex.RTM. COP or Udel.RTM. polysulfone (CAS Number 25135-51-7).
In this example the support is formed of TOPAS COC comprising 1%
Carbon black. The use of carbon black has the added advantage that,
during laser welding for example, the power requirement is reduced
increasing ease of manufacture of the device and protecting heat
labile components in the reaction chambers. The use of Carbon black
may also have benefits relating to heat dissipation/insulation.
[0111] The support surface of this embodiment is moulded but may be
formed by any standard moulding or machining techniques known in
the art. Whilst it is generally flat, it comprises profiled areas,
in this example of sunken-relief which form, in this case four,
capillary channels (3) arranged circumferentially around an
application area (4). In this case each of the four channels are
equidistantly spaced from one another at an angle of around
90.degree..
[0112] The moulded support surface forms an application area (4),
fluid flow paths (5) and detection zones (6).
[0113] Each end of the fluid flow pathways (5) farthest from the
application area (4) is characterised by a gradual taper of one or
more of the capillary channel walls. This taper increases the width
and/or depth and by extension the cross-sectional area of the
capillary flow channel forming a stop flow junction (7) preventing
further capillary fluid flow. Here, the channel forms a bulbous end
but may take a variety of other forms. The structure of the
capillary channel which defines the application area, fluid flow
pathway, detection zone and stop-flow junction is shown more
closely in FIG. 4. It will be apparent to one skilled in the art
that the channels may be arranged in any number of formats or be of
differing shapes to suit a particular assay. For example, and by
way of non-limiting example, the channels may be ovoid or
`tear-drop` shaped, trapezoidal, triangular, columnar or tubular.
Further examples, are shown in FIG. 4. For hospital, laboratory or
large volume use, it is conceivable that 10-20, 20-40, 40-60 or
50-100 capillary channels could be arranged on an assay device the
size and shape of a compact disc.
[0114] FIG. 4 illustrates further embodiments of stop flow
junctions. In this figure the fluid flow pathway (5) maintains an
even cross-sectional area along substantially its entire length
before narrowing or constricting to form a section of fluid flow
pathway of reduced cross-sectional area (13) towards the distal
end. This section of fluid flow pathway is in turn adjacent to, and
in fluid communication with a chamber (14). The chamber is divided
into regions by a low wall or barrier (15)--in this case into two
parts (15a) and (15b). In use, a fluid enters the chamber (14)
filling part (15a). The wall (15), or more accurately the capillary
forces acting on the sample fluid in the region of the wall, stops
flow of fluid into the second part of the chamber (15b). In other
embodiments the wall is replaced by a raised portion (16). Again,
capillary forces acting on the sample fluid in the region of the
raised portion (16), prevents fluid from filling the whole of the
chamber. One skilled in the art will realise that the configuration
(for example size, shape, height) of the chamber (14) and wall (15)
or raised portion (16) may take a number of forms including
different geometric shapes such as circles, trapezoids and the
like. A vent port located adjacent to the second part or distal
region of the chamber (15b) allows for movement of air from within
the chamber to the atmosphere equalising pressures within the fluid
flow pathways of the device.
[0115] The support surface also comprises a number of alignment
posts (8) to align the support surface with the second surface--the
cover member (2).
[0116] The cover member (2) is also formed from a suitable medical
grade polymer such as those described above. In this example the
cyclic olefin copolymer (Topas COC), CAS number 26007-43-2 was used
again but in this case without carbon black. As a result the cover
member is optically clear/transparent--at least to the
excitation/emission wavelengths of the assay reader.
[0117] The cover member (2) is also moulded and generally flat
comprising a number of apertures.
[0118] The cover member is mountable with the support member,
optionally comprising a gasket or spacer element positioned there
between. Aperture (9) is aligned with the application area and
allows a user to apply a fluid sample to the application area. A
further series of smaller apertures function as vent openings
(10).
[0119] In the embodiments shown, alignment holes (11) orientate the
cover member with respect to the support member by accommodating
the alignment posts (8). In alternative embodiments the apertures
and alignment holes are provided in the support member, the cover
member simply fulfilling the role of a cover.
[0120] The Alignment elements may simply be raised members with
corresponding alignment holes or grooves. The support and cover
members may be fixed or bonded together for example by mechanical
means such as by use of screws, rivets, bolts or tabs.
Alternatively the support and cover members may be held together by
friction fit. In other embodiments the cover and support members
are bonded together, for example by use of glues, solvents,
adhesive tape and the like. In this case the cover and support
members are bonded together by use of heat and laser welding.
[0121] One or both of the support and cover members may comprise
identification means, to provide a unique identifier and/or to
provide a means for an assay reader, for example, to monitor the
number of times an assay device has been used or when sample was
applied so that an appropriate assay time passes. Such
identification means may also contain instructions or data such as
calibration or quality control data. Identification means may take
a variety of forms including, but not limited to, text, Braille,
numerical data, linear bar code(s), 2D bar code(s), RFID tag(s) and
the like.
[0122] A filter (12) is held firmly between the support and cover
members adjacent to and in fluid communication with the application
area (4) and aperture (9) and the fluid flow pathways.
[0123] In use, either an undiluted or diluted aqueous sample is
applied directly to the filter via aperture (9).
[0124] Fluid in the sample migrates through the filter and as it
does so large particulates are removed by passive filtration. In
the case of whole blood, such particulates include red blood cells.
As a result background interference from such large particulates is
reduced.
[0125] When used with blood, for example whole, venous, blood, the
filter may further comprise an HSA extraction means comprising
anti-HSA antibodies which remove HSA from the sample as previously
discussed, again reducing background. Blood may be obtained by
finger prick or in the case of neonates, a heel prick. This is a
way of opening a small wound, for example in the finger tip, which
produces no more than a few drops of blood (.about.less than 50
.mu.l such as from about 10 .mu.l to 20 .mu.l). After a blood
droplet has formed it can be applied directly to the application
area of the assay device or sucked up by a pipette and then applied
accordingly. Other means of obtaining a blood sample are known in
the art. The ability to utilise blood obtained by, for example a
finger prick, directly represents a significant advantage over
assays known in the art.
[0126] Alternatively the assay device may be utilised to test a
sample that is derived from plasma, in which case a 1 in 80
dilution of the sample may be performed. Generally the diluent that
is used is phosphate buffered saline (PBS) and it may comprise at
least one ligand binding inhibitor. The diluent may also comprise
two ligand binding inhibitors, one for the hydrophobic binding
site(s) and one for the drug binding site(s).
[0127] The fluid travels by means of capillary flow from the
application area (4) into the fluid flow pathways (5) with which
the application area is in fluid communication. As it travels the
liquid hydrates reagents in dry form, such as fluorescent dyes and
enzymes, and mixes with them. Thus, the sample moves by capillary
or lateral flow not requiring the application of external forces,
such as pumps, to move the aqueous sample. The amphipathic or
non-ionic polymer enhances capillary flow and the efficiency of
reagent mixing.
[0128] Use of the term `dry form` refers to components that are
maintained in a form in which they are generally substantially free
from, or depleted of, liquid or moisture; that is they are not in
solution until reconstituted by the performance of the assay
itself, rather than being reconstituted prior to and separate from
the assay procedures. Thus, the aqueous sample itself reconstitutes
the dry reagent or reagents, thereby eliminating the need for
separate reconstitution buffers and steps. As described above, use
of the amphipathic or non-ionic polymer assists mixing of dry
reagents with the aqueous sample.
[0129] Where enzymes or other reagents are used, particularly where
they are used in dry form, it is preferred that such enzymes or
other reagents are stabilised. In the context of this invention, a
`stabilised reagent` is a reagent that has improved stability with
respect to, for example, storage stability, thermal stability etc.
Thus, in particular embodiments the reagents that are used comprise
a stabilising agent. Particular stabilising methods are disclosed
in International Patent Application numbers WO90/005182 and
WO91/014773 the contents of which are hereby incorporated by
reference. Other suitable stabilising reagents include co-polymers
of vinylpyrrolidone and dimethylaminoethyl methacrylate, for
example, sold by International Speciality Products under the trade
name Gafquat.RTM.. Gafquat (CAS Registry Number: 53633-54-8;
7732-18-5) is the name for a range of water-soluble copolymers such
as Polyquatemium-11.
[0130] As the fluid front moves forward, an equivalent volume of
air is displaced through the vent openings equalising pressure
within the device. Once the fluid reaches the stop-flow junction,
surface tension prevents further capillary flow.
[0131] At this stage the device can be placed within a suitable
assay reader and the levels of analytes, for example cholesterol
and blood lipids, are measured.
[0132] The housing of the assay device is usually adapted to enable
it to be placed in functional communication with an assay reader.
For example, the assay device may be inserted into, placed on or
attached to the reader and the reader may comprise docking means,
such as a slot, or alignment means to enable the assay device to be
inserted, placed or attached appropriately. In this embodiment, the
assay device has a `V`-shaped cut out in the cover member which
facilitates alignment of the assay device with a reader. Generally
the assay device of the present invention is a disposable whilst
the reader will usually be reusable.
[0133] The assay device may be used for a variety of assay
processes or reactions such as immunoassays and fluorometric assays
including cholesterol, lipoprotein or triglyceride assays.
[0134] An immunoassay is a biochemical test that measures the
concentration of a substance in an aqueous sample, for example
serum or urine. The assay utilises the reaction of an antibody or
antibodies to its antigen taking advantage of the specific binding
of an antibody to its antigen. Preferably monoclonal antibodies are
used since they bind to one site of a particular molecule providing
specific and accurate tests. Both the presence of antigen or
antibodies may be measured, for example, when detecting infection
the presence of antibody against the pathogen may be measured.
Alternatively when measuring biological molecules such as hormones,
and the like, the hormone biological molecule may acts as the
antigen. The response of the aqueous fluid being measured may be
compared to standards of a known concentration, for example,
plotting of a standard curve on a graph. Detecting the quantity of
antibody or antigen may be achieved by a variety of methods such as
labelling either the antigen or antibody. By way of non-limiting
example, the label may consist of an enzyme (EIA or ELISA), a
radioisotope such as I-125, a magnetic label or a luminescent or
fluorescent label.
[0135] Advantageously, the device of the present invention relies
on capillary flow for fluid transport of low volume samples aided
by use of an amphipathic or non-ionic polymer and there is
therefore no need to use moving parts. Thus, the device overcomes
problems of scale, economy, manufacture and mechanical failure
encountered in the prior art.
[0136] An assay reader for use with the assay device of the present
invention may be adapted to receive two or three assay devices, for
example, from multiple patients or for multiple tests of an aqueous
sample from an individual patient. Such a reader may comprise two
(or more) excitation means that can be aligned with the detection
zones of the assay device. `Excitation means` are operable to
excite the sample in the detection, for example, so that it
fluoresces. The apparatus will also comprise at least one detection
means which are operable to detect, for example, the fluorescence
emitted by the sample at the detection zone(s).
[0137] Generally the excitation means comprises an illumination
source operable to illuminate the sample at about 400 nm-600 nm.
Accordingly the light source is preferably capable of illuminating
the sample at between about 400 nm-600 nm. The illumination source
may comprise one or more a bulbs, or one or more LEDs, or other
sources such as a one or more lasers. Excitation wavelengths may be
varied utilising at least one interference filter. The excitation
means may also comprise polarising means operable to polarise light
produced by the illumination source. The excitation means may also
further comprise focussing means adapted to focus the light on to
the sample. The focussing means may comprise a lens, or light guide
such as a fibre-optic filament or optical light film (3M).
[0138] The detection means may comprise a photodiode, CCD, or
photomultiplier or optical sensor, which is preferably yellow-red
sensitive. Fluorescence emitted by the sample may be detected
within a range including about 440 nm-650 nm depending on the dye
or dyes used. The detection means should be able to detect
fluorescence emitted at about 490 nm, about 495 nm, about 570 nm,
about 600 nm and about 610 nm. The fluorescence may be collected by
a second lens, and may pass through a polariser. Scattered
excitation light may be removed by a cut-off filter(s) or band pass
filter(s). For measurement of the fluorescence intensity, the
current from the photodiode or the count rate from the
photomultiplier may be read from an ammeter, voltmeter, or
rate-meter module. Other means will be apparent to one skilled in
the art.
[0139] The reader may also comprise an excitation correction system
so that fluctuations of in the light source may be accounted for.
The apparatus may comprise at least one fluorescence standard for
use in calibrating prior to determining the concentration of an
analyte or analytes. The standard may be an internal standard.
[0140] In particular embodiments the assay reader is configured to
detect and measure the fluorescence intensities of a single or a
plurality of assays simultaneously or in turn as the assay device
enters the reader or at some time thereafter.
[0141] The reader may also comprise processing means adapted to
determine the concentration of an analyte or analytes in the sample
based on the fluorescence detected.
[0142] The reader may further comprise display means for displaying
measurements determined from the sample, preferably as a read-out.
For example, the display means may comprise an LCD screen, or may
rely on a computer for powering and/or computing and/or display. In
its most basic form, the display means may simply be a window in
which an indication or measurement is displayed.
[0143] Usually, the assay reader is portable and advantageously,
the assay device and reader may be used to carry out assays simply,
rapidly and simultaneously to determine the presence/absence or
concentration of an analyte from an aqueous sample such as a
biological fluid. For example, a clinician with knowledge of
cholesterol, lipoprotein and HDL concentrations can use the device
to decide on an effective course of treatment. In addition, the
assay device and reader is portable and may be used by GPs, or
nurses who carry out home visits, or even as test kits for home
use.
[0144] In particular embodiments, the processing means are adapted
to directly determine the concentration of one or more analytes
such as, cholesterol, triglycerides, HDL, LDL, VLDL and IDL in the
sample based on fluorescence analysis. Alternatively, the
processing means may be adapted to calculate the concentration of
LDL, VLDL and IDL, in the sample based on the concentrations of
total lipoprotein, cholesterol and HDL.
Use of the Device in Analysis of Cholesterol and Lipids
[0145] The following example describes a method of measuring
lipoproteins in an aqueous biological sample using an assay device
of the present invention.
[0146] In this example, the fluid flow pathways are coated with an
amphipathic polymer, PEG, to speed up fluid transport. For lipid
profiling the amphipathic polymer is also combined with a
fluorescent dye such as Amplex Red, K37 or Nile Red and/or other
reagents such as enzymes, laid down or printed within the fluid
flow pathway(s) or within the detection zone.
[0147] The fluorescent dye(s) and/or other reagents may be printed
as one or more arrays of pico-litre droplets. Suitably such an
array may comprise between about 150 to 4500 droplets along a first
axis by 25 to 100 droplets along a second axis. Particular sizes of
array include about 3400.times.65 droplets, about 3000.times.65
droplets, about 1500.times.65 droplets, about 1900.times.65
droplets, 600.times.65 droplets, 450.times.65 droplets or about
400.times.65 droplets per mm.sup.2. It will be apparent to one
skilled in the art that, whilst these droplet densities may be
varied with little effect on assay performance, it will also be
apparent that conversely such densities can also be optimised to
improve assay performance. For example, the droplets could also be
applied as nano- or femto-litre droplets, applied as overlapping
arrays, applied one (or more) on top of another, applied as
discrete individual arrays, spaced apart or applied as blocks of
several arrays forming an array of a larger size (i.e. additive).
The array size may also be optimised with respect to a particular
reagent or dye concentration for example.
[0148] When `printing`, droplets of fluorescent dye may be applied
to the device at concentrations of between about 0.1 mM to 3.0 mM,
more suitably, between about 0.3 to 2.5 mM, and even more suitably,
between about 0.5 to 2.0 mM. Alternatively, when the device is for
use with diluted blood samples, the concentration of fluorescent
dye may be between about 0.8 to 1.2 mM. A useful concentration of
fluorescent dye is 1.0 mM and for undiluted blood samples a useful
concentration is about 2.0 mM. The dye is subsequently dried before
use.
[0149] In use the sample fluid hydrates the amphipathic polymer and
dilutes the fluorescent dye which can then bind to lipoproteins in
the sample. When so bound the dye fluoresces under appropriate
excitation. The total lipoprotein concentration in the sample may
be determined using fluorescence analysis.
[0150] The method generally comprises:
(i) contacting an aqueous biological sample with at least one dye
or luminophore and at least one amphipathic polymer wherein, the at
least one dye or luminophore binds to at least one lipoprotein in
the aqueous biological sample and when bound thereto fluoresces
under appropriate excitation; (ii) exciting the product(s) from
step (i) at an excitation wavelength of between about 400 nm-620
nm; (iii) measuring the fluorescence emission following step (ii)
at a wavelength of between about 440-650 nm.
[0151] The method may be used to prepare a lipid profile from an
aqueous biological sample.
[0152] By use of the term "total lipoprotein", is meant the
collective concentration of at least VLDL, HDL, LDL, IDL and
chylomicrons in other words, the sum of the concentrations of
triglyceride and total cholesterol in a sample. By use of the term
"total cholesterol", is meant the total concentration of
cholesterol in a sample. By use of the term "lipid profile", is
meant the concentration(s) or relative concentration(s) of lipid
components (i.e. total lipoproteins and total cholesterol and
triglyceride) in a sample.
[0153] Most lipids present in a blood or serum sample are bound to
lipoproteins. Conventional tests conducted in clinical labs do not
measure total lipoprotein. Hence, conventionally, it is required to
first determine, and then add the concentration of cholesterol and
cholesterol esters, to that of triglyceride to determine the total
lipoprotein concentration. Conventional measurement of triglyceride
in a clinical lab is subject to substantial errors because it
relies on the measurement of glycerol, which circulates naturally
in the blood. Advantageously, because the number (volume) of
lipoprotein particles is measured directly to determine the
concentration of total lipoprotein (which equates to the total
lipid concentration) the cholesterol assay according to the present
invention is not subject to this error. Thus, errors such as in the
triglyceride concentration caused by circulating glycerol in the
sample are obviated.
[0154] In their previous International patent application
PCT/GB2005/004757, published as WO2006/061646, the inventors
developed a simplified assay based on the use of, for example, K37
for measuring lipoproteins in a biological macromolecule that is
particularly useful when a clinician wishes to quickly and
efficiently obtain a lipid profile. For determining the
concentration of total lipoprotein (i.e. HDL, LDL, IDL and VLDL) in
a blood sample using K37 fluorescence measurements, the inventors
realised that the fluorescence response from K37 bound to the
various lipoprotein classes can be made substantially the same for
a given total lipoprotein concentration, i.e. total lipoprotein
concentration, irrespective of its composition (i.e. the ratio of
HDL:LDL:IDL:VLDL in the sample). Accordingly, it is preferred that
K37 is used in such a manner that the response of fluorescence
intensity is substantially linear across the range of
concentrations of lipoprotein molecules that would be expected from
samples that would be encountered in clinical tests.
[0155] Not wishing to be bound by any hypothesis, it is believed
that the intensity of fluorescence from the fluorescent dye depends
on its affinity for a particular lipoprotein molecule (HDL, LDL,
IDL or VLDL) in the sample. The quantum yield of fluorescence
depending on the environment within that lipoprotein molecular
complex, and also the degree of fluorescence quenching caused by
energy transfer between probe molecules packed closely together.
Hence, in their previous application, the inventors reasoned that
it would be possible to select a suitable concentration of the
probe substance and excitation and emission wavelengths that may be
used to make an accurate measurement of total lipoprotein by simple
fluorescent measurement. The inventors further realised that such a
concentration of probe would preferably balance K37's higher
quantum yield in HDL compared to VLDL and LDL with its higher
affinity for HDL, and therefore a higher degree of quenching within
HDL to produce a constant fluorescence signal response over all
lipoprotein particles.
[0156] The inventors have conducted a series of experiments to
investigate whether it was possible to obtain a linear and equal
relationship between the fluorescence of the probe substance, K37,
and the lipoprotein concentration for each lipoprotein particle
type (HDL, LDL, and VLDL), across the range of lipoprotein
concentrations that would be encountered in real serum clinical
samples. To their surprise, they found that there was a defined
concentration of K37 and particular excitation and emission
wavelengths at which there was a linear relationship between the
fluorescence of K37 and lipoprotein concentration. Thus, using the
methodology of their previous patent application
(PCT/GB2005/004757) the skilled person may identify other suitable
dyes that also demonstrate such a relationship with lipoprotein
concentration.
[0157] The use of enzymes in a cholesterol assay is advantageous
because cholesterol is often found in an esterified state, thus
preferably cholesterol esterase is used to hydrolyse cholesterol
ester to free cholesterol. Free cholesterol may then be converted
to cholest-4-ene-3-one ketone by the action of cholesterol oxidase,
generating hydrogen peroxide in the process. Advantageously, Amplex
Red and hydrogen peroxide are converted to resorufin and water by
horseradish peroxidase. Resorufin may then be detected as a
fluorescent compound with an absorption maxima of about 563 nm and
a peak emission wavelength of 587 nm. The total cholesterol content
can be measured by exciting the sample at around 485 nm and
measuring the resulting fluorescence at about 600 nm.
[0158] When an enzyme is utilised it may be provided at a level
many times in excess of the ratio that would be used for measuring,
for example, the enzymes Km, for example. Such a ratio may be
extremely high when compared to those used in the art which are
around 1:1000. Surprisingly, the combination of a difference in
surface energies of the device coupled with the use of dry
stabilised enzymes and optionally an amphipathic polymer enables
far smaller quantities of both sample and reagent to be used. Not
wishing to be bound by theory, it is believed that the difference
in surface energies of the two at least partially opposing surfaces
creates a circular motion within the laminar flow of the aqueous
sample leading to more efficient mixing. As a result of this
increase in efficiency, larger levels of enzyme may be used in
smaller reaction volumes leading to yet more efficient and faster
reactions than those previously possible in the methods of the
prior art.
[0159] The second step for the method may comprise:
(ii) exciting the product(s) from step (i) at an excitation
wavelength of between about 400 nm-520 nm.
[0160] The excitation wavelength may be between about 420 nm-480 nm
or between about 440 nm-470 nm. The excitation wavelength or
wavelengths used will depend on the specific fluorescent dyes being
used in the assay. For Amplex red the excitation wavelength is
about 480 nm, for K37 the excitation wavelength is about 440 nm and
for Nile Red the excitation wavelength is about 580 nm.
[0161] A third step of the method comprises:
(iii) measuring the fluorescence emission at a wavelength of
between about 490-650 nm.
[0162] Alternatively the fluorescence emission can be measured at a
wavelength of between about 520 nm-620 nm. At emission wavelengths
of about 540 nm, or higher, more accurate readings for determining
the total lipoprotein concentration (i.e. the concentration of HDL,
IDL, LDL and VLDL, but also chylomicrons if present) may be
observed. However, the preferred fluorescence emission
wavelength(s) that is/are measured will depend on the specific
fluorescent dyes being used in an assay. For Amplex Red the
fluorescence is measured at about 600 nm, for K37 fluorescence is
measured at about 495 nm and for Nile Red fluorescence is measured
at about 610 nm.
[0163] It should be appreciated that the excitation and emission
wavelengths need not be measured at the optimal wavelengths for
each specific dye. Wavelengths may be selected that give the best
separation or performance when the dyes are used either in
combination or in parallel, for example when the assays are
performed at the same time in a single assay device. It will be
apparent that steps (ii) and (iii), excitation and detection, may
also be carried out substantially simultaneously.
[0164] In addition, the concentration of triglyceride may be
calculated by subtracting total cholesterol concentration from the
total lipoprotein concentration. Hence, a more detailed lipid
profile of the sample is thereby generated consisting of total
lipoprotein concentration, total cholesterol concentration, and
also triglyceride concentration, which would be useful to the
clinician.
[0165] The inventors have previously discovered that a number of
dyes will bind to lipoproteins and exhibit different fluorescent
responses that are dependant on the particular lipoprotein bound.
Fluorescent measurement of these dyes makes it possible to
distinguish between the types of lipoprotein present in a sample.
This is done by comparing the enhanced or reduced fluorescence
caused by one type of lipoprotein in a lipoprotein mixture with the
fluorescence expected from the other lipoproteins (in the absence
of the specific propertied lipoprotein) as determined from a
calibration curve and a known value of the total lipoprotein
content. For example the fluorescent dye, Nile Red, exhibits a
significantly higher fluorescence in HDL than in other
lipoproteins, such as LDL and VLDL. Therefore, other fluorescent
dyes (e.g. Nile Red, K37 or any other lipophilic probe that shows
specificity, or fluorescence enhancement or reduction towards a
particular lipoprotein), may be used to discriminate between
classes or subclasses of lipoproteins in the sample.
[0166] Accordingly the method of the invention enables the
determination of the concentration of a particular class, or
sub-class of lipoprotein in a sample using fluorescence analysis.
Generally this involves determining the concentration of a
particular class or sub-class of lipoprotein by the shift in
fluorescence response of a dye specific to that lipoprotein using a
second and/or third fluorescent dye.
[0167] By way of example, in order to determine the HDL
concentration in a sample using Nile Red, a calculation is made of
the excess fluorescence from Nile Red due to the presence of HDL.
Firstly, the total lipoprotein concentration (measurement "A") is
measured by the linear correlation of K37 fluorescence with
lipoprotein concentration (as determined by step (i)).
[0168] Secondly, Nile Red fluorescence is then calibrated with LDL
(and/or VLDL as the fluorescence to concentration response must be
essentially the same) at various concentrations to obtain a
calibration curve with slope "X" and intercept "Y". A skilled
technician would know how to prepare a range of concentrations of
LDL (and/or VLDL), and determine the respective fluorescence for
each concentration.
[0169] Calibration curves may be constructed for a series of
concentrations of HDL and a constant concentration of LDL to give
slope "Z". Knowing the total lipoprotein concentration from the K37
measurement "A" and the excess Nile Red fluorescence of the unknown
sample "B", the concentration of HDL "C" in the unknown sample can
be determined by the following equation: C=(B-(AX-Y))/Z
[0170] It will be appreciated that, in practice, pre-prepared or
standard calibration curves may be used. Furthermore assay devices
of the present invention or assay readers for use with such devices
developed to generate lipid profiles may comprise internal
standards and/or have processing means that will allow for
automatic calculation of lipoprotein concentrations without user
intervention.
[0171] Therefore, it will be appreciated that fluorescent
measurements may be used for determining the concentration of HDL,
VLDL (by calculation), LDL (by calculation), total lipoprotein,
triglycerides (by calculation) and also total cholesterol. All
these parameters may be determined simultaneously, in parallel, by
exciting and measuring fluorescence over a similar range of
wavelengths. As discussed above, this is a considerable improvement
over conventional assays, which have to be carried out separately,
and often in dedicated laboratories, causing a delay in the
generation of results. In addition, the fact that multiple lipid
parameters can be measured at the same time considerably simplifies
the instrumentation required to carry out the measurements.
[0172] The lipid profile generated includes the determination of
the concentration of cholesterol bound to LDL in the sample. It is
especially advantageous to know the LDL cholesterol concentration
as it is highly atherogenic. Hence, the method provides a
multi-readout of at least three, preferably four or five, or more
parameters of the lipid composition in the sample. Furthermore, it
is possible to calculate/estimate the concentration of
Cholesterol-VLDL-Cholesterol from the triglyceride concentrations,
as it is generally assumed that most of the triglycerides are
carried in VLDL and the cholesterol component of VLDL is 20%. This
is particularly advantageous for helping the clinician to decide on
a suitable course of treatment.
EXAMPLES
[0173] The invention will now be described with reference to the
following examples which describe the complete exposition of
embodiments of the invention as described above.
Example 1
Total Cholesterol Assay
[0174] The assay utilizes a triple enzyme system capable of
converting one molecule of cholesterol or cholesterol ester into a
molecule of hydrogen peroxide (H.sub.2O.sub.2). The hydrogen
peroxide generated is then used to oxidize the dye Amplex Red
(non-fluorescent) to generate the highly fluorescent product
Resorufin.
Stabilization of Enzymes onto Plastic The total cholesterol assay
uses the following enzymes and dye:
Cholesterol Esterase (3.1.1.13)
Cholesterol Oxidase (1.1.3.6)
Horseradish Peroxidase (1.11.1.7)
[0175] Amplex Red: 10-acetyl-3,7-dihydroxyphenoxazine
[0176] Enzymes were stabilized onto plastic using Gafquat as a
stabilizing agent. The three enzymes were added to a solution of
0.01M potassium Phosphate buffer, pH 7.0. Final activity of each
enzyme was measured at 200 U/ml of buffer. The solution was then
diluted 1:1 with Gafquat (highly positively charged polymer)
formulation and 5 ul of the resulting solution was deposited onto a
plastic surface and dried at 30.degree. C. in the presence of
silica gel for 2 hours. The process resulted in a dry enzyme
bio-surface, with 0.5 U of each enzyme deposited.
Assay Procedure--Utilising 1/80 Sample Dilution
[0177] Two approaches were taken, a) reaction of cholesterol sample
(plasma) with dried enzymes and Amplex Red in solution, b) reaction
of cholesterol sample with both dried enzymes and dried stabilized
Amplex Red dye:
(a) Reaction of Cholesterol Sample (Plasma) with Dried Enzymes and
Amplex Red in Solution.
[0178] Dilution buffer A: 4.16 mM Amplex Red, 10 mM Cholic acid,
0.2% Triton X-100 in Dulbecco phosphate buffered saline pH 7.2.
[0179] The sample to be assayed was first diluted 1 part into 80
parts of dilution buffer A, then 50 ul of the diluted sample was
used to reconstitute and activate the dried tri-enzyme mix
(previously stabilized as described above) in the sample assay
chamber.
[0180] The cholesterol content was measured by exciting the sample
mix at 480 nm and measuring the resulting fluorescence at 600 nm.
Cholesterol concentration was determined directly through
measurement of the steady state fluorescence after 40 seconds, or
Vmax (maximal rate of substrate generation). Each evaluation was
made by reference to assay standard data.
(b) Reaction of Cholesterol Sample (Plasma) with Both Dried Enzymes
and Amplex Red.
[0181] Dilution buffer B: 10 mM Cholic acid, 0.2% Triton X-100 in
Dulbecco phosphate buffered saline pH 7.2.
[0182] This procedure is similar to the above process. However, in
this process the Amplex Red dye was dried in the flow path along
with the tri-enzyme mix. Firstly, one defined region of the assay
chamber was coated with 0.5 U Cholesterol Esterase (3.1.1.13), 0.5
U Cholesterol Oxidase (1.1.3.6) and 0.5 U Horseradish Peroxidase
(1.11.1.7) as described above. A second and separate region of the
assay chamber was then coated with 10 .mu.l of Amplex Red/PEG2000
solution and dried at 30.degree. C. in the presence of silica gel
for 2 hr. The dye coating solution was composed of 5.35 mg/ml
Amplex Red, 5% w/v PEG2000 in dimethylsulphoxide (DMSO).
[0183] The sample to be assayed was diluted, 1 part into 80 parts
of dilution buffer B, and 50 ul of the diluted sample was used to
reconstitute and activate the dried Amplex Red dye and tri-enzyme
mix in the sample assay chamber.
[0184] The cholesterol content was measured by exciting the sample
mix at 480 nm and measuring the resulting fluorescence at 600 nm.
The cholesterol concentration was again determined directly through
either the measurement of Vmax (maximal rate of substrate
generation) or steady state fluorescence after 40 seconds. Each
evaluation was made by reference to assay standard data.
[0185] Both assays were determined to be capable of determining
clinically relevant cholesterol levels of between 2-11 mM.
Assay Procedure--Utilising Undiluted Sample
[0186] Neat biological samples were assayed by applying the sample
to a borosilicate filter impregnated with anti-HSA antibody located
within the consumable device, which serves to filter and direct the
sample to an appropriate 200 .mu.m deep read or detection area.
Pre-coating of the read area firstly with 400.times.65 pico-litre
droplets of enzyme reagent per mm.sup.2, followed by 600.times.65
pico-litre droplets of dye reagent per mm2 and 450.times.65
pico-litre droplets of inhibitor reagent per mm2 facilitates rapid
flow of the sample into the read area. Subsequent excitation of the
sample at 480 nm (10 nm band pass) generates fluorescence that can
be detected through a 600 nm (10 nm band pass) filter to allow the
Total Cholesterol content of the sample to be determined by
reference to suitable standard measurements.
[0187] Enzyme Reagent: Cholesterol Esterase, Cholesterol Oxidase
and Horseradish Peroxidase, each dissolved in a Gafquat stabiliser
mix at 200 units per ml.
[0188] Detergent solution: 1.63 g Cholic Acid, 10 g Polyethylene
glycol 2000, 800 ul Triton X-100 and 253.3 ul of Diethyl Maleate is
dissolved in dimethylformamide (DMF) and the final volume adjusted
to 40 ml.
[0189] Dye Reagent: 5 mg of Ampliflu Red solid added to 480 .mu.l
of detergent solution.
[0190] Inhibitor Reagent: 260 mg sodium azide and 912.92 mg
potassium phosphate dibasic trihydrate is dissolved in water and
the final volume adjusted to 40 ml.
Example 2
Total Lipid Assay
[0191] The K37 dye was dissolved in DMF to a final concentration of
1.0 mM. Next 5% w/v PEG 2000 was dissolved into the dye solution
and 60 nanolitres of the resulting solution was deposited onto a
plastic surface and dried by removing the solvent under vacuum for
1 hour at room temperature in the dark.
Assay Procedure--Utilising Diluted Sample
[0192] The sample to be assayed (plasma) was first diluted 1 part
into 80 parts phosphate buffered saline containing 50 mM Sodium
Octanoate, pH 7.4.
[0193] 5 .mu.l of diluted plasma sample was applied to the dried
dye which spontaneously hydrated. The Total lipid content was
measured by exciting the sample at 440 nm (10 nm bandpass) and
measuring the resulting fluorescence passing through a 495 nm
filter (10 nm bandpass). The total lipid content was determined
empirically by reference to known standards.
Assay Procedure--Utilising Undiluted Sample
[0194] Neat biological samples are assayed by applying the sample
to a borosilicate filter impregnated with anti-HSA antibody located
within a consumable device, which serves to filter and direct the
sample to an appropriate 200 .mu.m deep read area. Pre-coating of
the read area with 3000.times.65 pico-litre droplets of 2 mM K37/5%
(w/v) PEG2000 in DMF per mm.sup.2 facilitates rapid flow of the
sample into the read area and spontaneous partitioning of dye into
the lipoproteins contained within the sample. Subsequent excitation
of the sample at 440 nm (10 nm band pass) generates fluorescence
that can be detected through a 495 nm (10 nm band pass) filter to
allow the total lipid content of the sample to be determined by
reference to suitable standard measurements.
Example 3
HDL Cholesterol Assay
[0195] Nile Red was dissolved in DMF to a final concentration of
0.5 mM. Next 5% w/v PEG 2000 was dissolved into the dye solution
and 60 nanolitres of the resulting solution was deposited onto a
plastic surface and dried by removing the solvent under vacuum for
1 hour at room temperature in the dark.
Assay procedure--Utilising Diluted Sample
[0196] The sample to be assayed (plasma) was first diluted 1 part
into 80 parts phosphate buffered saline containing 50 mM Sodium
Octanoate, pH 7.4.
[0197] 5 .mu.l of diluted plasma sample was applied to the dried
dye which spontaneously hydrated. The HDL cholesterol content was
measured by exciting the sample at 580 nm (10 nm bandpass) and
measuring the resulting fluorescence passing through a 610 nm
filter (10 nm bandpass). HDL cholesterol content was calculated by
use of the algorithm described in the main specification. An
equivalent assay may also be performed utilising whole, undiluted
blood.
Assay procedure--Utilising Undiluted Sample
[0198] Neat biological samples are assayed by applying the sample
to a borosilicate filter impregnated with anti-HSA antibody located
within a consumable device, which serves to filter and direct the
sample to an appropriate 200 .mu.m deep read area. Pre-coating of
the read area with 3400.times.65 pico-litre droplets of 0.5 mM Nile
Red/5% (w/v) PEG2000 in DMF per mm.sup.2 facilitates rapid flow of
the sample into the read area and spontaneous partitioning of dye
into the lipoproteins contained within the sample. Subsequent
excitation of the sample at 580 nm (10 nm band pass) generates
fluorescence that can be detected through a 610 nm (10 nm band
pass) filter to allow the HDL-c content of the sample to be
determined by reference to suitable standard measurements.
[0199] Manipulating the data from the three tests described in
examples 1, 2 and 3 provides the following:
Measurement of total cholesterol--ie test (1) Measurement of total
lipid concentration--ie test (2) Measurement of HDL cholesterol--ie
test (3) Calculated value of triglyceride--ie tests (2) less (1)
Calculated value of VLDL--ie value of triglyceride/2.2 Calculated
value of LDL--ie determined by Friedwald equation
Example 4
Use of PEG to Enhance Lateral Fluid Flow in Near Horizontal
Capillaries
[0200] Glass capillaries 100 mm long and having internal diameters
of 2 mm, 1 mm and 0.5 mm were either left untreated,
detergent-treated or coated with PEG. The detergent treated
capillaries were prepared by washing with a solution of virkon and
Triton X100 5% followed by drying. PEG treated capillaries were
prepared by flowing 5% (w/v) PEG in chloroform through the
capillaries, allowing the surplus to drain followed by drying.
[0201] Treated and un-treated capillaries were fixed in a
near-horizontal position (about 10.degree. upward flow angle) and
the tips of the capillaries were submerged in water. The distance
traveled and flow rate of water moving into each capillary was
measured:
Untreated Capillaries:
[0202] 2 mm--reached 20 mm in .about.30 seconds 1 mm--reached 90 mm
in 15 seconds 0.5 mm--reached end of tube (100 mm) in 18
seconds
Detergent Treated Capillaries
[0203] 2 mm--reached 20 mm in .about.20 seconds 1 mm--reached 90 mm
in 12 seconds 0.5 mm--reached end of tube (100 mm) in 15
seconds
PEG-Coated Capillaries:
[0204] 2 mm--reached 80 mm in .about.20 seconds 1 mm--reached end
in 1-2 seconds 0.5 mm--reached end of tube (100 mm) in 1-2
seconds
[0205] This data demonstrates that capillary fluid flow in
PEG-coated capillaries is approximately four times faster than in
detergent treated capillaries and approximately six times faster
than in untreated capillaries.
[0206] Capillaries were made hydrophobic by treating with a
siliconising agent (dimethyl dichlorosilane) and baking at
120.degree. C. Water did not enter the lumen of these capillaries.
Coating the hydrophobic capillaries with PEG (as above) restored
the fluid flow conditions comparable to PEG coated capillaries
without silation. In some of the experiments where the surface
coating of PEG was discontinuous the fluid flow stopped at the
break in the PEG coating.
Experiment 5
Use of PEG to Enhance Fluid Flow in Vertical Capillaries
[0207] Capillaries having a length of 100 mm and diameters of 2 mm,
1 mm, or 0.5 mm were treated as described above. The capillaries
were fixed in a vertical position and the tips of the capillaries
were submerged in water. The Height reached by water in the
vertical capillary tubes was measured:
Untreated Tubes:
[0208] 2 mm diameter--9 mm 1 mm diameter--22 mm 0.5 mm diameter--51
mm
Detergent Treated Tubes
[0209] 2 mm diameter--10 mm 1 mm diameter--22 mm 0.5 mm
diameter--53 mm
PEG-Coated Tubes
[0210] 2 mm diameter--11 mm 1 mm diameter--25 mm 0.5 mm
diameter--54 mm
[0211] Water did not enter hydrophobic silated capillary tubes at
all. By coating the silated capillaries with PEG the capillary flow
heights were restored and similar to those of unsilated PEG-coated
capillaries.
[0212] The theoretical maximum heights at sea level using the
equation h=2.gamma. Cos .theta./pgr, where h is the height (m);
.gamma. is the surface tension; .theta. the contact angle; .rho. is
the density; g is acceleration due to gravity; and r is the radius
of the tube (m) are:
14 mm at 2 mm ID
28 mm at 1 mm ID
56 mm at 0.5 mm ID
Example 6
Reproducibility of Transfer and Long-Term Stability
[0213] The reproducibility of transfer and long-term stability of
hydrophobic molecule (i.e. dye)/amphipathic polymer mixture was
measured as follows:
[0214] PEG with a molecular weight of 2000 Da was dissolved into a
solution of hydrophobic dye (either Nile Red or K37) in
dimethylformamide (DMF), at a concentration of 5% w/v. PEG/dye
films were made by depositing 25 .mu.l of PEG/dye solution in DMF
in a 5 ml glass vial. The solution was spread over the base of the
vial and then placed in a vacuum chamber for one hour to evaporate
the solvent.
[0215] Reproducibility of transfer was measured by comparing the
fluorescence intensity of dye in DMF added to a lipoprotein
solution with the fluorescence intensity of an identical
lipoprotein solution in which the dye/PEG film was re-dissolved.
Reproducibility was calculated by obtaining the Coefficients of
variation (CVs) of fluorescence intensities from ten dye/PEG films
in lipoprotein solutions.
[0216] Stability was evaluated by laying down films for long-term
storage, and measuring the fluorescence intensity of these films
when re-dissolved in lipoprotein solutions after varying storage
times. Films were stored in the dark under a range of conditions:
in air with no desiccating agent, in air in the presence of silica
gel, and under vacuum in the presence of silica gel. Films were
stored under these conditions at temperatures of both 20 and
37.degree. C.
[0217] The fluorescence intensities for dyes in lipoprotein
solutions dissolved in DMF or in PEG films were determined:
TABLE-US-00001 K37 Nile Red From DMF 268000 297000 From PEG film
269000 296000
[0218] Identical fluorescence intensity readings (within 0.5%) were
obtained for PEG films as were obtained when adding dye dissolved
in DMF. The reproducibility of fluorescence readings from dye/PEG
films with lipoprotein was calculated:
TABLE-US-00002 K37 Nile Red Film 1 6.23E+05 4.27E+05 Film 2
6.25E+05 4.27E+05 Film 3 6.24E+05 4.29E+05 Film 4 6.25E+05 4.26E+05
Film 5 6.24E+05 4.27E+05 Film 6 6.26E+05 4.30E+05 Film 7 6.23E+05
4.30E+05 Film 8 6.25E+05 4.28E+05 Film 9 6.22E+05 4.28E+05 Film 10
6.22E+05 4.26E+05 Mean 6.24E+05 4.28E+05 SD 1370.32 1475.73 CV (%)
0.22 0.34
[0219] CVs of less than 0.5% were obtained for both dyes.
Stability Measurements
TABLE-US-00003 [0220] K37 in K37 in K37 in PEG 20 K37 in PEG 20 K37
in K37 in PEG 37 K37 in PEG DMF 20 PEG 20 C. in air over C. in vac
over PEG 37 C. in air over 37 C. in vac Time C. C. in air silica
gel silica gel C. in air silica gel over silica gel Zero 138630
139320 137241 138624 137440 138317 136942 1 week 137500 137113
137840 138801 137640 136922 136817 4 weeks 134890 134641 133980
134202 135016 135412 134097 8 weeks 135254 136021 135972 136671
135926 136201 135552 % CV 1.31 1.44 1.25 1.56 0.92 0.90 0.98
TABLE-US-00004 NR in NR in NR in PEG 20 NR in PEG 20 NR in NR in
PEG 37 NR in PEG DMF 20 PEG 20 C. in air over C. in vac over PEG 37
C. in air over 37 C. in vac Time C. C. in air silica gel silica gel
C. in air silica gel over silica gel Zero 495197 496130 494590
495760 494118 494241 494829 1 week 495002 495600 494802 494400
494636 493907 494621 4 weeks 493128 492686 493440 493662 494017
492506 492906 8 weeks 493492 494620 494525 493986 493920 492500
493650 % CV 0.21 0.31 0.12 0.19 0.06 0.19 0.18
Conclusions:
[0221] (1) Dyes incorporated into PEG films fully re-dissolve when
reconstituted with aqueous solutions, and give the same
fluorescence intensities as dyes added in organic solvents. (2) The
dye/PEG films are reproducible and films made up in the same way
result in the same fluorescence intensity. (3) The dye/PEG films
are stable for at least 52 weeks when stored under the harshest
storage conditions tested (37.degree. C., no desiccant).
Example 7
Comparison of Amphipathic/Non-Ionic Polymers
[0222] Borosilicate capillaries (100 mm long, 1 mm ID from
Composite Metal Services Ltd--CV1012) were coated by drawing up 7
mm of a 5% polymer solution in various solvents and rocked until
dry. The solvent used was dependent on solubility with chloroform
favoured because of its high evaporation rate.
[0223] Experiments on the coated capillaries were performed in
triplicate and an uncoated capillary was run each time as a
reference. The 4 capillaries were held vertical on a lined
reference card by 2 slots cut into the card for each capillary. The
ends of the capillaries extended exactly 7 mm past the bottom of
the card.
[0224] The experiment was videoed using a Sunkwang High Resolution
Low Lux Color Camera connected to a Winnov 500050G V1000+OV PC card
and the program Videum Capture was used to capture and analyse the
data. The frame rate was set at 5 frames per second (to avoid
dropped frames) unless otherwise stated. The flow liquid (10-4M
Rose Bengal in water) was contained in a flat bottomed watch glass
and the card simply lowered end-on until the bottom of the card
came to rest on the walls of the watch glass. Capillaries were
submerged in the flow liquid to a depth of 2 mm. This produced a
gap of 5 mm to the bottom of the card, 9 mm from the bottom of the
card to the first line and a further 6 mm to the second line up the
card. Flow rates were calculated by counting how many frames
elapsed for the 9 mm of travel between the bottom of the card and
the first line.
TABLE-US-00005 TABLE 1 Performance of polymer coatings/solvent
systems in water column height and capillary flow rate. Average
water STDEV on Average STDEV on Fastest column height column flow
rate average flow rate Coating Solvent mm height mm/s rate mm/s
mm/s PEG2000 Chloroform 17.8 0.76 21.9 3.3 25.7 PEG2000 DMF 19.8
1.89 27.6 28.1 60 PEG2000 Water 13.5 5.77 29.7 37.2 72 PEG6000
Chloroform 19.3 0.76 21.5 4.8 25.7 PEG12,000 Chloroform 16.5 0.87
7.6 3.3 9.5 PEG20,000 Chloroform 15.3 1.04 3.2 1.6 4.9 Triton X100
Water 14.2 1.04 7.9 2.2 10 Carboxymethyl cellulose Water 18.0 * 180
* 180 0,0'-Bis(2-aminoethyl) Chloroform 17.0 3.5 38.8(52.5)
25.0(10.6) 60 polyethylene glycol 2000 PEG methyl ether 5000
Chloroform 17.8 1.26 2.4 0.2 2.6 * = a result from a single
capillary ( ) = a result from 2 out of 3 capillaries as capillary
had a column height below the first line of the card and was
therefore estimated.
[0225] FIG. 5 illustrates the effect of molecular weight of PEG on
vertical flow rate.
[0226] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
* * * * *