U.S. patent application number 15/508182 was filed with the patent office on 2018-11-08 for methods and apparatus for detecting compounds in liquids.
This patent application is currently assigned to Undercover Colors, Inc.. The applicant listed for this patent is Undercover Colors, Inc.. Invention is credited to Tyler Confrey-Maloney, Catherina Gomes, Michael Gorczynski, Stephen Gray, Aly Khalifa, Nicolas Letourneau, Sarah Paluskiewicz, Ronald Smith.
Application Number | 20180321212 15/508182 |
Document ID | / |
Family ID | 59398943 |
Filed Date | 2018-11-08 |
United States Patent
Application |
20180321212 |
Kind Code |
A1 |
Letourneau; Nicolas ; et
al. |
November 8, 2018 |
METHODS AND APPARATUS FOR DETECTING COMPOUNDS IN LIQUIDS
Abstract
Described herein are apparatus and methods for detecting
substances of abuse or other analytes in liquids. For example, the
apparatus and methods described herein can be used for real-time
detection of analytes, such as substances of abuse. The methods
comprise providing a detection area comprising a chromatographic
membrane capable of receiving the liquid and allowing for migration
of the liquid, the chromatographic membrane comprising an
anti-analyte antibody-particle conjugate, an analyte-conjugate
protein at a test line; exposing at least the first location of the
apparatus to the liquid; and determining whether an interaction
between the analyte-conjugate protein and the liquid occurs to
detect the presence of the analyte. The chromatographic membrane
may further comprise an anti-species antibody at a control line.
Specific buffers are disclosed, and these buffers may be used in
the preparation of the apparatus to overcome challenges associated
with miniaturization and challenges associated with exposure to
beverages.
Inventors: |
Letourneau; Nicolas;
(Raleigh, NC) ; Khalifa; Aly; (Raleigh, NC)
; Gorczynski; Michael; (Raleigh, NC) ; Gomes;
Catherina; (Raleigh, NC) ; Smith; Ronald;
(Raleigh, NC) ; Paluskiewicz; Sarah; (Raleigh,
NC) ; Gray; Stephen; (Raleigh, NC) ;
Confrey-Maloney; Tyler; (Raleigh, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Undercover Colors, Inc. |
Raleigh |
NC |
US |
|
|
Assignee: |
Undercover Colors, Inc.
Raleigh
NC
|
Family ID: |
59398943 |
Appl. No.: |
15/508182 |
Filed: |
January 27, 2017 |
PCT Filed: |
January 27, 2017 |
PCT NO: |
PCT/US17/15489 |
371 Date: |
March 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62287677 |
Jan 27, 2016 |
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62287623 |
Jan 27, 2016 |
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62287643 |
Jan 27, 2016 |
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62337603 |
May 17, 2016 |
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62337558 |
May 17, 2016 |
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62337608 |
May 17, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2021/7766 20130101;
G01N 33/525 20130101; G01N 33/53 20130101; G01N 33/146 20130101;
A61F 13/84 20130101; A61B 5/6826 20130101; G01N 33/531 20130101;
G01N 33/54386 20130101; A61F 2013/8473 20130101; G01N 33/54366
20130101; G01N 33/54393 20130101; A61F 13/00055 20130101; G01N
2021/775 20130101; G01N 2001/007 20130101; A61F 2013/429 20130101;
A61F 13/15 20130101; G01N 33/54313 20130101; A61F 2013/422
20130101; A61F 13/42 20130101; G01N 21/78 20130101; G01N 2021/7763
20130101; G01N 33/558 20130101; G01N 33/5306 20130101; G01N 33/94
20130101; G01N 33/52 20130101; G01N 33/9426 20130101 |
International
Class: |
G01N 33/14 20060101
G01N033/14; G01N 21/78 20060101 G01N021/78; G01N 33/53 20060101
G01N033/53; G01N 33/52 20060101 G01N033/52; G01N 33/543 20060101
G01N033/543; G01N 33/558 20060101 G01N033/558; G01N 33/94 20060101
G01N033/94; G01N 33/531 20060101 G01N033/531 |
Claims
1. An apparatus for detecting the presence of an analyte in a
liquid, the apparatus comprising a lateral flow assay capable of
receiving a sample of a beverage and analyzing the beverage for the
presence of the analyte.
2. The apparatus of claim 1, wherein the lateral flow assay
comprises: a sample area, a conjugate area, and a detection area,
wherein at least one of the sample area, conjugate area, or
detection area comprises at least one residual buffer
composition.
3. The apparatus of claim 2, wherein the conjugate area comprises
at least one anti-analyte antibody-particle conjugate or
anti-analyte aptamer-particle conjugate; wherein the detection area
comprises a chromatographic membrane and at least one
analyte-conjugate protein, and wherein the sample area is
configured for receiving a liquid.
4. The apparatus of claim 2, wherein the detection area further
comprises at least one anti-species antibody or anti-species
aptamer.
5. The apparatus of claim 2, wherein the sample area, conjugate
area, and the detection area are located on a single pad.
6. The apparatus of claim 2, wherein at least one of the sample
area, conjugate area, and the detection area is located on a
separate pad.
7. The apparatus of claim 3, wherein the chromatographic membrane
comprises one or more of cellulose, nitrocellulose, polyester
fiber, and/or glass fiber.
8. The apparatus of claim 2, wherein the apparatus further
comprises a cover defining a pattern, wherein the cover is disposed
over the detection area.
9. The apparatus of claim 2, wherein the detection area is
positioned on or within or under a natural fingernail, an
artificial fingernail, a layer of fingernail polish, a fingernail
sticker, a fingernail decal, a sticker, a cup, a drink coaster, a
drink stirrer, a toothpick, a drink ornament, a pencil, or a
pen.
10. The apparatus of claim 2, wherein the residual buffer
composition comprises at least one surfactant.
11. The apparatus of claim 10, wherein the surfactant comprises a
poloxamer, a fatty alcohol, a polyethylene glycol alkyl ether, a
polypropylene glycol alkyl ether, a glucoside alkyl ether,
polyethylene glycol octyl, a glycerol alkyl ester, a phenyl ether,
polyoxyethylene (20) oleyl ether, octylphenol ethoxylate, a
polyethylene glycol alkylphenyl ether, polyethoxylated tallow
amine, N,N-bis[3-(D-gluconamido)propyl]cholamide, polyoxyethylene
(20) cetyl ether, dimethyldecylphosphine oxide, branched
octylphenoxy poly(ethyleneoxy)ethanol, a
polyoxyethylene-polyoxypropylene block copolymer,
t-octylphenoxypolyethoxyethanol, polyoxyethylene (20) sorbitan
monooleate, or a combination thereof.
12. The apparatus of claim 2, wherein the residual buffer
composition comprises at least one buffer salt.
13. The apparatus of claim 12, wherein the buffer salt comprises
monosodium phosphate, disodium phosphate, sodium tetraborate,
Tris(hydroxymethyl)methylaminopropanesulfonic (TAPS),
N-cyclohexyl-2-aminoethanesulfonic acid (CHES),
N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
bis-tris methane (Bis TRIS), tris(hydroxymethyl)aminomethane
(TRIS),
2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic
acid (TES), 2-(N-morpholino)ethanesulfonic acid (YMS),
N-(carbamoylmethyl)iminodiacetic acid (ADA),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), bis tris
propane, piperazine-N,N'-bis(2-ethanesulfonic acid)(PIPES),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),
3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), cholamine
chloride, (3-(N-morpholino)propanesulfonic acid) (MOPS),
N,N-bis(2-hydroxyethyl)taurine (BES),
N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid
(DIPSO), 4-(N-morpholino)butanesulfonic acid (MOBS),
3-[N-rris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid
(TAPSO), acetamidoglycine, triethanolamine (TEA),
piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) POPSO,
4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)
hydrate (HEPPSO),
3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS),
tricine, tris(hydroxymethyl)aminomethane, trometamol (TRIZMA),
glycinamide, glycyl-glycine,
N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
bicine, 2-amino-2-methyl-1-propanol (AMP),
N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid
(CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS),
4-(cyclohexylamino)-1-butanesulfonic acid (CABS), and
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES),
carnitine, gamma-aminobutyric acid, taurine, or salts of amino
acids alanine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, valine, or a combination thereof.
14. The apparatus of claim 2, wherein the sample area comprises a
residual sample area buffer composition, and wherein the residual
sample area buffer composition comprises a potassium salt of a weak
acid and at least one surfactant.
15. The apparatus of claim 2, wherein the conjugate area comprises
a residual conjugate area buffer composition, and wherein the
residual conjugate area buffer composition comprises a Good's
buffer salt and one or more of a protein, an oligomer, a polymer,
and a surfactant.
16. The apparatus of claim 2, wherein the detection area comprises
a residual detection area buffer composition, and wherein the
residual detection area buffer composition comprises a phosphate
salt and one or more of a saccharide, a protein, an oligomer, and a
polymer.
17. The apparatus of claim 1, wherein the lateral flow assay is
configured to detect multiple analytes.
18. The apparatus of claim 2, wherein the residual buffer
composition is configured to reduce the acidity of a test
liquid.
19. The apparatus of claim 2, wherein the residual buffer
composition is configured to increase the viscosity of a test
liquid.
20. The apparatus of claim 2, wherein the residual buffer
composition is configured to substantially reduce or substantially
remove the appearance of colored components of a test liquid.
21. A method of detecting an analyte in a liquid, said method
comprising: providing the apparatus of claim 1; exposing a portion
of the apparatus to the liquid; and observing an indication to
determine presence or absence of the analyte in the liquid.
22. The method of claim 21, wherein the liquid is a beverage.
23. The method of claim 21, wherein the liquid is a food
extract.
24. The method of claim 21, wherein the liquid has a pH from about
4.5 to about 6.8.
25. A method of making an apparatus for detecting the presence of
an analyte in a liquid, the method comprising: applying a buffer
solution to at least one of a conjugate area; a detection area; or
a sample area for receiving a liquid; drying the buffer solution;
applying at least one anti-analyte antibody-particle conjugate or
anti-analyte aptamer-particle conjugate to at least a portion of
the conjugate area; applying at least one analyte-conjugate protein
to at least a portion of the detection area; and assembling the
conjugate pad, detection area, sample area and a wick so that the
sample area is in contact with a portion of the conjugate area,
another portion of the conjugate area is in contact with a proximal
end of the detection area, and the wick is in contact with a distal
end of the detection area.
Description
CROSS-REFERENCE TO PRIORITY APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/287,677, filed on Jan. 27, 2016; U.S.
Provisional Application No. 62/287,623, filed on Jan. 27, 2016;
U.S. Provisional Application No. 62/287,643, filed on Jan. 27,
2016; U.S. Provisional Application No. 62/337,603, filed on May 17,
2016; U.S. Provisional Application No. 62/337,558, filed on May 17,
2016; and U.S. Provisional Application No. 62/337,608, filed on May
17, 2016, each of which is incorporated herein by reference in its
entirety.
FIELD
[0002] Described herein are apparatus and methods for detecting
compounds in liquids. Also described are buffer solutions useful in
the apparatus and methods. For example, the apparatus and methods
described herein can be used for real-time detection of substances
of abuse.
BACKGROUND
[0003] The demand and need for persons to be able to detect
different substances on a real-time basis has increased with
increased frequency of drug use and abuse. In some embodiments, a
need may arise to determine if a drug has been added to a beverage
without the consumer's knowledge, and to make this determination
discretely.
[0004] For example, an increased misuse of various psychotropic
and/or sedating drugs for recreational or criminal purposes has
become more problematic in the present culture. A particularly
troubling form of misuse is the surreptitious introduction of these
drugs into ordinary beverages for the purpose of rendering the
consumer of the beverage disoriented or unconscious. The
unknowingly sedated individual may then be taken advantage of,
e.g., become the victim of robbery or sexual assault.
Drug-facilitated sexual assault has become increasingly common,
particularly among younger members of the population, to the degree
that most universities have warning and prevention programs and
policies in place to prevent drug-facilitated sexual assault.
Conventional apparatus to detect such drugs prior to ingestion
often are insufficient as they may be too cumbersome to use, take
too long to detect the targeted substance, detect only a limited
substance, and lack selectivity and sensitivity to many other
compounds.
[0005] As another example, an increased frequency of diagnoses of
auto-immune disorders or highly sensitive allergies has occurred in
the general population. For example, Celiac's disease, peanut
allergies, lactose allergies or other conditions triggered by
different ingested substances have become more common in the
general population. If the particular harmful substance is ingested
by persons having these types of conditions, significant and severe
consequences for the person may result.
[0006] In view of these trends, conventional testing methods and
devices often are too cumbersome or take too long to evaluate a
particular liquid for a targeted substance. In some embodiments, no
specific miniaturized apparatus for real-time detection of certain
targeted substances or compounds exist. In some embodiments,
beverage components may interfere with testing methods.
SUMMARY
[0007] The terms "invention", "the invention", "this invention" and
"the present invention" used herein are intended to refer broadly
to all of the subject matter described herein and the claims below.
Statements containing these terms do not to limit the subject
matter described herein or limit the meaning or scope of the claims
below. This summary is a high-level overview of various aspects of
the invention and introduces some of the concepts that are further
described in the Detailed Description section below. This summary
is not intended to identify key or essential features of the
claimed subject matter, nor is it intended to be used in isolation
to determine the scope of the claimed subject matter. The subject
matter should be understood by reference to appropriate portions of
the entire specification, any or all drawings, and each claim.
[0008] Disclosed herein are apparatus and methods for detecting the
presence of a targeted substance, analyte, or drug in a liquid. In
some embodiments, the apparatus is a lateral flow device for a
lateral flow assay, whereby a liquid to be analyzed migrates along
a fluid path from a sample area, across a conjugate area, across a
chromatographic membrane, and into a wick. The target substance or
analyte, if present, reacts with an anti-analyte antibody and the
reaction results in a visual indication of whether the target
analyte is present in the liquid. In some examples, aptamers may be
used instead of or in addition to antibodies.
[0009] As one example, the methods and apparatus described herein
can be used for real-time point-of-contact detection of drugs, such
as date rape drugs or other sedating drugs, in beverages or bodily
fluids. In other examples, the methods and apparatus described
herein can be used for real-time point-of-contact detection of
heavy metals in foodstuffs or nutritional supplements; for the
detection of contaminants in cosmetics; or for detection of
contaminants or nutrients in soils. In some embodiments, the
methods and apparatus described herein can be used for real-time
detection of any analyte for which an anti-analyte antibody exists
or may be manufactured.
[0010] Accordingly, described here are viable methods and apparatus
for the safe, real-time detection of targeted substances. Examples
of such target substances include compounds within abused
substances such as drugs, allergens, and biological and
environmental toxins. Non-limiting specific examples of target
substances include benzodiazepines; amine-containing compounds,
including but not limited to, narcotics; alcohol; and other abused
drugs, e.g., club drugs. Current "club drugs" include ketamine,
4-hydroxybutanoic acid (GHB), ephedrine, methamphetamine,
amphetamine, flunitrazepam, 3,4-methylenedioxy-methamphetamine
(MDMA), also known as ecstasy or molly, benzodiazepines such as
clonazepam, tetrahydrocannabinol (THC), and many more. Drugs that
impair memory or sedating drugs such as zolpidem, eszopiclone,
ramelteon, zaleplon, doxepine, triazolam, temazepam, and alprazolam
may be detected.
[0011] Other examples of such target substances include proteins,
sugars, steroids and their metabolites. Still other examples of
target substances include poisons, pesticides, toxins, chemical
warfare agents, environmental poisons, explosives and the starting
materials used to make them. Further, the target substances may
include small molecules or mixtures of small molecules. In some
embodiments, an apparatus may detect multiple analytes.
[0012] Further, a single-analyte or a multi-analyte apparatus may
incorporate a signaling mechanism, which in some embodiments is a
visual indication, a vibration, or a sound that indicates presence
or absence of the target compound. For example, the visual
indication can comprise the appearance, or lack thereof, of a
colored dot, pattern, or region; the printing of words, such as
"SAFE," "OK," "YES," or "NO"; checkmarks; emoticons or symbols such
as a ""; or fluorescence. In some examples, the signaling mechanism
comprises completing lines, logos, patterns or symbols.
[0013] In some embodiments, the apparatus described herein are very
small. For example, the apparatus may have a length of 20 mm or
less, 15 mm or less, 12 mm or 10 mm or less. In some embodiments,
the apparatus comprises a length ranging from about 10 mm to about
150 mm, for example, from about 10 mm to about 25 mm, or from about
10 mm to about 20 mm. In some embodiments, the apparatus can have a
length of about 10 mm or less, 11 mm or less, 12 mm or less, 13 mm
or less, 14 mm or less, 15 mm or less, 16 mm or less, 17 mm or
less, 18 mm or less, 19 mm or less, 20 mm or less, 21 mm or less,
22 mm or less, 23 mm or less, 24 mm or less, 25 mm or less, 26 mm
or less, 27 mm or less, 28 mm or less, 29 mm or less, or 30 mm or
less In some embodiments, the apparatus has a fluid path length
greater than the length of the apparatus.
[0014] In other embodiments, the apparatus may have other
dimensions. The invention, however, overcomes the significant
hurdles associated with the miniaturization of lateral flow assay
technology. For example, miniaturization can lead to an undesirable
increase in the flow rate of the liquid across a chromatographic
membrane. In addition to challenges posed by miniaturization, some
target liquids (e.g., beverages) include acidic components, high
ethanol concentration, high sugar concentrations, and/or other
components that may interfere with accurate test results. In some
examples, the apparatus is miniaturized to be convenient for
discreet use and/or to be wearable. Specific embodiments of
wearable apparatus consistent with the present apparatus and
methods are described and set forth in a PCT patent application
entitled "Wearable Apparatus for Detecting a Target Substance in
Liquids," applied for by Undercover Colors, Inc. and filed on the
same day as the present application, which is incorporated by
reference in its entirety. Additional benefits of miniaturization
of a lateral flow assay include reduced raw material usage, the
ability to pack more assays into shipping containers, having an
increased number of assays multiplexed in a single apparatus,
significantly reducing sample volume, and providing rapid test
results due to shorter flow distances.
[0015] The apparatus and methods described herein overcome
challenges associated with miniaturization and challenges
associated with exposure to beverages and other mixtures. In some
embodiments, the apparatus and methods moderate liquid flow rate by
modifying the chromatographic membrane and/or modifying the
viscosity of the liquid. In some embodiments, buffer solutions may
be used to prepare the apparatus, leaving residual buffer
components at desired locations on the apparatus, so that in use
the buffer components are reconstituted and neutralize acidic
components in a test liquid, slow the progression of the test
liquid through the device, or otherwise facilitate the test method.
In some embodiments, buffers or buffer solutions may be customized
for treatment of various components of an apparatus, such as the
sample area, the conjugate area, and/or the chromatographic
membrane.
[0016] In some embodiments, the buffer solution can comprise a
viscosity of at least 2 centiPoise (cP). In some embodiments, the
buffer or buffer additives increase the viscosity of the liquid
being analyzed, thereby adjusting the flow rate of the liquid
across the chromatographic membrane. In some such embodiments, when
residual buffer components on an apparatus are reconstituted by a
test liquid, the reconstituted buffer solution increases the
viscosity of the test liquid and slows the flow rate of the
liquid.
[0017] In some embodiments, a buffer solution may be utilized in
preparing reagents or in applying reagents to the apparatus. In
some embodiments, the solution or residual buffer composition may
render the test compatible with liquids that may contain an
analyte. In some embodiments, unique buffer components may resolve,
bind, or eliminate incompatible substances, such as acids
(including alpha-hydroxy acids such as lactic acid, malic acid, or
citric acid), found in the liquid. In some examples, buffer
additives may be introduced to components of the apparatus in a
buffer solution or by themselves. In some embodiments, the buffer
liquid may be evaporated after the buffer solution is applied. In
this case, a residual buffer composition is left on the area of the
apparatus that was exposed to the buffer solution.
[0018] The sample pad or area, in some embodiments, is the
component or portion of the apparatus that is exposed to a test
liquid. In some embodiments, a sample pad buffer may neutralize the
test liquid when the liquid is an acidic beverage. In some
embodiments, the sample pad buffer may comprise salts of weak
acids. In some embodiments, the sample pad buffer may be
concentrated to provide sufficient neutralization to the beverage,
even with a miniaturized sample pad.
[0019] In one aspect, a method of detecting an analyte in a liquid
described herein comprises the steps of providing an apparatus
comprising sample pad, a conjugate pad, and a detection layer,
wherein the conjugate pad comprises at least one marker, such as an
anti-analyte antibody-particle conjugate, and wherein the detection
layer comprises an analyte-conjugate protein and a chromatographic
membrane capable of receiving the liquid and allowing for migration
of the liquid; exposing at least the sample pad to the liquid; and
determining whether an interaction between the analyte-conjugate
protein and the liquid occurs to detect the presence of the
analyte. In some embodiments, the detection layer further comprises
an anti-species antibody. In some embodiments, buffers or buffer
solutions may be customized for deposition into or onto the sample
pad, conjugate pad, or detection layer. Thus, in some embodiments,
the sample pad, conjugate pad, or detection layer comprises
residual buffer components. For example, a buffer solution may be
deposited onto a detection layer and then dried to form test and/or
control lines or areas. In some embodiments, the chromatographic
membrane of the detection layer may comprise activated carbon,
silica gel, ionic exchange resins, polyelectrolyte polymers,
hydrogels, and/or size exclusion chromatography matrices. In some
embodiments, the chromatographic membrane may comprise cellulose,
nitrocellulose, glass fiber, similar materials or a combination of
these materials. For the purposes of this application, "detection
layer" and "detection area" may be used interchangeably.
[0020] In some embodiments, the marker comprises an anti-analyte
antibody conjugated to a particle. In other embodiments, the marker
comprises an anti-analyte aptamer conjugated to a particle. For
ease of discussion herein the term anti-analyte antibody-particle
conjugate may be used to refer to either (or both) anti-analyte
antibody-particle conjugates and anti-analyte aptamer-particle
conjugates. The particle may be a nanoparticle, microbead,
macromolecule, small molecule, or other visualization means. A
visualization means may be any composition that contributes to a
visible indication when the anti-analyte antibody-particle
conjugate experiences an interaction, such as with an
analyte-conjugate protein or an anti-species antibody, to develop
color at a test or control line. In some examples, the particle is
a colored particle, which may be a gold nanoparticle, a magnetic
nanoparticle, or a dye-infused polymer microbead. In other
embodiments, the particle may be a fluorescent label conjugate, or
a radiolabel. Optionally, the particle includes carboxyfluorescein,
2,7-dichlorofluorescein, Eosin B, Eosin Y, erythrosine,
fluorescein, fluorescein amidite, fluorescein isocyanate,
merbromin, phloxine B, Rose Bengal, derivatives or salts thereof,
or combinations thereof. The term anti-analyte antibody-particle
conjugate and marker are used interchangeably herein. Unless
otherwise specified, either term encompasses conjugates including
any suitable particles, such as those listed above. In some
embodiments, the marker is present on the conjugate pad, which is
generally positioned between the sample pad and the chromatographic
membrane.
[0021] In some embodiments, the conjugate pad may be pretreated
with a conjugate pad buffer. In some embodiments, the conjugate pad
buffer may be quite concentrated because the conjugate pad may be
very small, such as 4 mm.times.4 mm. In some embodiments, the
conjugate pad buffer may neutralize compounds found in the liquid
that might otherwise interfere with test results. In some examples,
the conjugate pad buffer may comprise organic polyols and/or
amines, polyelectrolyte polymers, surfactants, and combinations
thereof. Further, in some embodiments, the conjugate pad buffer is
formulated to be compatible with the anti-analyte antibody-particle
conjugate, or in other words, the conjugate pad buffer will not
denature the anti-analyte antibody-particle conjugate.
[0022] In some embodiments, the anti-analyte antibody-particle
conjugate is dissolved in a conjugate dilution buffer before being
printed or otherwise deposited on the conjugate pad. For example,
the conjugate dilution buffering agent can comprise organic salts,
proteins, sugars, and combinations thereof.
[0023] In some embodiments, an analyte-conjugated protein deposited
on the chromatographic membrane defines a test line or test area,
and an anti-species antibody deposited defines a control line or
control area. Both the test line and the control line may be
immobilized on the chromatographic membrane. As explained further
below, the "line" does not have to be linear, and may take various
predetermined shapes that inform the user of the presence or
absence of the target substance. In some embodiments, the test line
or lines define a pattern, which may comprise an indication such as
"yes", "no", "safe", "OK", or "". In some embodiments, if the
pattern has been created to detect multiple analytes, only a
certain portion of the pattern may change color. For example, the
word "SAFE" may appear as SAFE, where the color of the letter "A"
has not developed in the cross-bar region. In some examples, the
pattern is formed with a stencil to create letters, symbols, or
words. In some examples, the pattern is formed such that the test
results complete a design, word, symbol, or number.
[0024] In some examples, the liquid comprises a consumable liquid.
For example, the consumable liquid can be beer, cider, an energy
drink, a flavored drink, a fruit drink, liquor or another alcoholic
beverage, milk or a milk-containing beverage, soda, a sports drink,
a vegetable drink, water, wine, or a combination thereof. In some
examples, the liquid comprises a non-consumable liquid. For
example, the non-consumable liquid can be blood, non-potable water,
an organic solvent, potable water, serum, treated waste water,
untreated waste water, urine, vomit, or a combination thereof. The
liquid can comprise a solution, a suspension, or an emulsion. In
some embodiments, the liquid comprises solid particles or ice
suspended therein. In other embodiments, the liquid is used to
extract an analyte from a solid material, such as extracting
allergens from non-liquid food products, prior to detection.
[0025] In some embodiments, the detection layer is positioned on
the surface of an inert substrate. In some examples, the
chromatographic membrane may be pretreated with a chromatographic
membrane buffering agent, which many interchangeably be called a
detection layer buffering agent or a detection area buffering
agent. The chromatographic membrane buffering agent may comprise
proteins, dibasic sodium phosphate, polyelectrolyte polymers,
saccharides, or combinations thereof. In some examples, the
chromatographic membrane may be buffered at a pH ranging from 7 to
8.
[0026] In another aspect, an apparatus described herein for
detecting the presence of an analyte in a liquid comprises a
detection layer. In some embodiments, the apparatus comprises a
sample pad capable of receiving the liquid, a conjugate pad
comprising an anti-analyte antibody-particle conjugate, and a
detection layer comprising a chromatographic membrane allowing for
migration of the liquid and comprising an analyte-conjugate protein
at a test location. In some embodiments, the chromatographic
membrane further comprises an anti-species antibody at a control
location. In some embodiments, the detection layer further
comprises an absorbent, and/or is pre-treated with a desiccant. The
absorbent can include chromatography paper, silica gel, or alumina.
In some examples, the detection layer comprises a lateral flow
assay, which may be multiplexed for testing for the detection of
multiple compounds.
[0027] In some examples, the sample pad is capable of receiving the
liquid, and in some embodiments, the liquid moves from the sample
pad to the conjugate pad to the chromatographic membrane. In some
embodiments, the liquid moves from the chromatographic membrane to
a wick. The wick serves a fluid reservoir to keep fluid from
stalling on the chromatographic membrane. In some embodiments, in
order to miniaturize the assay, the wick is comprised of a folded
layer (e.g., the layer is folded back upon itself). In some
embodiments, the wick is U-shaped or S-shaped. In some cases, the
fluid path in the apparatus may be curved through multiple planes
and/or in multiple directions according to the shape of the
components of the apparatus. As a result, in some examples, the
overall length of the apparatus may be shortened without impeding
the detection ability of the apparatus. In some embodiments, the
apparatus has a fluid path length greater than the length of the
apparatus.
[0028] A particular advantage of miniaturization of a lateral flow
assay is timeliness of test results. For example, a conventional
lateral flow assay with an 80 mm long chromatographic membrane
requires a minimum of 5 minutes to display test results. In
contrast, some embodiments of the miniaturized assays described
herein display test results much faster. For example, a 12 mm
detection layer comprising a residual buffer formulation as
described herein requires only about 30 seconds to display test
results. An additional advantage of a miniaturized lateral flow
assay is reduced test fluid volume. In some examples, a sample
volume of no more than 15 .mu.L is required for an apparatus
described herein, compared to 80 .mu.L for a conventional 80 mm
lateral flow assay. In some embodiments, sample volume is less than
40 .mu.L, less than 30 .mu.L, less than 20 .mu.L, less than 10
.mu.L, or less than 5 .mu.L. In some embodiments test results are
displayed in less than 1 minute, less than 30 seconds, less than 15
seconds, less than 10 seconds, or less than 5 seconds.
[0029] In some embodiments, the apparatus further comprises a cover
over the chromatographic membrane. The cover may comprise one or
more openings to permit gas to escape, or the cover may be
gas-permeable. In some embodiments, the cover is an opaque cover, a
tinted cover, a transparent cover, or a translucent cover. In some
embodiments, the cover defines a stencil pattern, which may
comprise an indication such as "yes", "no", "safe", "OK", or "".
The stencil pattern may be placed over the second position of the
chromatographic membrane. Such a pattern may be helpful to the user
by making the test results easy to understand.
[0030] Optionally, the apparatus can be positioned on, within, or
below an object. In some embodiments, the object can be a
fingernail, an artificial fingernail, a layer of fingernail polish,
a fingernail sticker, a fingernail decal, a sticker, a cup, a drink
coaster, a drink stirrer, a toothpick, a drink ornament, a pencil,
a pen, a ring, a bracelet, a bracelet charm, a necklace, a lanyard
pendant, a coaster, a swizzle stick, or another appropriate object.
In some embodiments, the apparatus may be positioned on skin or on
a fingernail.
[0031] In some embodiments, the miniaturized apparatus has a
thickness of up to 4 mm. In some embodiments, the length of the
apparatus is up to 14 mm. In some examples, the width of the
apparatus is up to 4 mm. In some embodiments, the miniaturized
apparatus is configured to detect the presence of multiple drugs in
many varieties of beer, white wines, red wines, neat liquors, mixed
drinks, soda, fruit juices, and water.
[0032] In some embodiments, an apparatus for detecting the presence
of an analyte in a liquid comprises a conjugate pad comprising a
conjugate area comprising at least one anti-analyte
antibody-particle conjugate or anti-analyte aptamer-particle
conjugate; a detection layer comprising a chromatographic membrane
and at least one analyte-conjugate protein, and a sample area for
receiving a liquid, wherein the sample area is a separate sample
pad or is a portion of the conjugate pad separate from the
conjugate area. In some examples, combining the sample pad and
conjugate pad into a single membrane reduces the number of
components in the apparatus and improves manufacturability, in
particular for a miniaturized lateral flow assay apparatus. In some
examples, at least one of the detection layer, the sample area, or
the conjugate area comprises a residual buffer composition. In
other examples, the apparatus comprises a single pad comprising
separate areas, such as a sample area, a conjugate area, a
detection layer (or chromatographic membrane area), and a wick
area. In still other examples, the apparatus comprises a single pad
comprising separate areas, such as a sample area, a conjugate area,
and a detection layer (or chromatographic membrane area).
[0033] In some embodiments, the detection layer further comprises
at least one anti-species antibody. In some embodiments, the
chromatographic membrane comprises one or more of cellulose,
nitrocellulose, polyester fiber, and/or glass fiber. In some
embodiments, the apparatus further comprises a cover defining a
pattern, wherein the cover is disposed over the detection layer. In
some embodiments, the detection layer is positioned on or within or
under a natural fingernail, an artificial fingernail, a layer of
fingernail polish, a fingernail sticker, a fingernail decal, a
sticker, a cup, a drink coaster, a drink stirrer, a toothpick, a
drink ornament, a pencil, or a pen.
[0034] In some embodiments, the sample area comprises a first
residual buffer composition. The first residual buffer composition
may comprise a potassium salt of a weak acid and at least one
surfactant. In some embodiments, the conjugate area comprises a
second residual buffer composition. The second residual buffer
composition may comprise a Good's buffer salt and one or more of a
protein, an oligomer, a polymer, and a surfactant. In some
embodiments, the detection layer comprises a third residual buffer
composition. The third residual buffer composition may comprise a
phosphate salt and one or more of a saccharide, a protein, an
oligomer, and a polymer.
[0035] In some embodiments, a method of detecting an analyte in a
liquid comprises providing an apparatus as described herein;
exposing a portion of the apparatus to the liquid; and observing a
visual indication to determine presence or absence of the
analyte.
[0036] In some embodiments, a method of making an apparatus for
detecting the presence of an analyte in a liquid comprises (1)
applying a buffer solution to at least one of (a) a conjugate pad
comprising a conjugate area comprising at least one anti-analyte
antibody-particle conjugate or anti-analyte aptamer-particle
conjugate, (b) a detection layer comprising a chromatographic
membrane and an analyte-conjugate protein, or (c) a sample area for
receiving a liquid, wherein the sample area is a separate sample
pad or is a portion of the conjugate pad separate from the
conjugate area; (2) drying the buffer solution; and (3) assembling
the conjugate pad, detection layer, sample area and a wick so that
the sample area is in contact with one portion of the conjugate
area, another portion of the conjugate area is in contact with a
proximal end of the detection layer, and the wick is in contact
with a distal end of the detection layer.
[0037] The details of one or more embodiments are set forth in the
drawings and description below. Other features, objects, and
advantages will be apparent from the drawings, the description, and
from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0038] FIG. 1 is an exploded cross-sectional view of an apparatus
according to some embodiments described herein.
[0039] FIG. 2 is a cross-sectional view of an apparatus according
to some embodiments described herein.
[0040] FIG. 3 shows test results of comparative assays and
inventive assays according to some embodiments described
herein.
[0041] FIG. 4 shows test results of comparative assays and
inventive assays according to some embodiments described
herein.
[0042] FIG. 5 shows test results of comparative assays and
inventive assays according to some embodiments described
herein.
[0043] FIG. 6 shows test results of comparative assays and
inventive assays according to some embodiments described
herein.
DETAILED DESCRIPTION
[0044] The subject matter of embodiments of the present invention
is described herein with specificity to meet statutory
requirements, but this description is not intended to limit the
scope of the claims. The claimed subject matter may be embodied in
other ways, may include different elements or steps, and/or may be
used in conjunction with other existing or future technologies.
This description should not be interpreted as implying any
particular order or arrangement among or between various steps or
elements except when the order of individual steps or arrangement
of elements is explicitly described. The illustrative examples are
given to introduce the reader to the general subject matter
discussed herein and are not intended to limit the scope of the
disclosed concepts. The following sections describe various
additional embodiments and examples with reference to the drawings
in which like numerals indicate like elements and directional
descriptions are used to describe illustrative embodiments but,
like the illustrative embodiments, should not be used to limit the
present invention.
[0045] Unless specifically stated to the contrary, the numerical
parameters set forth in the following specification are
approximations that can vary depending upon the desired properties
sought to be obtained by the present invention. At the very least,
and not as an attempt to limit the application of the doctrine of
equivalents to the scope of the claims, each numerical parameter
should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding
techniques.
[0046] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in its respective testing measurements.
Moreover, all ranges disclosed herein encompass any and all
subranges subsumed therein. For example, a stated range of "1 to
10" includes any and all subranges between (and inclusive of) the
minimum value of 1 and the maximum value of 10; that is, all
subranges beginning with a minimum value of 1 or more, e.g. 1 to
6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to
10.
[0047] Described herein are apparatus and methods, and buffer
solutions for use therein, for detecting a targeted substance. In
some embodiments, the methods and apparatus can detect a targeted
compound in a liquid. In some embodiments, the methods and
apparatus can detect a targeted substance in a solid. As one
example, the methods and apparatus described herein can be used for
real-time point-of-contact detection of drugs, such as date rape
drugs or other sedating drugs, in beverages or bodily fluids. In
other examples, the methods and apparatus described herein can be
used for real-time point-of-contact detection of heavy metals in
foodstuffs or nutritional supplements, contaminants in cosmetics,
contaminants or nutrients in soils, or other analytes of interest.
As another example, the methods and apparatus described herein can
be used for real-time detection of certain proteins or sugars,
e.g., gluten, peanut proteins, or lactose. In some embodiments, the
methods and apparatus described herein can be used for real-time
detection of other materials, for example, bacteria, pathogens,
fungi, metals, or volatile organics and other targeted compounds.
In some examples, sedating drugs or date-rape drugs may be
detected.
[0048] In some embodiments, benzodiazepine drugs may be detected.
Benzodiazepines, or "benzos," are a class of drugs having a
chemical structure containing a benzene ring fused to a diazepine
ring, as shown in formula (1) below. Benzodiazepines have sedating
properties, and thus are used by criminals to incapacitate
victims.
##STR00001##
[0049] Benzodiazapines that may be detected include, but are not
limited to, adinazolam, alprazolam, bentazepam, bretazenil,
bromazepam, brotizolam, camezepam, chlordiazepoxide, cinazepam,
cinolazepam, clobaxam, clonazepam, chorazepate, clotiazaepam,
diazepam, flunitrazepam, lorazepam, lormetazepam, medazepam,
midazolam, nitrazepam, oxaepam, temazepam, and
thielnalprazolam.
[0050] In some embodiments, amine-containing compounds (e.g.
amine-containing drugs) may be detected. An "amine-containing"
compound or drug, as referred to herein, includes species having at
least one primary, secondary, and/or tertiary amine, and/or salts
thereof. The amine formula can be represented by
NR.sup.1R.sup.2R.sup.3, wherein R.sup.1, R.sup.2, and R.sup.3 can
be the same or different from one another, and R.sup.1, R.sup.2 and
R.sup.3 can include, but are not limited to, hydrogen, substituted
or unsubstituted straight-chained or branched C.sub.1-C.sub.6
alkyls (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl),
substituted or unsubstituted C.sub.6-C.sub.10 aryls (e.g., benzyl),
substituted or unsubstituted straight-chained or branched
C.sub.1-C.sub.6 alkanols (e.g., methanol, ethanol, propanol,
butanol, pentanol, hexanol), substituted or unsubstituted
C.sub.6-C.sub.10 aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted C.sub.4-C.sub.8 cycloalkyl, or
combinations thereof, with the proviso that R.sup.1, R.sup.2 and
R.sup.3 cannot all be hydrogen. The amine salts as described herein
can be represented as (HNR.sup.1R.sup.2R.sup.3)+X.sup.-, where
X.sup.- is a counterion and R.sup.1, R.sup.2 and R.sup.3 are as
defined above for amine. An amine-containing compound as described
herein does not include ammonia or uronium compounds or salts
thereof, such as urea and derivatives and salts thereof, e.g., urea
nitrate.
[0051] Examples of amine-containing compounds that may be detected
include, for example, amphetamine, cathinone, cyclobenzaprine,
diphenhydramine, doxylamine, ephedrine, ketamine, lysergic acid
diethylamide (LSD), methamphetamine, 3,4-methylenedioxyamphetamine
(MDA), 3,4-methylenedioxy-methamphetamine (MDMA), methcathinone,
tetrahydrozoline and salts thereof, and combinations thereof.
[0052] In some embodiments, other drugs may be detected by the
apparatus and methods described herein. For example, steroids such
as estrogens, gestogens, androgens, andrenocortical steroids, bile
acids, cardiotonic glycosides and aglycones such as digoxin and
digoxigenin, spaonins and sapogenins, their derivatives and
metabolites may be detected. Barbiturates such as phenobarbital may
be detected. Amphetamines; catecholamines such as ephedrine,
L-dopa, epinephrine, carcene, papverin, and metabolites thereof may
be detected. Alkaloids such as morphine alkaloids may be detected.
Purines such as theophylline, caffeine, and metabolites and
derivative thereof may be detected. Marijuana derivatives such as
cannabinol and tetrahydrocannabinol may be detected. Vitamins such
as A, B (such as B.sub.12), C, D, E, K, folic acid and thiamine may
be detected. Drugs that impair memory or sedating drugs such as
zolpidem, eszopiclone, ramelteon, zaleplon, doxepine, triazolam,
temazepam, and alprazolam may be detected. Antibiotics such as
penicillin, chloromycetin, actinomycetin, tetracycline, terramycin,
and metabolites and derivative thereof may be detected. In
addition, antihistamines, methadone, and other drugs may be
detected.
[0053] The apparatus and methods described herein can detect a
target substance in a liquid. The liquid may comprise a solution, a
suspension, or an emulsion. In some examples, the liquid has solid
particles or ice suspended therein.
[0054] In some embodiments, the liquid comprises a consumable
liquid. For example, the consumable liquid can include beer, cider,
energy drinks, flavored drinks, fruit drinks, liquor or other
alcoholic beverages, milk, milk-containing beverages, soda, sports
drinks, vegetable drinks, water, wine, or combinations thereof. In
some embodiments, the liquid has a high concentration of ethanol.
In some embodiments, the liquid has a high concentration of sugar.
In some embodiments, the liquid is acidic.
[0055] In some examples, the liquid comprises a non-consumable
liquid. For example, the non-consumable liquid can include blood,
non-potable water, organic solvents, potable water, serum, treated
waste water, untreated waste water, urine, vomit, sweat, tears,
reproductive fluids, other bodily secretions, or combinations
thereof.
[0056] If the desired targeted substance is suspected to be in a
non-liquid, for example, in a solid food, a suitable solvent may be
used to extract at least some of the targeted substance, and that
solvent may serve as the liquid tested using apparatus and methods
described herein. Similarly, nutritional supplements, cosmetics, or
soil may be tested for presence of heavy metals or undesirable
chemicals by contacting the solid with a solvent to extract any
target analyte and testing the solvent. Further, soluble air
quality contaminants may be extracted for testing. In some
examples, the extraction employs a solvent or water.
[0057] The apparatus and methods described herein can provide
preliminary forensic analyses that can be of assistance to law
enforcement or forensic experts, for example, by providing quick
confirmation of the presence or absence of a targeted analyte in
the blood, sweat, tears, urine, vomit, or beverage of a person who
may have ingested a target compound. Advantageously, the apparatus
and methods described herein allow for real-time determination of
any of the above-mentioned analytes. The methods described herein
require no expensive equipment or scientific training to identify
presence or absence of an analyte.
[0058] In some embodiments, an apparatus according to embodiments
described herein is a lateral flow device for a lateral flow assay,
whereby a liquid being analyzed migrates along a fluid path from a
sample area, across a conjugate area, and then across a
chromatographic membrane to a wick. The target substance or
analyte, if present, reacts with an anti-analyte antibody, and the
reaction results in a visual indication of whether the target
analyte is present in the liquid.
[0059] I. Buffer Solutions
[0060] Buffer solutions described herein may be applied to parts of
a lateral flow apparatus as described herein to enhance performance
of the apparatus. For example, a buffer solution may render a test
apparatus and method as described herein compatible with liquids
that contain components that otherwise would interfere with the
analysis. Buffer solutions may also be used to slow liquid travel
time across a membrane to ensure sufficient reaction time between
any target analyte and a corresponding anti-analyte antibody or
analyte-conjugate protein.
[0061] The buffer solutions described herein include salts, acids,
proteins, excipients, viscosity modifiers, and/or surfactants. In
this application, "buffer," "buffer solution," and "buffer
formulation" may be used interchangeably to describe a solution
comprising at least one buffering compound and water. Optionally,
the buffer solution may further comprise buffer additives. Buffer
additives are compounds that do not necessarily contribute to the
buffering ability of the buffer solution (e.g., they do not
substantially affect the acid-base chemistry of the buffer
solution). In some embodiments, the buffering compounds may
comprise buffer salts and optionally additional acids or bases,
such as hydrochloric acid or sodium hydroxide. In some embodiments,
buffer additives comprise shielding agents such as proteins, e.g.
Bovine Serum Abumin (BSA); viscosity modifying polymers such as
poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP),
poly(ethylene glycol) (PEG), or oligomers or copolymers thereof;
excipients or stabilizing agents such as saccharides (for example,
dextran, trehalose, maltodextrose); and surfactants such as
non-ionic surfactants (for example, polysorbate 20 or 80, Triton
X-100, Triton X-305, or Pluronic F-68).
[0062] Buffer salts useful in the buffer solutions described herein
include weak acids and bases. The buffer salts may be mono-basic,
di-basic, tri-basic, or have higher-order basicity, depending on
how many protons the buffer salt can accept. For example, a
mono-basic buffer salt will be able to accept one proton, whereas a
di-basic buffer salt can accept two protons. Similarly, acids
useful in the buffer solutions described herein can be mono-protic,
di-protic, tri-protic, and so on, depending on how many protons
they can donate.
[0063] Traditional buffering acids and bases, such as boric acid,
carbonic acid, and phosphoric acid, and their corresponding borate,
carbonate, and phosphate salts may be used in the buffer solutions
described herein. In addition, Good's buffer salts, as described by
Norman Good and colleagues, and similar salts commonly used in
biochemical applications may also be used. Non-limiting examples of
buffer salts useful in the buffer solutions described herein are
monosodium phosphate, disodium phosphate, sodium tetraborate,
tris(hydroxymethyl)methylaminopropanesulfonic (TAPS),
N-cyclohexyl-2-aminoethanesulfonic acid (CHES),
N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
bis-tris methane (Bis TRIS), tris(hydroxymethyl)aminomethane
(TRIS),
2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic
acid (TES), 2-(N-morpholino)ethanesulfonic acid (MES),
N-(carbamoylmethyl)iminodiacetic acid (ADA),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), Bis tris
propane, piperazine-N,N'-bis(2-ethanesulfonic acid)(PIPES),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),
3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), cholamine
chloride, (3-(N-morpholino)propanesulfonic acid) (MOPS),
N,N-bis(2-hydroxyethyl)taurine (BES),
N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid
(DIPSO), 4-(N-morpholino)butanesulfonic acid (MOBS),
3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid
(TAPSO), acetamidoglycine, triethanolamine (TEA),
piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) POPSO,
4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)
hydrate (HEPPSO),
3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS),
tricine, tris(hydroxymethyl)aminomethane, trometamol (TRIZMA),
glycinamide, glycyl-glysine,
N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
bicine, 2-amino-2-methyl-1-propanol (AMP),
N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid
(CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS),
4-(cyclohexylamino)-1-butanesulfonic acid (CABS), and
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). Other
potentially useful buffer salts are salts of amino acids such as:
alanine, arginine, asparagine, aspartic acid, cysteine, glutamic
acid, glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, valine. Further, buffer salts could include carnitine,
gamma-aminobutyric acid, taurine.
[0064] The following examples disclose buffering compounds
according to some embodiments. In some examples, a buffer solution
comprises TRIS (tris(hydroxymethyl)aminomethane) and water. The
concentration of TRIS in the buffer solution can be up to 0.3 M, up
to 0.4 M, up to 0.5 M, up to 0.60 M, up to 0.7 M, up to 0.8 M, or
up to 1.0 M. In some embodiments the TRIS concentration is about
0.5 M.
[0065] In some embodiments, a buffer solution useful in the methods
and apparatus described herein comprises a dibasic salt, a
monobasic salt, and water. In some embodiments, the dibasic salt
and monobasic salt have a common anion. For example, the common
anion may be a phosphate anion, although any other known dibasic
anion may be used. The cation may be any monovalent or divalent
cation, such as potassium, sodium, or calcium. In some examples,
the buffer solution comprises sodium phosphate monobasic and/or
sodium phosphate dibasic.
[0066] In other embodiments, a buffer solution useful in the
methods and apparatus described herein comprises one or more
phosphate salts, one or more chloride salts, or a combination
thereof, and water. Examples of useful chloride salts include
sodium chloride and/or potassium chloride. Optionally, the one or
more phosphate salts include disodium phosphate and/or potassium
dihydrogen phosphate. Optionally, the salts used to prepare the
phosphate buffer solution are hydrates. The hydrate may be, for
example, a monohydrate, a dihydrate, a trihydrate, a tetrahydrate,
a pentahydrate, a hexahydrate, or a heptahydrate. In some
embodiments, the disodium phosphate used to prepare the phosphate
buffer may be disodium phosphate heptahydrate
(Na.sub.2HPO.sub.4.7H.sub.2O). A phosphate salt may be present in
the buffer in an amount of up to 25 mM, up to 25 mM, up to 50 mM,
up to 100 mM, up to about 200 mM, from about 5 to about 15 mL, from
about 20 to about 25 mM, from about 40 to about 60 mM, or from
about 80 to about 120 mM.
[0067] In some embodiments a buffer solution useful in the methods
and apparatus described herein comprises a borate salt and water.
Optionally, the borate salt is a hydrate. As described above, the
hydrate may be, for example, a monohydrate, a dihydrate, a
trihydrate, a tetrahydrate, a pentahydrate, a hexahydrate, or a
heptahydrate. For example, the borate salt used to prepare the
borate buffers can be tetraborate heptahydrate
(Na.sub.2B.sub.4O.sub.7.10H.sub.2O). Borate salts may be present in
the buffer in amounts of up to 25 millimolar, up to 50 mM, or up to
100 mM.
[0068] In some embodiments, a buffer solution useful in the methods
and apparatus described herein comprises one or more carboxylates
such as succinate and citrate and water. In some embodiments, a
buffer solution comprises a monovalent cation carbonate, such as
potassium carbonate (K.sub.2CO.sub.3) or divalent cation carbonate,
such as calcium carbonate (CaCO.sub.3). For example, potassium
carbonate may be present in the buffer solution in amounts of up to
500 mM, up to 750 mM, up to 1.0 M, up to 1.1 M, up to 1.2 M, up to
1.3 M, up to 1.4 M, or up to 1.5 M.
[0069] In some embodiments, a buffer solution useful in the
apparatus and methods described herein includes acids or bases in
addition to any weak acid or base buffering compounds. These
additional acids and bases may be used to adjust the final pH of
the buffered solutions. In some embodiments, hydrochloric acid
(HCl) or sodium hydroxide (NaOH) may be used as such additional
acid or base, although other acids or bases could be used
instead.
[0070] The following examples disclose buffer additives according
to some embodiments. In some embodiments, a buffer solution useful
in the methods and apparatus described herein may include one or
more shielding agents. In some embodiments a shielding agent is a
protein. Non-limiting examples of shielding agents include gelatin,
casein, and Bovine Serum Albumin (BSA). In some examples, a buffer
solution may comprise BSA in any amount from less than 1 percent to
more than 10 percent based on the weight of the buffer solution. In
some embodiments, the buffer solution comprises BSA in about 0.1%,
about 0.2 wt %, about 0.5 wt %, about 1 wt %, about 3 wt %, about 5
wt %, about 8 wt %, about 10 wt %, about 12 wt %, or about 15 wt %
based on the weight of the buffer solution.
[0071] In some embodiments, a buffer solution useful in the
apparatus and methods provided herein may include at least one
excipient (e.g., one, two, three, four, or more excipients). Useful
excipients include, but are not limited to, saccharides and amino
acids. Useful saccharides include, for example, monosaccharides and
disaccharides, such as but not limited to sucrose, mannitol,
sorbitol, lactose, dextrose, fructose, glucose, maltose and
combinations thereof. In some examples, the buffers are
substantially free of saccharides other than sucrose (for example,
the buffers are substantially free of non-sucrose polyols).
Substantially free of non-sucrose polyols means including less than
0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, or 0%
of non-sucrose polyols based on the weight of the buffer.
[0072] In some embodiments, buffer solutions useful in the
apparatus and methods provided herein comprise viscosity modifiers.
Suitable viscosity modifiers include, but are not limited to,
saccarides, such as sucrose, and polymers, such as poly(vinyl
alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), poly(ethylene
glycol) (PEG), or oligomers or copolymers thereof. Any molecular
weight polymer or oligomer may be used, provided the polymer or
oligomer is soluble in the buffer solution. In some embodiments,
poly(vinylpyrrolidone)-40 (PVP-40) is used to increase the
viscosity of the buffer solution. In some embodiments, the
viscosity modifier is present at up to 0.1 wt %, up to 0.2 wt %, up
to 0.3 wt %, up to 0.4 wt %, up to 0.5 wt %, up to 0.6 wt %, up to
0.7 wt %, up to 0.8 wt %, up to 0.9 wt %, up to 1.0 wt %, up to 2.0
wt %, up to 3.0 wt %, up to 4.0 wt %, or up to 5.0 wt % based on
the total weight of the buffer solution.
[0073] In some embodiments, buffer solutions useful in the
apparatus and methods provided herein comprise at least one
detergent or surfactant. Detergent and surfactant refer to a
substance having both a hydrophilic moiety and a hydrophobic
moiety. Useful surfactants include ionic and non-ionic surfactants.
In some examples, Triton X-35, Triton X-100, and/or Pluronic F-68
is optionally included as the non-ionic surfactant in a buffer
solution. One or more surfactants can be present in the buffers,
optionally in an amount of less than 1% by weight based on the
weight of the buffer. For example, the surfactant(s) can be present
in the buffers in an amount of up to 3%, up to 2%, up to 1%, up to
0.75%, up to 0.5% by weight, up to 0.25% by weight, up to 0.1% by
weight, or up to 0.05% by weight (e.g., 0.25% by weight or 0.5% by
weight).
[0074] In some embodiments, the surfactant may be one or more
non-ionic surfactant such as fatty alcohols, polyethylene glycol
alkyl ethers, polypropylene glycol alkyl ethers, glucoside alkyl
ethers, polyethylene glycol octyl, glycerol alkyl esters, phenyl
ethers (such as Triton X-100), polyoxyethylene (20) oleyl ether
(such as Brig 98), octylphenol ethoxylate (such as Triton X-305),
polyethylene glycol alkylphenyl ethers, polyethoxylated tallow
amine, N,N-bis[3-(D-gluconamido)propyl]cholamide, polyoxyethylene
(20) cetyl ether, dimethyldecylphosphine oxide, branched
octylphenoxy poly(ethyleneoxy)ethanol, a
polyoxyethylene-polyoxypropylene block copolymer,
t-octylphenoxypolyethoxyethanol, polyoxyethylene (20) sorbitan
monooleate, alkyl polyglycosides, polysorbates (such and Tween), or
poloxamers (such Synperonics, Pluronics, or Kolliphor).
[0075] In some embodiments, the surfactant may be an anionic
surfactant such as 2-acrylamido-2-methylpropane sulfonic acid,
ammonium lauryl sulfate, ammonium perfluorononanoate, docusate,
perfluorobutanesulfonic acid, perfluorononanoic acid,
perfluorooctanesulfonic acid, perfluorooctanoic acid, sodium alkyl
sulfate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate,
sodium laurate, sodium laureth sulfate, sodium lauroyl sarcosinate,
sodium myreth sulfate, sodium nonanoyloxybenzenesulfonate, sodium
pareth sulfate, or sodium stearate.
[0076] In some embodiments, the surfactant may be a cationic
surfactant such as behentrimonium chloride, benzalkonium chloride,
benzethonium chloride, cetrimonium bromide, cetrimonium chloride,
dimethyldioctadecylammonium bromide, dimethyldioctadecylammonium
chloride, lauryl methyl gluceth-10 hydroxypropyl dimonium chloride,
octenidine dihydrochloride, N-oleyl-1,3-propanediamine,
stearalkonium chloride, or tetramethylammonium hydroxide.
[0077] In some embodiments, the surfactant may be a zwitterionic
surfactant such as CHAPS detergent, cocamidopropyl betaine,
cocamidopropyl hydroxylsultaine, lauryldimethylamine oxide, and
Sodium lauroamphoacetate. These surfactant examples are
non-limiting, as other nonionic, anionic, cationic, or zwitterionic
surfactants may be used.
[0078] Buffer solutions provided herein may be used to pretreat one
or more specific areas of an apparatus to deposit buffering
compounds and buffer additives in one or more desired locations of
the apparatus. In some embodiments, a buffer solution is applied to
the respective portion of the apparatus as a solution or
suspension, then the liquid portion of the buffer solution is
removed (e.g. by evaporation) leaving a residual buffer component
comprising one or more residual buffering compounds and, any
residual buffer additives present in the buffer solution. The
residual buffer component includes any buffer components that
remain after the liquid medium (e.g. water) is removed. As a test
liquid traverses the apparatus, it comes into contact with the
deposited buffer components and any residual buffer additive and
dissolves them, essentially reconstituting the buffer solution. The
term "reconstituted" does not imply that the buffer solution formed
in the test liquid has the same concentration as the buffer
solution used to pre-treat the apparatus or that the solutions are
identical. The reconstituted buffer solution may have a different
concentration than the buffer solution used to pre-treat the
apparatus.
[0079] For example, a first buffer solution may be applied to a
sample area to deposit buffering compounds and buffer additives
selected to neutralize or counteract beverage components that might
interfere with a test result. Another buffer solution may be
applied to the chromatographic membrane to increase the viscosity
of the beverage or liquid, for example to slow its migration across
the chromatographic membrane. In some embodiments specific
combinations of buffer solutions may be used in an apparatus where
a first buffer solution is applied to the sample area, a second
buffer solution is applied to the chromatographic membrane, and the
first and second buffer solutions are different. Such combinations
of buffer solutions can be used synergistically to improve the
performance of the apparatus and methods across a wide range of
test liquids.
[0080] In some embodiments, specific combinations of neutralizing
agents, buffering agents, and surfactants are used synergistically
to improve the performance of the assay across a wide range of
sample matrices. Neutralizing agents can be used alone or in
combination with buffering agents to improve assay performance
across a diverse set of test liquids. Neutralizing reagents may
include traditional buffering agents, such as Good's buffer salts,
and other acidic or basic components which treat the sample prior
to the sample encountering the detection means. Neutralizing
reagents may consist of carboxylate salts such as sodium citrate or
potassium carbonate. Buffering reagents create a stable and
consistent environment for the detection means to function within
and may consist of ionic or zwitterionic buffer salts. Buffering
agents alone may not provide adequate neutralization of all sample
types. Neutralizing agents alone may be too acidic or basic to be
compatible with the detection means. For example, one potential
combination of neutralizing agent and buffering agent is potassium
carbonate (0.1 to 3M) and tris (0.1M to 3M), respectively, at any
combination of neutralizing and buffering agent concentrations
within the specified ranges. In some embodiments, the ratio of
neutralizing agent to buffering agent is 2:1.
[0081] The neutralizing agent may be located in an assay component
such as the sample pad or area which is separate from the buffering
agent located in the conjugate pad or area. In some cases, the
neutralizing agent is K.sub.2CO.sub.3 (0.1 to 3M) or other
carboxylate salt. In some cases, the buffering agent is Tris (0.1M
to 3M) or other Good's buffer agent. Separation of the neutralizing
agent from the conjugate pad is of particular importance when the
neutralizing agent is not compatible with the antibody-particle
conjugate as is the case with K.sub.2CO.sub.3 and antibody-gold
nanoparticle conjugates. The neutralizing agent may deposited on
the same assay component but in a separate area from the detection
means. In some cases, the neutralizing agent is K.sub.2CO.sub.3
(0.1 to 3M) or other carboxylate salt. In some cases, the buffering
agent is Tris (0.1M to 3M) or other Good's buffer agent.
[0082] In some embodiments, certain combinations of non-ionic
surfactants are particularly useful for ensuring an apparatus
described herein is compatible with a wide range of test liquids.
These non-ionic surfactants may be used alone or in conjugation
with neutralizing and buffering agents. In some examples, a first
non-ionic surfactant is Pluronic F68 (0.1% to 2%) or other
poloxamer and a second non-ionic surfactant is Triton X-100 (0.1%
to 2%) or other polyethylene oxide phenyl ether at any combination
of concentrations within the stated ranges for each compound.
Buffer formulations and residual buffer formulation may comprise a
first and a second non-ionic surfactant at any combination of
concentrations within the stated ranges for each surfactant. The
non-ionic surfactants may be located in the conjugate pad. The
non-ionic surfactants may be located in the sample pad. One
non-ionic surfactant may be located in the sample pad and one
non-ionic surfactant may be located in the conjugate pad.
[0083] In some embodiments, combinations of neutralizing agents,
buffering agents, and non-ionic surfactants were found to improve
assay performance. For example, a useful combination includes the
neutralizing agent K2CO3 (0.1 to 3M), buffering agent Tris (0.1M to
3M), the non-ionic surfactant Triton X-100 (0.1 to 2%), and a
second non-ionic surfactant Pluronic F68.
[0084] In some embodiments a buffer solution described herein
comprises a dibasic salt and water. In some embodiments, the buffer
solution comprises sodium phosphate monobasic and sodium phosphate
dibasic. In one specific example, a buffer solution comprises
sodium phosphate monobasic in an amount of 5-20 grams per liter and
sodium phosphate dibasic in an amount of 540-80 grams per liter. In
some embodiments, the water is molecular biology reagent grade
water. The buffer solution is prepared by placing 0.7-0.9 liter of
water in a container, adding the prescribed amounts of monobasic
and dibasic salts, and then adding water to one liter. The pH of
the buffer solution is adjusted to the desired pH using NaOH or HCl
as needed, and the buffer solution is filtered using a micron
filter.
[0085] In some embodiments, the monobasic salt may be present in
11.4, 11.2, 11.0, 10.8, 10.6, 10.4, 10.2, 10.0, 9.8, 9.6, 9.4, or
9.2 grams times the batch volume. In some embodiments, the dibasic
salt may be present in 70, 65, 60, 55, 50, or 45 grams times the
batch volume. If salts other than sodium phosphate monobasic and
sodium phosphate dibasic are used, these amounts may be adjusted as
understood by one of skill in the art. In some embodiments, the
buffer solution has a pH of 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8, or any
number in this range. A sodium phosphate monobasic/sodium phosphate
dibasic buffer solution is useful for applying reagents to an
apparatus. Specifically, in some embodiments, a sodium phosphate
monobasic/sodium phosphate dibasic buffer solution may be used as
an elution buffer to desalt an antibody that has been stored.
[0086] A buffer solution described herein may comprise a borate
salt, boric acid, and water. In some embodiments, the buffer
solution may comprise a borate salt, boric acid, BSA, water, and
optionally sodium hydroxide or hydrochloric acid. In some examples,
the buffer solution comprises sodium tetraborate decahydrate in an
amount of 5-20 grams per liter of buffer solution and boric acid in
an amount of 0.5-2 grams per liter of buffer solution. In some
embodiments, BSA is present in an amount of 5-20 grams per liter of
buffer solution. In some embodiments, the water is molecular
biology reagent grade water. In some embodiments, the buffer
solution is prepared by measuring the dry reagents into a
container, and adding water to final volume. In some embodiments,
the pH of the buffer solution is adjusted to the desired pH using
NaOH or HCl as needed. In some examples the buffer solution is
filtered using a 0.2 micron filter.
[0087] In some embodiments, the sodium tetraborate decahydrate may
be present in 16, 14, 12, 11.8, 11.6, 11.4, 11.2, 11.0, 10, 9, or 8
grams per liter of buffer solution, or any number within this
range. In embodiments, the boric acid may be present in 2.0, 1.8,
1.6, 1.4, 1.2, 1.0, 0.8, 0.6, or 0.4 grams per liter of buffer
solution, or any number within this range. In some examples, BSA
may be present at 12, 11, 10, 9, or 8 grams per liter of buffer
solution. In some embodiments, the buffer solution has a pH of 9.5,
9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6, or 8.5, or any number
within this range. This borate buffer solution is useful for
applying reagents to an apparatus. Specifically, in some
embodiments, this borate buffer solution may be used as a
conjugation blocking buffer in the preparation of an
antibody-particle conjugate.
[0088] A buffer solution described herein may comprise a borate
salt, boric acid, and water. In some embodiments, the buffer
solution may comprise borate salt, boric acid, BSA, water, and
optionally sodium hydroxide or hydrochloric acid. In some examples,
the buffer solution comprises sodium tetraborate decahydrate in an
amount of 5-20 grams per liter of buffer solution and boric acid in
an amount of 0.5-20 grams per liter of buffer solution. In some
embodiments, BSA is added in an amount of 50-200 grams per liter of
buffer solution. In some embodiments, the water is molecular
biology reagent grade water. In some embodiments, the buffer
solution is prepared by measuring the dry reagents into a
container, and adding water to final volume. In some embodiments,
the pH of the buffer solution is adjusted to the desired pH using
NaOH or HCl as needed. In some examples the buffer solution is
filtered using a 0.2 micron filter.
[0089] Other borate buffers comprising different concentrations of
BSA may also be useful. In other embodiments, the sodium
tetraborate decahydrate may be present in 16, 14, 12, 11.8, 11.6,
11.4, 11.2, 11.0, 10, 9, or 8 grams per liter of buffer solution.
In some other embodiments, the boric acid may be present in 2.0,
1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, or 0.4 grams per liter of buffer
solution, or any number within this range. In some other examples,
BSA may be present at 120, 110, 100, 90, or 80 grams per liter of
buffer solution. In some other embodiments, the buffer solution has
a pH of 9.5, 9.4, 9.3, 9.2, 9.1, 9.0, 8.9, 8.8, 8.7, 8.6, or 8.5,
or any number within this range. This other borate buffer solution
is useful for applying reagents to an apparatus. Specifically, in
some embodiments, this buffer solution may be used as a conjugate
dilution buffer in applying an antibody-particle conjugate to an
apparatus.
[0090] A buffer solution described herein may comprise a dibasic
salt and water. In some embodiments, a useful buffer solution
comprises sodium phosphate monobasic and sodium phosphate dibasic;
one or more of a saccharide, a protein, and a viscosity modifier;
one or more of sucrose, BSA, and poly(vinylpyrrolidone)-40
(PVP-40); and optionally sodium hydroxide or hydrochloric acid. In
some examples, the buffer solution comprises sodium phosphate
monobasic in an amount of 0.1-0.5 grams per liter of buffer
solution and sodium phosphate dibasic in an amount of 0.5-3 grams
per liter of buffer solution. In some examples, the buffer solution
further comprises 0.5-2 g sucrose per liter of buffer solution,
0.5-2 g BSA per liter of buffer solution, and 1-5 g PVP-40 per
liter of buffer solution. In some embodiments, the water is
molecular biology reagent grade water. In some embodiments, the
buffer solution is prepared by adding the prescribed amounts of
monobasic and dibasic salts, adding the sucrose, BSA, and PVP-40,
and then adding water to final volume. In some embodiments, the pH
of the buffer solution is adjusted to the desired pH using NaOH or
HCl as needed. In some examples the buffer solution is filtered
using a 0.2 micron filter.
[0091] In some embodiments, the monobasic salt may be present in
0.3, 0.2, or 0.1 grams per liter of buffer solution. In some
embodiments, the dibasic salt may be present in 2, 1.8, 1.6, 1.4,
1.2, 0.8, 0.6, or 0.5 grams per liter of buffer solution. If salts
other than sodium phosphate monobasic and sodium phosphate dibasic
are used, these amounts may be adjusted as understood by one of
skill in the art. Sucrose may be present in 2, 1, or 0.5 grams per
liter of buffer solution. BSA may be present in 2, 1, or 0.5 grams
per liter of buffer solution. PVP-40 may be present in 4, 2, or 1
grams per liter of buffer solution. In some embodiments, the buffer
solution has a pH of 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8, or any number
in this range. This buffer solution is useful for slowing the flow
of a liquid in an apparatus. Specifically, in some embodiments,
this buffer solution may be used as a pretreatment buffer solution
for a chromatographic membrane in an apparatus.
[0092] A buffer solution described herein may comprise a Good's
buffer salt and water. In some embodiments a buffer solution
comprises a Good's buffer salt, a protein, a viscosity modifier,
optionally one or more surfactants, and optionally one or more
additional acids or bases. In some examples, the buffer solution
comprises TRIS. In some embodiments, the buffer solution comprises
TRIS, BSA, PVP-40, optionally one or more of Triton X-100 and
Pluronic F-68, optionally sodium hydroxide or hydrochloric acid. In
some examples, the buffer solution comprises TRIS in an amount of
50-200 grams per liter of buffer solution. In some examples, the
buffer solution further comprises 20-40 g BSA per liter of buffer
solution, and 5-20 g PVP-40 per liter of buffer solution. In some
embodiments, the buffer solution further comprises 1-5 g Triton
X-100 per liter of buffer solution and 2-10 g Pluronic F-68 per
liter of buffer solution. In some embodiments, the water is
molecular biology reagent grade water. In some embodiments, the
buffer solution is prepared by adding the prescribed amounts of
TRIS, adding the BSA, and PVP-40, Triton X-100 and Pluronic F-68,
and then adding water to final volume. In some embodiments, the pH
of the buffer solution is adjusted to the desired pH using NaOH or
HCl as needed. In some examples the buffer solution is filtered
using a 0.2 micron filter.
[0093] In some embodiments, the TRIS may be present in about 150,
140, 130, 120, 115, 110, 100, 90, or 80 grams per liter of buffer
solution. BSA may be present in 40, 35, 32, 30, 28 or 25 grams per
liter of buffer solution. PVP-40 may be present in about 20, 15,
12, 10, 8 or 55 grams per liter of buffer solution. Triton X-100
may be present in about 5, 4, 3, 2.8, 2.5, 2.3, or 2 grams per
liter of buffer solution. Pluronic F-68 may be present in about 8,
7, 6, 5.5, 5, 4.5, 4, 3.5, 3, or 2 grams per liter of buffer
solution. In some embodiments, the buffer solution has a pH of 7.6,
7.8, 8.0, 8.2, or 8.4, or any number in this range. This buffer
solution is useful for pretreating a component of an apparatus.
Specifically, in some embodiments, this buffer solution may be used
as a pretreatment buffer solution for a conjugate pad in an
apparatus.
[0094] A buffer solution described herein may comprise a
carboxylate and water. In some examples, the buffer solution
comprises potassium carbonate. Optionally, the buffer solution may
comprise one or more surfactants. In some embodiments, the buffer
solution comprises a carboxylate (such as potassium carbonate),
water, optionally one or more surfactants, and optionally one or
more additional acids or bases. In some embodiments, the surfactant
is Triton X-305. In some examples, the buffer solution comprises
potassium carbonate in an amount of 50-200 grams per liter of
buffer solution. In some examples, the buffer solution further
comprises 1-10 g Triton X-305 per liter of buffer solution. In some
embodiments, the water is molecular biology reagent grade water. In
some embodiments, the buffer solution is prepared by adding the
prescribed amounts of potassium carbonate and Triton X-305 to a
container, and then adding water to final volume. In some
embodiments, the pH of the buffer solution is adjusted to the
desired pH using NaOH or HCl as needed. In some examples the buffer
solution is filtered using a 0.2 micron filter.
[0095] In some embodiments, the potassium carbonate may be present
in about 150, 140, 138, 136, 130, 120, 110, 100, or 80 grams per
liter of buffer solution. Triton X-100 may be present in about 5,
4, 3.8, 3.6, 3.4, 3, 2.5, or 2 grams per liter of buffer solution.
In some embodiments, the buffer solution has a pH of 7.4, 7.2, 7.0,
6.8, or 4.4, or any number in this range. This buffer solution is
useful for pretreating a component of an apparatus. Specifically,
in some embodiments, this buffer solution may be used as a
pretreatment buffer solution for a sample pad in an apparatus.
[0096] II. Methods of Detecting Targeted Compounds
[0097] Methods of detecting targeted compounds, including
substances of abuse and/or drugs, are described herein.
[0098] Examples of non-limiting methods of detecting an analyte
according to embodiments described herein include providing an
apparatus comprising a sample pad, a conjugate pad, a detection
layer comprising a chromatographic membrane, and a wick. The
apparatus is described in greater detail in Section III. The
chromatographic membrane of the invention is capable of receiving
the liquid being tested and also allows for migration of the liquid
through the chromatographic membrane, in some embodiments by
capillary action. The sample pad is exposed to the liquid, for
example by direct contact with the liquid, the liquid migrates from
the sample pad to the conjugate pad (or from the sample area to the
conjugate area), and then the liquid advances through the conjugate
pad (or area), and then through the chromatographic membrane. As
the liquid advances through the apparatus, the conjugate pad (where
the anti-analyte antibody-particle conjugate is located) is exposed
to the liquid, and the anti-analyte antibody-particle conjugate
becomes at least partially dissolved in the liquid.
[0099] In some embodiments, aptamers may be used instead of or in
addition to antibodies. For ease of discussion the term antibodies
is used throughout this application, but throughout the
specification should be understood to encompass both antibodies and
aptamers.
[0100] The method further includes determining whether an
interaction between the anti-drug antibody-particle conjugate and
the liquid occurs to detect the presence of the analyte. The
determining step comprises monitoring the test line (where the
analyte-conjugate protein is located) to observe whether the test
line develops color, and optionally whether a control line develops
color, as described below. Thus, the determining step comprises
observing a visual indication to determine presence or absence of
the analyte.
[0101] If the liquid contains an analyte that matches the
anti-analyte antibody of the anti-analyte antibody-particle
conjugate, the analyte will bind to the anti-analyte antibody
portion of the conjugate, and because the anti-analyte portion of
the conjugate is bound to the analyte, the anti-analyte antibody
portion cannot not bind to the analyte-conjugate protein, and no
color will be deposited at the test line. However, if the liquid is
substantially free from an analyte that matches the anti-analyte
antibody of the anti-analyte antibody-particle conjugate, the
anti-analyte antibody-particle conjugate will bind to the
analyte-conjugate protein and color will be deposited at the test
line.
[0102] Optionally, the chromatographic membrane may comprise an
anti-species antibody at a control line. Regardless of whether an
analyte is bound to the anti-analyte antibody-particle conjugate,
the control line will develop color when the anti-analyte
antibody-particle conjugate is drawn by the liquid to the control
line.
[0103] Using a competitive (indirect) immunoassay format, a result
indicating that no analyte is present consists of two lines (test
and control lines are visible) while a result indicating that
analyte is present consists of one line (control line is visible)
in some examples. In other examples where a control line is not
employed, a result indicating that no analyte is present consists
of one line (test line is visible) while a result indicating that
analyte is present consists of no line. Areas of color deposition
are not limited to lines, and may comprise symbols or patterns. The
phrases "test line, test location, test pattern, test symbol, and
test area" may be used interchangeably. The phrases "control line,
control location, control pattern, control symbol, and control
area" may be used interchangeably. Alternately, a direct
immunoassay format may be used, where the visible presence of a
test line indicates the target analyte is present.
[0104] In some embodiments, the method comprises observing a visual
indication or signal mechanism as to whether a particular compound
is present. For example, the indication can comprise the appearance
of a colored dot, pattern, or region, the absence of any appearance
of a colored region, the printing of words, such as "SAFE," "OK,"
"YES," or "NO," checkmarks, emoticons or symbols such as a ","
fluorescence, vibration, or sounds. In some examples, the signaling
mechanism comprises completing lines, logos, patterns or symbols.
In some embodiments, if the pattern has been created to detect
multiple analytes, only a certain portion of the pattern may change
color. For example, the word "SAFE" may appear as SAFE, where the
color of the letter "A" has not developed in the cross-bar
region.
[0105] The method described herein does not rely on the observation
or measurement of color change of the anti-analyte
antibody-particle conjugate to detect the presence of an analyte in
a liquid. The method described herein does not rely on other
techniques, such as electrophoresis. The method relies on observing
color deposition (or lack thereof) at the test and/or control
lines, area, or patterns, or regions.
[0106] Any of the apparatus described in the section below may be
used in the methods described herein.
[0107] III. Apparatus
[0108] Certain embodiments described herein provide an apparatus
for detecting the presence of a targeted substance, analyte, or
drug in a liquid, wherein the apparatus comprises a sample pad, a
conjugate pad, and a detection layer, and in some cases, a wick. In
some embodiments, the detection layer can detect the presence of a
particular substance upon receiving a liquid to be tested for the
particular targeted substance. For example, the sample pad can be
exposed to the liquid in question and then the apparatus may be
monitored by a user to determine whether there is a particular
interaction between the detection layer and the liquid to indicate
the presence of the targeted substance. In some embodiments, the
particular substance is a benzodiazepine or an amine-containing
compound. In other embodiments, the particular substance is a
protein or sugar.
[0109] In other examples, the apparatus comprises a single pad
comprising separate areas, such as a sample area, a conjugate area,
a detection layer (or chromatographic membrane area), and a wick
area. In still other examples, the apparatus comprises a single pad
comprising separate areas, such as a sample area, a conjugate area,
and a detection layer (or chromatographic membrane area).
[0110] In some embodiments, the apparatus can be configured to
minimize, substantially reduce, or eliminate backflow. This
backflow or potential flow of components from the apparatus to the
test liquid may be undesirable, especially for testing of
consumable liquids. In some embodiments, the potential
backflow/reverse flow may comprise the test liquid and chemicals
from the apparatus. To address the potential for backflow, in some
embodiments, the detection layer may further comprise an untreated
pad at the sample port or opening in the top layer to substantially
eliminate backflow. The untreated pad may minimize, substantially
reduce, or eliminate potential flow of material back to the test
liquid due to saturation of the untreated pad upon introduction of
the apparatus into the test liquid. Once introduced into the test
liquid, the saturated untreated pad may serve as a constraint on
backflow by minimizing the gradient and motive force of flow from
the sample pad to the test liquid. This constraint of the saturated
untreated pad may help ensure that essentially none of the chemical
additives or buffers from the apparatus come in contact with the
test liquid. In some examples, preventing backflow can be achieved
by using an untreated sample pad and/or by designing the apparatus
to encase the components of apparatus (other than the untreated pad
and/or sample pad) in a plastic housing.
[0111] In some embodiments, the apparatus is a lateral flow device
for a lateral flow assay, whereby a liquid to be analyzed migrates
along a fluid path from a sample area, across a conjugate area,
across a chromatographic membrane, and into a wick. The target
substance or analyte, if present, reacts with an anti-analyte
antibody and the reaction results in a visual indication of whether
the target analyte is present in the liquid. In some examples,
aptamers may be used instead of or in addition to antibodies.
Lateral flow assays typically have a fluid path along the length of
the apparatus. In some examples disclosed herein the length of the
fluid path is the same as the length of the apparatus. In other
examples, the length of the fluid path is greater than the length
of the apparatus, Although the term "lateral flow" is used
throughout this specification, in some cases the fluid path may
vary in the x-y plane or in the z direction in order to achieve
detection results in an apparatus with a confined length. In such
embodiments, the length of the fluid path typically is greater than
the length of the apparatus.
[0112] An apparatus for detecting the presence of a targeted
substance in a liquid comprises a sample pad; a conjugate pad that
includes a binder-particle conjugate, such as an anti-analyte
antibody-particle conjugate; and a chromatographic membrane,
optionally including an analyte-conjugate protein. In some
embodiments, the sample pad and the conjugate pad are formed from a
single material comprising a sample area and a conjugate area; the
sample area and the conjugate area do not overlap. In some
embodiments, the anti-analyte antibody-particle conjugate can be
included in the conjugate pad by depositing a composition
comprising an anti-analyte antibody-particle conjugate on the
chromatographic membrane from a conjugate dilution buffer. The
analyte-conjugate protein can be included in the chromatographic
membrane at a test location by depositing a composition comprising
an analyte-conjugate protein on the chromatographic membrane in a
line or desired pattern. Optionally, an anti-species antibody can
be included in the chromatographic membrane at a control location
by contacting the chromatographic membrane with a composition
comprising an anti-species antibody.
[0113] The wick serves a fluid reservoir to keep fluid moving
through the chromatographic membrane. In some embodiments, in order
to miniaturize the assay, the wick is comprised of a folded layer
(e.g., the layer is folded back upon itself). In some embodiments,
the wick is U-shaped or S-shaped. In some cases, the fluid path in
the wick may be curved through multiple planes and/or in multiple
directions according to the shape of the wick. As the fluid flows
from the chromatographic membrane to the wick, it continues along a
fluid path within the wick. In some examples, the fluid path
through the wick is not aligned with the path of the fluid through
the chromatographic membrane, as the wick may direct the fluid path
to the sides of and/or around or under the chromatographic
membrane.
[0114] In some embodiments, the apparatus can be configured to
direct flow of a liquid through the detection layer (or
chromatographic membrane) in a generally horizontal orientation,
e.g., substantially along a single horizontal plane from a first
end of the detection layer to the second end of the detection
layer. In other embodiments, the detection layer can be configured
to direct flow of a liquid through the detection layer in a
generally vertical orientation, e.g., substantially through a
plurality of horizontal planes, for example, from the bottom of the
detecting layer to the top of the detecting layer, resulting in the
length of the fluid path being greater than the length of the
apparatus.
[0115] In some examples, as a result of the length of the fluid
path being greater than the length of the apparatus, the length of
the apparatus may be shortened without impeding the detection
ability of the apparatus. In some cases, the length of the fluid
path is from 5-10% greater than the length of the apparatus, from
10-20% greater than the length of the apparatus, from 20-30%
greater than the length of the apparatus, from 30-40% greater than
the length of the apparatus, from 50-75% greater than the length of
the apparatus, from 75-100% greater than the length of the
apparatus, or from 100-200% greater than the length of the
apparatus.
[0116] The apparatus can be positioned on the surface of an object.
In some examples, the apparatus can be positioned on, integrated
in, or incorporated in an object. In other examples, the apparatus
can be positioned below, or below the surface of, an object.
Suitable objects include, for example, a natural fingernail, an
artificial fingernail, a layer of fingernail polish, a fingernail
sticker, a fingernail decal, a cup, a drink coaster, a drink
stirrer, a toothpick, a drink ornament (e.g., an umbrella), a
pencil, a pen, a test strip, a sticker, a decal, a nail decal, a
mesh nail wrap, or any other appropriate surface or structure. In
other embodiments, the apparatus may be positioned directly on
skin, such as on a finger.
[0117] In some embodiments, the apparatus comprises a thickness
ranging from about 0.2 micrometers (.mu.m) to about 5 millimeters
(mm). In some embodiments, the apparatus comprises a thickness
ranging from about 20 .mu.m to about 5 mm. In some embodiments, the
detection layer can have a thickness of about 0.6 .mu.m or less, 1
.mu.m or less, 10 .mu.m or less, 25 .mu.m or less, 50 .mu.m or
less, 100 .mu.m or less, 1 mm or less, 2 mm or less, 3 mm or less,
4 mm or less, or 5 mm or less.
[0118] In some embodiments, the apparatus comprises a length
ranging from about 0.5 mm to about 15 mm. In some embodiments, the
apparatus comprises a length ranging from about 1 mm to about 10
mm, or from about 3 to about 8 mm. In some embodiments, the
detection layer can have a length of about 0.4 mm or less, 0.5 or
less, 1 mm or less, 2 mm or less, 3 mm or less, 4 mm or less, 5 mm
or less, 6 mm or less, 7 mm or less, 8 mm or less, 9 mm or less, 10
mm or less, 12 mm or less, or 15 mm or less.
[0119] In some embodiments, the apparatus comprises a width ranging
from about 0.5 mm to about 5 mm. In some embodiments, the apparatus
comprises a width ranging from about 1 mm to about 10 mm, or from
about 3 to about 8 mm. In some embodiments, the detection layer can
have a width of about 0.4 mm or less, 0.5 or less, 1 mm or less, 2
mm or less, 3 mm or less, 4 mm or less, 5 mm or less, 6 mm or less,
7 mm or less, 8 mm or less, 9 mm or less, or 10 mm or less.
[0120] In some embodiments, the apparatus can be laminated to
provide protection from the external environment without
compromising the integrity of the test by permitting gas
permeability during use.
[0121] Turning now to the Figures, FIG. 1 is an exploded
cross-section view of an apparatus 100 according to one embodiment
described herein. Apparatus 100 comprises a sample pad 110, a
conjugate pad 120, a detection layer 130 and an absorption pad or
wick 160. The sample pad 110 is adjacent to a first portion 122 of
the conjugate pad 120 so that in use a liquid is absorbed into the
conjugate pad 120 from the sample pad 110. A second portion 124 of
the conjugate pad is adjacent to the chromatographic membrane 130
at a proximal end 132 of the chromatographic membrane 130 so that
in use a liquid is absorbed into the chromatographic membrane at
the proximal end 132 and moves through the chromatographic membrane
toward the distal end 134 of the chromatographic membrane 130.
Between the proximal and distal ends the chromatographic membrane
includes at least one test line 140 where an analyte-conjugated
protein is deposited and at least one control line 150 where an
anti-species antibody is deposited. The apparatus also comprises an
absorption pad or wick 160 adjacent to the chromatographic membrane
130 so that in use liquid is absorbed into the wick from the
chromatographic membrane 130. In some embodiments multiple test
lines may be present to test for a plurality of targeted
substances. Optionally, the apparatus may have a clear cover layer
170.
[0122] FIG. 2 is a cross-section view of an apparatus 200 according
to one embodiment described herein. Apparatus 200 comprises a
combined sample pad-conjugate pad 250 which has a sample area 252
and a conjugate area 254 that do not overlap. Apparatus 200 further
comprises a detection layer 230 and a wick 260. The conjugate area
254 of the sample-conjugate pad 250 is adjacent to a first portion
232 of the detection layer 230 so that in use a liquid is absorbed
into the sample-conjugate pad 250, migrates into a proximal end 232
of the chromatographic membrane 230, and flows toward the distal
end 234 of the chromatographic membrane 230. The apparatus also
comprises an absorption pad or wick 260 adjacent to the distal end
234 chromatographic membrane 230 so that in use a test liquid is
absorbed into the wick from the chromatographic membrane 230.
Optionally, the apparatus may have a clear cover layer 270.
[0123] In some embodiments, the sample pad is pretreated with a
sample pad buffer solution. Pretreatment involves contacting the
sample pad with the sample pad buffer solution and then drying the
sample pad. The dried sample pad comprises a residual buffer
composition. In use, when a test liquid contacts the residual
buffer composition, the buffer is reconstituted in the test liquid.
In some embodiments, the sample pad may be pretreated with a sample
pad buffer solution consisting essentially of biocompatible
materials so that any potential interaction of the sample pad
buffer ingredients with a beverage will not introduce material
unsuitable for ingestion into the beverage. In some embodiments,
the sample pad buffer aids in neutralization of the acidic
beverages, and may also reduce the impact of other components, such
as high sugar content, on test results. For purposes of this
application, "sample pad buffer solution" and "sample area buffer
solution may be used interchangeably.
[0124] In some embodiments, the sample pad buffer solution
comprises potassium carbonate and/or calcium carbonate. In some
embodiments, the sample pad buffer solution comprises a calcium
salt of a weak acid.
[0125] In some embodiments the sample pad buffer solution comprises
potassium carbonate. In some embodiments, potassium carbonate is
present in the sample pad buffer solution in a concentration of
from 200 to 3000 millimolar (mM), from 500 to 2000 mM, from 750 to
1500 mM, from 0.8 to 1.2 molar (M), from 0.9 to 1.1 M, or about 1
M. In some embodiments, potassium carbonate is present in the
sample pad buffer solution in a concentration of at least 800 mM,
at least 900 mM, at least 1.0 M, at least 1.1 M, at least 1.2 M, or
at least 1.3 M. Optionally, in some embodiments, the sample pad
buffer solution may comprise hydroxide, borate, and/or bicarbonate
salts.
[0126] In some examples, the conjugate pad may be pretreated with
conjugate pad buffer solution comprising a stabilizing agent and/or
a shielding agent. Examples of stabilizing agents include
saccharides such as sucrose, fructose, and trehalose. Examples of
shielding agents include gelatin, casein, and BSA.
[0127] In some examples, the conjugate pad buffer solution
comprises a buffer salt. Tris(hydroxymethyl) aminomethane (Tris)
was found to be particularly effective at neutralizing, and
therefore mitigating negative effects, of acidic beverages on test
results. In some embodiments, the conjugate pad buffer solution has
a pH from 7.5 to 8.5, from 7.75 to 8.25, from 7.9 to 8.1, or about
7.8, about 8.0, or about 8.2. Other buffer solutions, such as
sodium borate buffer solutions, in some examples could not be
prepared in concentrations high enough to provide the necessary
buffering capacity for the conjugate pad. An usually high
concentration of buffer solution is required due to the a very
limited area (such as a 4.times.4 mm conjugate pad) to be
impregnated with the conjugate pad buffer solution, which is dried,
and then is reconstituted as the sample liquid flows through the
conjugate pad. In some embodiments, the conjugate pad buffer
solution comprises Tris in a concentration of from 0.4 to 0.6 molar
(M), from 0.423 to 0.575 M, from 0.45 to 0.55 M. In some
embodiments, the conjugate pad buffer solution comprises Tris in a
concentration of about 0.4 M, about 0.5 M, or about 0.6 M.
[0128] In some embodiments, the conjugate pad buffer solution
further comprises one or more of a protein, a
poly(vinylpyrrolidone), and a surfactant. Some non-limiting
examples of surfactants are Aerosol OT, benzalkonium chloride,
BRIJ35, BRIJ 52, BRIJ98, CHEMAL-LA-9, Cremophore EL, IGEPAL CA210,
Merpol A, Pluronic F68, Pluronic F127, Pluronic L64, Silwet L7600,
Surfactant 10G, Synperonic F108,
2,4,7,9,-tetramethyl-5-decyn-4,7-diol ethoxylate, Tergitol,
Tetronic 90R4, Triton X-45, Triton X 100, Triton X-305, Tween-20,
Tween-60, and Tween-80. In some embodiments, the protein is BSA and
the poly(vinylpyrrolidone) is PVP-40. In some embodiments, the
surfactant comprises Triton X-100, Triton X-305 and/or Pluronic
F68. In some examples, BRIJ-98 was found to reduce background
coloration caused by beverages. Not intending to be bound by
theory, BRIJ-98 causes precipition of many colored red wine
components. In some embodiments, the residual buffer composition
can substantially reduce or substantially remove the appearance of
colored components of a test liquid. "Substantially reduce or
substantially reduce" means that the color of the test liquid does
not interfere with the indication of test results.
[0129] In some embodiments, the conjugate pad buffer composition is
compatible with the anti-analyte antibody-particle conjugate, for
example, the conjugate pad buffer composition, when reconstituted
by the test liquid, does not denature the anti-analyte
antibody-particle conjugate. Not intending to be bound by theory,
this may be due to the high pH of the conjugate pad buffer and the
resulting neutralization of acid in test liquids such as
beverages.
[0130] The conjugate is not bound to the conjugate pad; rather, it
is adhered or immobilized, so that the conjugate is released to the
test liquid and flows with the test liquid across the
chromatographic membrane. In some embodiments of the invention, the
conjugate pad and the sample pad are formed from a single piece of
material, such as nitrocellulose or a woven mesh of glass fibers or
polyester fibers.
[0131] In some embodiments, the anti-analyte antibody-particle
conjugate may be prepared from a commercially available monoclonal
antibody and a gold or dye nanoparticle material. In some
embodiments, the commercially available monoclonal antibody may be
desalted using a spin column to replace the antibody storage buffer
with an antibody desalting buffer. In some embodiments, the
antibody desalting buffer comprises a sodium phosphate buffer. A
sodium phosphate antibody desalting buffer may have a concentration
of 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, or 150 mM, or
from 50-150 mM, or from 75-125 mM, or from 90-110 mM. In some
embodiments, the antibody desalting buffer may have a slightly
basic pH, such as 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,
or 8.2. When the antibody has been desalted and is dissolved in the
antibody desalting buffer, it may be conjugated with a gold or dye
nanoparticle material. In some embodiments, the gold or dye
nanoparticle material may be dissolved or suspended in water with
the pH adjusted to slightly basic, such as pH 7.05, 7.1, 7.15, 7.2
or 7.25 by the addition of HCl or NaOH.
[0132] In some embodiments, after the antibody is dissolved in the
antibody desalting buffer and the gold or dye nanoparticle is
dissolved or suspended in slightly basic water, the two components
are mixed, and a conjugation blocking buffer may be added to the
resulting mixture. In some embodiments, the conjugation blocking
buffer comprises sodium tetraborate, boric acid, and BSA. In some
embodiments, the blocking buffer can have a sodium tetraborate
concentration of 10 mM, 25 mM, 50 mM, 75 mM, or 100 mM, or from
10-110 mM, or from 25-75 mM, or from 45-55 mM. In some embodiments,
the conjugation blocking buffer may have a basic pH, such as 8.2,
8.4, 8.6, 8.8, 9.0, 9.2, 9.4, or 9.6. The resulting anti-analyte
antibody-particle conjugate may be isolated as a pellet by
centrifugation.
[0133] In some embodiments, the anti-analyte antibody-particle
conjugate is applied to the conjugate pad from a conjugate dilution
buffer which may be used to reconstitute the anti-analyte
antibody-particle conjugate pellet. In some embodiments, the
anti-analyte antibody-particle conjugate is dissolved in a
conjugate dilution buffer prior to depositing the anti-analyte
antibody-particle conjugate on the conjugate pad.
[0134] In some embodiments, the conjugate dilution buffer comprises
a salt of a weak acid, a protein, and optionally, excipients such
as saccharides. In some embodiments, the conjugate dilution buffer
comprises a borate, such as sodium tetraborate, boric acid, BSA,
and optionally, sucrose and/or trehalose. In some examples, the
conjugate dilution buffer comprises 40-60 mM, 45-55 mM or about 50
mM borate, 1-3 weight percent (wt. %) BSA (e.g. 1 wt. %, 2 wt %, or
3 wt. %), 3-7 wt. % trehalose (e.g. 4 wt. %, 5 wt %, or 6 wt. %),
and 15-25 wt. % sucrose (e.g. 18 wt. %, 20 wt %, or 22 wt. %). In
some embodiments the conjugate dilution buffer has a pH of from 8.5
to 9.5, (e.g. 8.7, 9.0, or 9.3). In some embodiments, the pH of the
conjugate dilution buffer is titrated.
[0135] In some examples, the conjugate dilution buffer comprises
40-60 mM, 45-55 mM or about 50 mM borate, 1-3 weight percent (wt.
%) BSA (e.g. 1 wt. %, 2 wt %, or 3 wt. %), and sucrose and/or
trehalose are added after the conjugate has been dissolved in the
conjugate dilution buffer. In some embodiments, sucrose is added in
an amount of 10%, 20%, or 30%, where 20% is determined, for
example, according to the formula (volume of conjugate in conjugate
dilution buffer)(20 g sucrose)/(100 mL). Similarly, in some
embodiments, trehalose is added in an amount of 3%, 5%, or 7%,
where 5% is determined, for example, according to the formula
(volume of conjugate in conjugate dilution buffer)(5 g
trehalose)/(100 mL).
[0136] In some embodiments, any of the buffer solutions or buffer
additives described herein has a viscosity of at least 10 cP. In
other embodiments, the buffer or buffer additives can comprise a
viscosity of at least 0.5 cP, at least 1 cP, at least 5 cP, at
least 20 cP, at least 30 cP, at least 60 cP, at least 80 cP, at
least 100 cP, at least 1000 cP, at least 20,000 cP, or at least
50,000 cP. In other embodiments, the buffer or buffer additives can
comprise a viscosity of between 0.5-2 cP, between 2-5 cP, between
5-20 cP, between 20-30 cP, between 30-60 cP, between 1-100 cP,
between 2-80 cP, between 5-50 cP, between 10-1000 cP, between
10-20,000 cP, or between 10-50,000 cP. In some embodiments, the
chromatographic membrane may be pretreated with a chromatographic
membrane buffer solution comprising 10-20 mM or 12-18 mM or about
15 mM sodium phosphate, 0.5-1.5 wt. % or 0.7 to 1.3 wt. %, or about
1 wt. % sucrose, 0.05-0.15 wt. % or 0.7 to 1.2 wt. % or about 1 wt.
% BSA, and 0.1-0.3 wt. % or about 0.2 wt. % PVP-40. In some
embodiments, the chromatographic membrane buffer has a pH from 7.1
to 7.5. In some embodiments, the chromatographic membrane buffer
may have a pH of 7.1, 7.2, 7.3, 7.4, or 7.5, or from 7.1-7.5, or
from 7.2 to 7.4, or from 7.25-7.35.
[0137] The detection layer of certain embodiments described herein
can provide an visual indication or signal mechanism to a user as
to whether a particular compound is present. For example, the
visual indication can comprise the appearance, or lack thereof, of
a colored dot, pattern, or region; the printing of words, such as
"SAFE," "OK," "YES," or "NO"; checkmarks, emoticons or symbols such
as a "," fluorescence, vibration, or sounds.
[0138] In some embodiments, the detection layer can provide an
indication to a user by electrochemical detection. In some examples
the detection layer can provide a device-aided quantitation, for
example with the aid of smartphone application or other device. In
some embodiments, the detection layer can provide a
semi-quantitative or quantitative indication of the analyte in the
liquid. In some examples, the indication of the analyte present in
the liquid comprises a pattern of lines. For example, in one
embodiment, 6 test lines may be placed on the detection layer. When
a direct immunoassay format is used, if the analyte is present in
certain quantities, one test line may develop color. If the analyte
is present in certain, larger quantities, additional test lines may
develop color. If the analyte is present in an even greater amount,
all test lines may develop color.
[0139] Alternately, when an indirect immunoassay format is used, if
the analyte is present in certain quantities, one test line may
develop color. If the analyte is present in certain, lesser
quantities, additional test lines may develop color. If the analyte
is present in an even lesser amount, all test lines may develop
color. In some examples, during the assembly of the apparatus, the
test line is deposited from a buffer solution in a low-to-high
concentration gradient in the direction of liquid flow across the
chromatographic membrane to create a 2 mm test line with no leading
edge effect. "Leading edge effect" refers to the formation of a
color gradient at the test or control lines where the darkest area
is along the leading edge of the line and the color of the line
become progressively lighter as it nears the distal edge. The
"leading edge" of the test/control line is the side of the line
which contacts the fluid first as it flows through the
membrane.
[0140] In some embodiments, the detection layer comprises a
thickness ranging from about 50 microns to about 1000 microns. In
some embodiments, the detection layer comprises a thickness ranging
from about 200 microns to about 400 microns. In some embodiments,
the detection layer can have a thickness of about 100 microns or
less, 200 microns or less, 400 microns or less, 600 microns or
less, 800 microns or less, or 1000 microns or less.
[0141] In some embodiments, the detection layer can be configured
to detect the presence of a plurality of targeted substances. In
some embodiments, the detection layer can be physically divided to
permit the detecting of multiple analytes without inferring with
the detection of another analyte. As another example, a detection
layer can be multiplexed with certain components to test for
multiple analytes on a single detection layer. In some embodiments,
the apparatus can include a plurality of discrete, physical
sections positioned adjacent to each other to make up a single
detection layer. For example, a plurality of matrices can be
positioned side by side with each matrix configured to test for the
presence of a different compound in a liquid.
[0142] In some embodiments, the apparatus comprising a detection
layer can also include at least one additional layer. In some
embodiments, the apparatus can include at least one of a top
laminate layer, a bottom laminate layer, and a removable layer. In
some embodiments, the apparatus can include any combination of
layers described herein.
[0143] In some examples, an apparatus described herein includes a
specific combination of residual buffer formulations that can
render the apparatus compatible with a wide range of test fluids.
For example, a first residual buffer formulation may be used at a
location near the beginning of the liquid flow path, for example
the sample area, to interact with components in the test fluid that
could be detrimental to test results, such as acids, alcohol,
and/or colorants, and a second residual buffer formulation may be
used at a separate location further down the liquid flow path to
buffer the test liquid near a certain pH so as not to denature
proteins involved in the assay.
[0144] In addition, a specific combination of buffer formulations
can allow combining multiple detection means (such as using two or
more marker-test line combinations) for detecting multiple
analytes, whereas in the absence of the specific combination of
residual buffer formulations the different detection means would
not be compatible with the same scope of test fluids. In one
example, in the absence of a particular residual buffer
formulation, a first detection means for detecting a first analyte
is only compatible with test fluids A and B, and a second detection
means for detecting a second analyte is only compatible with test
fluids B and C. In that case, the first and second means could not
be used in combination to simultaneously detect the first and
second analytes in fluids A and C. But a single apparatus including
an appropriate combination of residual buffer formulations is
compatible with fluids A, B, and C, and can detect the first and
the second analytes in all three fluids. This "multiplexing" is
useful for the detection of multiple analytes with may require
different detection means (such as different antibodies, aptamers,
or markers) with a single apparatus. In some examples, an apparatus
described herein may detect the presence of both benzodiazepines
and ketamines.
[0145] Anti-Analyte Antibody Particle Conjugates
[0146] In some embodiments, the anti-analyte antibody-particle
conjugate described herein includes an anti-analyte antibody, a
colored nanoparticle, and optionally one or more additional
components.
[0147] An antibody is a large, Y-shaped protein produced mainly by
plasma cells that is used by the immune system to identify and bind
pathogens such as bacteria and viruses. Aptamers are
oligonucleotide or peptide molecules that bind to a specific target
molecule. The anti-analyte antibody or anti-analyte aptamer is an
antibody or aptamer that is formed to bind a specific analyte such
as a drug molecule. As understood by a person of ordinary skill in
the art, immunoassay techniques employ this "lock and key"
approach, using a specific antibody or aptamer to bind with a
targeted substance.
[0148] Particles to enhance visibility and/or detection are
frequently attached to the anti-analyte antibody to increase
visibility and/or detection of the anti-analyte antibody. Useful
particles may comprise colored compounds or fluorescent compounds.
In some embodiments, the particle comprises fluorescein, Rose
Bengal, derivatives and salts thereof, or combinations thereof, or
similar fluorophores. In other embodiments, nanoparticles are used
as particles. The nanoparticle may be any colored nanoparticle such
as gold and/or dye-infused polymer microbeads.
[0149] The anti-analyte antibody is joined, or conjugated, to the
particle by a linker. The linker may be any linker that is not
inconsistent with the objectives of the current invention.
Non-limiting examples of linkers are
(N-(.kappa.-maleimidoundecanoyloxy) sulfosuccinimide ester)
(sulfo-KMUS),
(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate-
)) (LC-SMCC), N-(e-maleimidcCaproyloxy)-N-HydroxySuccinimide ester
(KMUs), succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP),
(succinimidyl-6-((b-maleimidopropionamido)hexanoate) (SMPH),
4-(N-maleimidomethyl)cyclohexanecarboxylic acid
N-hydroxysuccinimide ester (SMCC),
4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid
3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC),
(N-succinimidyl (4-iodoacetyl)aminobenzoate) (SIAB),
N-(.gamma.-maleimidobutyryloxy)sulfosuccinimide sodium salt
(sulfo-GMBS), 4-maleimidobutyric acid N-hydroxysuccinimide ester
(GMBS), (succinimidyl 3-(bromoacetamido)propionate) (SBAP),
N-(2-carboxyethyl)maleimide (BMPA),
N-.alpha.-maleimidoacet-oxysuccinimide ester (AMAS), N-succinimidyl
3-(acetylthio)propionate (SATP), 3-maleimidobenzoic acid
N-hydroxysuccinimide ester (MBS),
(m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester) (sulfo-MBS),
N-.epsilon.-maleimidocaproic acid (EMCA),
N-(.epsilon.-maleimidocaproyloxy)succinimide, N-succinimidyl
6-maleimidocaproate (EMCS), succinimidyl-(4-vinyl sulfone)benzoate
(SVSB), N-succinimidyl 3-maleimidopropionate (BMPS), or
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).
In some embodiments, the gold nanoparticles are functionalized with
compounds such as 6-mercaptohexanoic acid, 8-mercaptooctanoic acid,
12-mercaptododecanoic acids, or other mercapto-carboxylic acids.
Reagents such as EDC may be used to couple the antibodies to these
reagents. Polymer microbeads may be functionalized with similar
carboxylic acids or amines.
[0150] The anti-analyte antibody-particle conjugate can be present
in an anti-analyte antibody-particle conjugate composition in an
amount of from about 0.05 wt. % to about 10 wt. % (e.g., from about
0.5 wt. % to about 8 wt. % or from about 0.8 wt. % to about 5 wt.
%). For example, the anti-analyte antibody-particle conjugate can
be present in the anti-analyte antibody-particle conjugate
composition in an amount of about 10 wt. % or less, about 9.5 wt. %
or less, about 9 wt. % or less, about 8.5 wt. % or less, about 8
wt. % or less, about 7.5 wt. % or less, about 7 wt. % or less,
about 6.5 wt. % or less, about 6 wt. % or less, about 5.5 wt. % or
less, about 5 wt. % or less, about 4.5 wt. % or less, about 4 wt.
%, about 3.5 wt. % or less, about 3 wt. % or less, about 2.5 wt. %
or less, about 2 wt. % or less, about 1.5 wt. % or less, about 1
wt. % or less, or about 0.5 wt. % or less. In some embodiments, the
anti-analyte antibody-particle conjugate can be present in the
anti-analyte antibody-particle conjugate composition in an amount
of about 0.05 wt. %, about 0.1 wt. %, about 0.15 wt. %, about 0.2
wt. %, about 0.25 wt. %, about 0.3 wt. %, about 0.35 wt. %, about
0.4 wt. %, about 0.45 wt. %, about 0.5 wt. %, about 0.55 wt. %,
about 0.6 wt. %, about 0.65 wt. %, about 0.7 wt. %, about 0.75 wt.
%, about 0.8 wt. %, about 0.85 wt. %, about 0.9 wt. %, about 0.95
wt. %, about 1 wt. %, about 1.5 wt. %, about 2 wt. %, about 2.5 wt.
%, about 3 wt. %, about 3.5 wt. %, about 4 wt. %, about 4.5 wt. %,
or about 5 wt. %.
[0151] A buffering agent can be present in the conjugate dilution
buffer such that pH of the total marker composition is from about 5
to about 9 (e.g., from about 6 to about 8 or from about 6.5 to
about 7.5). For example, the buffering agent can be added to the
composition to provide a pH of about 5, about 5.5, about 6, about
6.5, about 7, about 7.5, or about 8.
[0152] The composition can further include a solvent carrier. A
suitable solvent carrier includes water (e.g., deionized water).
Optionally, the solvent carrier can further include one or more
organic solvents, such as alcohols, glycols, and other liquids.
[0153] Analyte-Conjugate Protein
[0154] The analyte-conjugate protein comprises the analyte that is
to be detected conjugated or linked to a protein. Examples of
suitable proteins are BSA, CRM197, KLH, thyroglobulin, tetanus
toxoid, rabbit serum albumin, myoglobulins, tuberculin,
poly-lysine, and/or poly-glutamic acid.
[0155] Anti-Species Antibody
[0156] The anti-species antibody is capable of binding with the
anti-analyte antibody-particle conjugate. Because the anti-species
antibody will bind with the anti-analyte antibody-particle
conjugate whether or not an analyte is present in the liquid, the
location where the anti-species antibody has been deposited will
develop color when the liquid contains an anti-analyte
antibody-particle conjugate.
[0157] Chromatographic Membrane
[0158] As described above, the detection layer comprises a
chromatographic membrane. In some embodiments, the chromatographic
membrane comprises, may consist essentially of, or may be formed
from cellulose or cellulose derivatives, including
surface-functionalized cellulose, glass fiber, and/or other
materials. In some embodiments, the chromatographic membrane
comprises a porous chromatography medium. Typical pore size may
comprise a diameter of about 10 to 14 .mu.m, although other size
pores may be used. The chromatography medium may be hydrophobic or
hydrophilic, and may comprise inorganic powders such as silica,
magnesium sulfate, and alumina; natural polymeric materials,
particularly cellulosic materials and materials derived from
cellulose, such as fiber containing papers, for example, filter
paper or chromatographic paper; synthetic or modified naturally
occurring polymers, such as nitrocellulose, cellulose acetate;
poly(vinyl chloride), polyacrylamide, agarose, or polyacrylate;
alone or in combination with other materials. Ceramics may also be
used. Optionally, the chromatography medium can be bound to a
backing layer.
[0159] Optionally, the chromatographic membrane may comprise one or
more polymers. Optionally, the one or more polymers includes
polysaccharides. Suitable polysaccharides for use in the
chromatographic membrane include agar, agarose, alginate,
carrageenan, cellulose, chitosan, dextran, konjac, and mixtures
thereof. Exemplary agarose polymers include, for example,
carboxymethyl agarose, diethylaminoethyl agarose, and like
derivatives. Optionally, the agarose polymers for use in the
chromatographic membrane are commercially available from Pharmacia
Fine Chemicals, Inc. (Piscataway, N.J.). Exemplary cellulose
polymers include, for example, cellulose esters (e.g., cellulose
acetate, cellulose acetate butyrate, cellulose acetate propionate),
carboxymethyl cellulose, diethylaminoethyl (DEAE) cellulose,
nitrocellulose, phosphocellulose, quaternary ammonium substituted
cellulose, and sulfoxyethyl cellulose. Optionally, the cellulose
polymers for use in the matrix are commercially available from
Whatman Co. (Whatman Paper Co., Ltd., Maidstone, England) or BioRad
Corp. (Richmond, Calif.). In some embodiments, the chromatographic
membrane includes glass fiber. Glass and polyester fiber may be
acquired from Fusion 5 from GE Healthcare (GE Healthcare, Little
Chalfont, UK).
[0160] The chromatographic membrane can further include an
absorbent. For example, the absorbent can include chromatography
paper, filter paper, and other materials typically used for
chromatography, such as for paper chromatography or thin layer
chromatography (TLC). The chromatography paper and filter paper can
be qualitative or quantitative filter paper, such as the
chromatography paper and filter paper commercially available from
Whatman Co. (Whatman Paper Co., Ltd., Maidstone, England).
[0161] Optionally, the absorbent comprises silica gel, alumina,
high performance thin layer chromatography (HPTLC) silica gel,
polysilicic acid, aluminum oxide, cellulose, polyamide, reversed
phase silica gel C.sub.2 (dimethyl bonded), reversed phase silica
gel C.sub.2 (ethyl bonded), reversed phase silica gel C.sub.8
(octyl bonded), reversed phase silica gel C.sub.18 (octadecyl
bonded), acetylated cellulose, silica gel modified with amino
groups, silica gel modified with cyano groups, Kieselghur
impregnated with hydrocarbons, anionic and cationic anion exchange
resins, diethylaminoethyl cellulose, and mixtures of the listed
sorbents. The absorbent can be immobilized on an inert surface.
[0162] Optionally, the chromatographic membrane can be pre-treated
with a desiccant to integrate the desiccant into the
chromatographic membrane. The desiccant can be any desiccant as
known to those of skill in the art, including, but not limited to,
molecular sieves, silica gels, clays, synthetic polymers, and
starches. For example, suitable desiccants include alumina,
bauxite, anhydrous calcium sulfate, water-absorbing clays, silica
gel, zeolite, and mixtures thereof.
[0163] Optionally, the chromatographic membrane can be pre-treated
with a buffering agent, such as the buffering agents described
above. The matrix can be pre-treated with a buffering agent such
that the matrix is buffered at a pH ranging from about 3 to about 8
(e.g., from about 4 to about 6 or from about 4.5 to about 5.5). For
example, the buffering agent can be added to the composition to
provide a pH of about 3, about 3.5, about 4, about 4.5, about 5,
about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8.
[0164] The chromatographic membrane can be positioned on the
surface of an object. In some examples, the matrix can be
positioned within an object. In other examples, the chromatographic
membrane can be positioned below the surface of an object. Suitable
objects include, for example, a fingernail, an artificial
fingernail, a layer of fingernail polish, a fingernail sticker, a
fingernail decal, a cup, a bar coaster, a drink stirrer, a
toothpick, a drink ornament (e.g., an umbrella), a pencil, a pen, a
test strip, a sticker, a decal, a nail decal, a mesh nail wrap, or
any other appropriate surface. Other appropriate surfaces include
items that could easily and discreetly be brought into contact with
a suspect liquid, providing an improved degree of personal security
for the liquid consumer.
[0165] The apparatus described herein can also include a cover over
the chromatographic membrane. The cover can be an opaque cover, a
tinted cover, a transparent cover, or a translucent cover.
Optionally, the cover can include one or more perforations. These
perforations allow for the escape of gaseous materials during the
use of the apparatus. In examples where the cover is opaque,
tinted, or translucent, the cover can optionally include one or
more transparent windows on the cover. The cover can be attached to
the apparatus using any suitable binding material as known to those
of skill in the art, including, for example, an adhesive. Suitable
adhesives include but are not limited to acrylic and methacrylic
ester homo- or copolymers, butyl rubber based systems, silicones,
urethanes, vinyl esters and amides, olefin copolymer materials,
di-alkyl fumarates, natural or synthetic rubbers, and the like,
including hot-melt adhesives.
[0166] In some embodiments, the apparatus comprising a detection
layer can also include at least one additional layer. In some
embodiments, the apparatus can include at least one of a cover
layer, a support layer, and a removable layer. In some embodiments,
the apparatus can include any combination of layers described
herein.
[0167] In some embodiments, the detection layer comprises a lateral
flow assay. In some embodiments, the lateral flow assay can rely on
antibody-analyte interactions to determine the presence of drugs in
an alcoholic or non-alcoholic beverage. In some embodiments, the
lateral flow assay can include an anti-analyte antibody that is
conjugated to colored particles which can be carried through a
chromatographic membrane upon which a analyte-conjugated protein
(test line) and an anti-species antibody (control line) are
immobilized.
[0168] In some embodiments, the lateral flow assay can have an
extended storage life. In some embodiments, the detection layer
comprising a lateral flow assay (and apparatus) can be laminated to
provide protection from external environment without compromising
the integrity of the test by permitting gas permeability during
use.
[0169] In some embodiments, a first indicator signals a portion of
at least one of a word, symbol, or character and a second indicator
signals a portion of at least one of a word, symbol, or character.
In some embodiments, the signal of the first indicator only signals
to a user the presence of the targeted substance and wherein the
signal of both the first indicator and the second indicator signals
to a user the absence of a targeted substance. Non-limiting
embodiments include:
1. An apparatus for detecting the presence of an analyte in a
liquid, the apparatus comprising a lateral flow assay capable of
receiving a sample of a beverage and analyzing the beverage for the
presence of the analyte. 2. The embodiment of paragraph 1, wherein
the apparatus comprises: a sample area, a conjugate area, and a
detection area, wherein at least one of the sample area, conjugate
area, or detection area comprises at least one residual buffer
composition. 3. The embodiment of paragraph 2, wherein the
conjugate area comprises at least one anti-analyte
antibody-particle conjugate or anti-analyte aptamer-particle
conjugate; wherein the detection area comprises a chromatographic
membrane and at least one analyte-conjugate protein, and wherein
the sample area is configured for receiving a liquid. 4. The
embodiment of paragraph 2 or 3, wherein the detection area further
comprises at least one anti-species antibody or anti-species
aptimer. 5. The embodiment of any one of paragraphs 2-4, wherein
the sample area, conjugate area, and the detection area are located
on a single pad. 6. The embodiment of any one of paragraphs 2-4,
wherein at least one of the sample area, conjugate area, and the
detection area is located on a separate pad. 7. The embodiment of
any one of paragraphs 3-6, wherein the chromatographic membrane
comprises one or more of cellulose, nitrocellulose, polyester
fiber, and/or glass fiber. 8. The embodiment of any one of
paragraphs 1-7, further comprising a cover defining a pattern,
wherein the cover is disposed over the detection area. 9. The
embodiment of any one of paragraphs 2-8, wherein the detection area
is positioned on or within or under a natural fingernail, an
artificial fingernail, a layer of fingernail polish, a fingernail
sticker, a fingernail decal, a sticker, a cup, a drink coaster, a
drink stirrer, a toothpick, a drink ornament, a pencil, or a pen.
10. The embodiment of any one of paragraphs 2-9, wherein the
residual buffer composition comprises at least one surfactant. 11.
The embodiment of paragraph 10, wherein the surfactant comprises
Pluronic F-68, a fatty alcohol, a polyethylene glycol alkyl ether,
a polypropylene glycol alkyl ether, a glucoside alkyl ether,
polyethylene glycol octyl, a glycerol alkyl ester, a phenyl ether,
polyoxyethylene (20) oleyl ether, octylphenol ethoxylate, a
polyethylene glycol alkylphenyl ether, polyethoxylated tallow
amine, N,N-bis[3-(D-gluconamido)propyl]cholamide, polyoxyethylene
(20) cetyl ether, dimethyldecylphosphine oxide, branched
octylphenoxy poly(ethyleneoxy)ethanol, a
polyoxyethylene-polyoxypropylene block copolymer,
t-octylphenoxypolyethoxyethanol, polyoxyethylene (20) sorbitan
monooleate, or a combination thereof. 12. The embodiment of any one
of paragraphs 2-11, wherein the residual buffer composition
comprises at least one buffer salt. 13. The embodiment of paragraph
12 wherein the buffer salt comprises monosodium phosphate, disodium
phosphate, sodium tetraborate,
Tris(hydroxymethyl)methylaminopropanesulfonic (TAPS),
N-cyclohexyl-2-aminoethanesulfonic acid (CHES),
N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS),
bis-tris methane (Bis TRIS), tris(hydroxymethyl)aminomethane
(TRIS),
2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic
acid (TES), 2-(N-morpholino)ethanesulfonic acid (MES),
N-(carbamoylmethyl)iminodiacetic acid (ADA),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), bis tris
propane, piperazine-N,N'-bis(2-ethanesulfonic acid)(PIPES),
N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),
3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), cholamine
chloride, (3-(N-morpholino)propanesulfonic acid) (MOPS),
N,N-bis(2-hydroxyethyl)taurine (BES),
N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid
(DIPSO), 4-(N-morpholino)butanesulfonic acid (MOBS),
3-[N-rris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid
(TAPSO), acetamidoglycine, triethanolamine (TEA),
piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) POPSO,
4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)
hydrate (HEPPSO),
3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS),
tricine, tris(hydroxymethyl)aminomethane, trometamol (TRIZMA),
glycinamide, glycyl-glysine,
N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS),
bicine, 2-amino-2-methyl-1-propanol (AMP),
N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid (AMPSO), N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid
(CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS),
4-(cyclohexylamino)-1-butanesulfonic acid (CABS), and
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES),
carnitine, gamma-aminobutyric acid, taurine, or salts of amino
acids alanine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, or valine. 14. The embodiment of any one of
paragraphs of claims 2-13, wherein the sample area comprises a
residual sample area buffer composition, and wherein the residual
sample area buffer composition comprises a potassium salt of a weak
acid and at least one surfactant. 15. The embodiment of any one of
paragraphs 2-14, wherein the conjugate area comprises a residual
conjugate area buffer composition, and wherein the residual
conjugate area buffer composition comprises a Good's buffer salt
and one or more of a protein, an oligomer, a polymer, and a
surfactant. 16. The embodiment of any one of paragraphs 2-15,
wherein the detection area comprises a residual chromatographic
membrane buffer composition, and wherein the residual
chromatographic membrane buffer composition comprises a phosphate
salt and one or more of a saccharide, a protein, an oligomer, and a
polymer. 17. The embodiment of any one of paragraphs 2-16, wherein
the lateral flow assay is configured to detect multiple analytes.
18. The embodiment of any one of paragraphs 2-17, wherein the
residual buffer composition can reduce the acidity of a test
liquid. 19. The embodiment of any one of paragraphs 2-18, wherein
the residual buffer composition can increase the viscosity of a
test liquid. 20. The embodiment of any one of paragraphs 2-19
wherein the residual buffer composition can substantially reduce or
substantially remove the appearance of colored components of a test
liquid. 21. A method of detecting an analyte in a liquid, said
method comprising: providing the apparatus of any one of claims
1-20; exposing a portion of the apparatus to the liquid; and
observing a visual indication to determine presence or absence of
the analyte. 22. The embodiment of paragraph 21, wherein the liquid
is a beverage. 23. The embodiment of paragraph 21, wherein the
liquid is a food extract. 24. The embodiment of any one of
paragraphs 21-23, wherein the liquid has a pH from about 4.5 to
about 6.8. 25. A method of making an apparatus for detecting the
presence of an analyte in a liquid, the method comprising: applying
a buffer solution to at least one of a conjugate pad comprising a
conjugate area comprising at least one anti-analyte
antibody-particle conjugate or anti-analyte aptamer-particle
conjugate; a detection area comprising a chromatographic membrane
and an analyte-conjugate protein; or a sample area for receiving a
liquid; drying the buffer solution; and assembling the conjugate
pad, detection area, sample area and a wick so that the sample area
is in contact with a portion of the conjugate area, another portion
of the conjugate area is in contact with a proximal end of the
detection area, and the wick is in contact with a distal end of the
detection area.
[0170] The apparatus and methods of the appended claims are not
limited in scope by the specific apparatus and methods described
herein, which are intended as illustrations of a few aspects of the
claims and any apparatus and methods that are functionally
equivalent are intended to fall within the scope of the claims.
Various modifications of the apparatus and methods in addition to
those shown and described herein are intended to fall within the
scope of the appended claims. For example, additional specific
embodiments of apparatus, systems, and methods consistent with the
present apparatus and methods are described and set forth in a PCT
patent application entitled "Apparatus, System, and Method for
Detecting a Target Substance," applied for by Undercover Colors,
Inc. and filed on the same day as the present application, which is
incorporated by reference in its entirety.
[0171] Further, while only certain representative apparatus
materials and method steps disclosed herein are specifically
described, other combinations of the apparatus materials and method
steps also are intended to fall within the scope of the appended
claims, even if not specifically recited. Thus, a combination of
steps, elements, components, or constituents may be explicitly
mentioned herein; however, other combinations of steps, elements,
components, and constituents are included, even though not
explicitly stated. The term "comprising" and variations thereof as
used herein is used synonymously with the term "including" and
variations thereof and are open, non-limiting terms. Although the
terms "comprising" and "including" have been used herein to
describe various embodiments, the terms "consisting essentially of"
and "consisting of" can be used in place of "comprising" and
"including" to provide for more specific embodiments of the
invention and are also disclosed.
EXAMPLES
Example 1: Lateral Flow Immunoassay
[0172] A lateral flow immunoassay of the invention was prepared as
follows. A benzo test line solution was prepared using
(Benzodiazepine-BSA, 5:1 ratio) solution diluted to 2 mg/mL with pH
7.4 Phosphate Buffered Saline (lx). A benzo control line solution
was prepared using Goat Anti-Mouse Antibody solution diluted to 1
mg/mL with pH 7.4 Phosphate Buffered Saline (lx). A test line of
the diluted benzo test line solution was printed 12 mm from the
bottom of the FF120HP (GE Healthcare) nitrocellulose strip, which
has a capillary rise time of about 120 seconds for 4 cm. A control
line of diluted benzo control line solution was printed 1.5 mm
above the test line. The printed strip was placed in a forced air
oven to dry for 60 minutes at 10% humidity and 37.degree. C., and
then it was stored in a desiccator at 20% humidity until used. The
printed FF120HP nitrocellulose strip was treated with the Abcam
Immunoassay Buffer (BSA Free), and was placed in a forced air oven
to dry for 60 minutes at 10% humidity and 37.degree. C., and then
it was stored in a desiccator at 20% humidity until used.
[0173] Monoclonal Mouse Anti-Benzodiazepine Antibody-Gold NP
conjugate was prepared by first desalting the Monoclonal Mouse
Anti-Benzodiazepine Antibody solution using a Zeba spin columns
(Thermo Scientific, PN: 89882) to replace the stock buffer with 100
mM, pH 7.4 sodium phosphate buffer. The desalted Monoclonal Mouse
Anti-Benzodiazepine Antibody solution in PBS was then conjugated to
40 nm colloidal gold nanoparticles in 50 mM Sodium Borate Buffer.
During the conjugation process the 5 mM Sodium Borate buffer with
5% BSA is added to the conjugation solution. Upon completion of the
conjugation of the Monoclonal Mouse Anti-Benzodiazepine Antibody to
the 40 nm gold NP, the Monoclonal Mouse Anti-Benzodiazepine
Antibody-Gold NP conjugate was concentrated and subsequently
diluted to OD10 with 100 mM Sodium Borate buffer containing 0.5%
Fish Skin Gelatin and 0.1% Tween 80.
[0174] A conjugate pad was prepared by pretreating a strip of
Ahlstrom 8964 glass fiber pad with 50 mM Sodium Borate containing
1% BSA, 5% Sucrose, 2% Trehalose, 0.25% Tween 20, and 0.15M KCl,
and then the pretreated 6614 strip was placed in a forced air oven
for 60 minutes at 10% humidity and 37.degree. C., and then stored
in a desiccator at 20% humidity until used. The buffered diluted
conjugate solution was printed continuously across the 8864 strip
at a rate of 8 uL per centimeter, and then the strip was placed in
a forced air oven for 60 minutes at 10% humidity and 40.degree. C.,
and then stored in a desiccator at 20% humidity.
[0175] To prepare the sample pad a strip of CF4 (GE Healthcare) was
treated with 1M K.sub.2CO.sub.3, and then the strip was placed in a
forced air oven for 60 minutes at 10% humidity and 40.degree. C.,
and then stored in a desiccator at 20% humidity. The master card
was assembled by applying a printed strip of nitrocellulose to an
adhesive backing. The conjugate pad was applied so as to achieve an
overlap of 2 mm with the bottom of the nitrocellulose. The sample
pad was applied so as to achieve an overlap of 2 mm with the bottom
of the conjugate pad. An Ahlstrom 319 wicking pad was applied as to
achieve an overlap of 2 mm with the top of the nitrocellulose. The
master card was then cut into 4 mm wide strips.
Example 2: Preparation of Buffer Solutions
[0176] Buffer solutions were prepared as follows:
Antibody Desalting Buffer Solution:
[0177] A 100 mM sodium phosphate, pH 7.5 buffer was prepared by
combining, in order: Molecular Biology Reagent Water (Sigma, PN:
W4502) was added in the amount of: 80% of total batch volume Sodium
phosphate monobasic (Sigma, PN: S3139) was added in the amount of:
10.2 g/L.times.batch volume (L) Sodium phosphate dibasic (Sigma,
PN: S9763 was added in the amount of: 58.91 g/L.times.batch volume
(L) Molecular Biology Reagent Water (Sigma, PN: W4502) to final
volume pH was adjusted to 7.5 using NaOH or concentrated HCl.
Conjugation Blocking Buffer Solution:
[0178] A 50 mM sodium borate, 10% BSA, pH 9.0 buffer was prepared
by combining, in order: Sodium tetraborate decahydrate (Fisher, PN:
AC41945-0010): 11.4 g/L Boric acid (Fisher, PN: A74-1): 1 g/L
Bovine serum albumin (BSA, Equitech, PN: BAH64): 100 g/L Molecular
Biology Reagent Water (Sigma, PN: W4502) to final volume pH was
adjusted to 9.0 using NaOH or HCl, and then the buffer was filtered
using a 0.2 .mu.m filter (VWR, PN: 73520-994).
Conjugate Dilution Buffer Solution:
[0179] A 50 mM sodium borate, 1% BSA, 5% trehalose, and 20%
sucrose, pH 9.0 buffer was prepared by combining, in order: [0180]
a. Sodium tetraborate decahydrate (Fisher, PN: AC41945-0010): 11.4
g/L [0181] b. Boric acid (Fisher, PN: A74-1): 1 g/L [0182] c.
Bovine serum albumin (BSA, Equitech, PN: BAH64): 10 g/L [0183] d.
Sucrose (Sigma, PN: 84097) [0184] e. Trehalose (Sigma, PN: 90210)
[0185] f. Molecular Biology Reagent Water (Sigma, PN: W4502) to
final volume
Chromatographic Membrane Buffer Solution:
[0186] A 10 mM sodium phosphate, 0.1% sucrose, 0.1% BSA, 0.2%
PVP-40, pH 7.5 buffer was prepared by combining, in order, per
liter of buffer: Sodium phosphate monobasic (Sigma, PN: S3139),
0.204 g Sodium phosphate dibasic (Sigma, PN: S9763), 1.178 g
Sucrose (Sigma, PN: 84097) 1.0 g
[0187] Bovine serum albumin (BSA, Equitech, PN: BA H64). 1.0 g
Poly(vinylpyrrolidone)-40 (PVP-40, Sigma, PN: PVP-40): 2.0 g
[0188] Molecular Biology Reagent Water (Sigma, PN: W4502) to one
liter. pH was adjusted to 7.2 using NaOH or HCl, and then the
buffer was filtered using a 0.2 .mu.m filter (VWR, PN:
73520-994).
Conjugate Pad Buffer Solution:
[0189] A 0.5 M Tris, 3% BSA, 1% PVP-40, 0.25% Triton X-100, 0.5%
Pluronic F-68, pH 8.0 buffer was pre prepared by combining, in
order: Tris base (Sigma, PN: T1375): 114.8 g/L Bovine serum albumin
(BSA, Equitech, PN: BAH64): 30 g/L
Polyvinylpyrrolidone-40 (PVP-40, Sigma, PN: PVP-40): 10 g/L
Triton X-100 (Sigma, PN: T8787): 2.5 g/L
Pluronic F-68 (Thermo Fisher, PN: 24040032): 5 g/L
[0190] Add Molecular Biology Reagent Water (Sigma, PN: W4502) to
final volume
Sample Area Buffer Solution:
[0191] A 1.0 M Potassium Carbonate (K.sub.2CO.sub.3) buffer with
0.25% Triton X-305, pH 7.0 buffer was pre prepared by combining, in
order: Potassium carbonate (Sigma PN: P1472): 138.2 g/L
Triton X-305 (Sigma, PN: X305): 3.6 g/L
[0192] Add Molecular Biology Reagent Water (Sigma, PN: W4502) to
final volume
Example 3: Preparation of a Lateral Flow Assay with a Combined
Sample-Conjugate Pad
[0193] A lateral flow immunoassay of the invention was prepared as
follows. A benzo test line solution was prepared using
(Benzodiazepine-BSA, 5:1 ratio) solution diluted to 4 mg/mL with pH
7.4 Phosphate Buffered Saline (lx). A benzo control line solution
was prepared using Goat Anti-Mouse Antibody solution diluted to 1
mg/mL with pH 7.4 Phosphate Buffered Saline (lx). A test line of
the diluted benzo test line solution was printed 5 mm from the
bottom of the 8 mm wide CN095 (Sartorius) nitrocellulose strip,
which has a capillary rise time of about 85.+-.10 seconds for 4 cm.
A control line of diluted benzo control line solution was printed 2
mm above the test line. The printed strip was placed in a forced
air oven to dry for 30 minutes at 10% humidity and 40.degree. C.,
and then it was stored for 16 hours in a desiccator at 20%
humidity. The printed CN095 nitrocellulose strip (Sartorius) was
treated with the Chromatographic Membrane Buffer described above in
Example 2, and was placed in a forced air oven to dry for 30
minutes at 10% humidity and 40.degree. C., and then it was stored
for 16 hours in a desiccator at 20% humidity.
[0194] Monoclonal Mouse Anti-Benzodiazepine Antibody-Gold NP
conjugate was prepared by first desalting the Monoclonal Mouse
Anti-Benzodiazepine Antibody solution using a Zeba spin columns
(Thermo Scientific, PN: 89882) to replace the stock buffer with 100
mM, pH 7.4 sodium phosphate buffer. The desalted Monoclonal Mouse
Anti-Benzodiazepine Antibody solution in PBS was then conjugated to
40 nm colloidal gold nanoparticles. During the conjugation process
the Conjugation Blocking Buffer is added to the conjugation
solution. Upon completion of the conjugation of the Monoclonal
Mouse Anti-Benzodiazepine Antibody to the 40 nm gold NP, the
Monoclonal Mouse Anti-Benzodiazepine Antibody-Gold NP conjugate was
concentrated and subsequently diluted to OD15 with the Conjugate
Dilution Buffer as described above in Example 2.
[0195] A combined sample-conjugate pad was prepared by pretreating
a strip of Ahlstrom 6614 polyester fiber pad with Conjugate Pad
Buffer described above in Example 2, and then the pretreated 6614
strip was placed in a forced air oven for 60 minutes at 10%
humidity and 40.degree. C., and then stored for 16 hours in a
desiccator at 20% humidity. To prepare the sample area of the
combined sample-conjugate pad, only the sample area of an Ahlstrom
6614 polyester fiber pad was treated with the Sample Area Buffer
described above in Example 2. The buffered diluted conjugate
solution was printed continuously across the strip on 6614 in only
the conjugate area at a rate of 5 per centimeter, and then the
strip was placed in a forced air oven for 60 minutes at 10%
humidity and 40.degree. C., and then stored for 16 hours in a
desiccator at 20% humidity.
[0196] The master card was assembled by applying a printed strip of
nitrocellulose to an adhesive backing. The sample/conjugate pad was
applied so as to achieve an overlap of 2 mm with the bottom of the
nitrocellulose. An Ahlstrom 319 wicking pad was applied as to
achieve an overlap of 2 mm with the top of the nitrocellulose. The
master card was then cut into 4 mm wide strips.
Example 4: The Effect of K.sub.2CO.sub.3 and TRIS Sample Pad
Treatment on Lateral Flow Assay Results
[0197] Lateral flow assays were prepared by the process of Example
3, except that the assays of Table 1 had no sample pad/area
pretreatment, and the assays of Table 2 were pretreated with a
sample area buffer solution comprising potassium carbonate. A check
indicates no assay failure. An X equals false negative results due
to non-specific binding of conjugate to test line. The false
negative results were overcome by pre-treatment, with one exception
of hot coffee.
[0198] Procedure:
[0199] 1.) Prepare assays according to the procedure described in
Example 3. Prepare half of the assays without the addition of the
Sample Area Buffer.
[0200] 2.) Prepare individual spiked solutions of each beverage
listed. The beverages are spiked with either Alprazolam, Diazepam,
or Flunitrazepam to a final concentration of 1000 ng/mL.
[0201] 3.) Deposit 20 .mu.L of the designated blank beverage on the
untreated sample area of three assays per designated beverage and
record the results at 1 minute.
[0202] 4.) Deposit 20 .mu.L of the designated spiked beverage on
the untreated sample area of three assays per designated beverage
and record the results at 1 minute.
[0203] 5.) Deposit 20 .mu.L of the designated blank beverage on the
treated sample area of three assays per designated beverage and
record the results at 1 minute.
[0204] 6.) Deposit 20 .mu.L of the designated spiked beverage on
the treated sample area of three assays per designated beverage and
record the results at 1 minute.
TABLE-US-00001 TABLE 1 No potassium carbonate pre-treatment of
sample pad/area. Valium Xanax Rohypnol (diazepam) (alprazolam)
(flunitrazepam) Beer/ Sam Adams Boston Lager Other Guinness Blue
Moon Big Boss Bad Penny Lonerider Shotgun Betty Hefeweizen
Foothills People's Porter Duck-Rabbit Amber Sweetwater IPA Sierra
Nevada Pale Ale Bell's Oberon Mike's Hard Lemonade X X X Angry
Orchard Cider X X X White Yellowtail Pinot Grigio X Wine Barefoot
Moscato X X X Gallo Chardonnay Mondavi Woodbridge X X X Sauvignon
Blanc Barefoot Riesling X X Rose Gallo White Merlot X X X Wine
Sutter Home Pink Moscato X X Yellowtail Pink Moscato X X X Barefoot
Red Moscato Mondavi Woodbridge X X X White Zinfandel Red Yellowtail
Merlot X X X Wine Sutter Home Pinot Noir X X X Barefoot Shiraz
Mondavi Woodbridge Zinfandel Gallo Cabernet Sauvignon Mixed Rum and
Coke Drinks Martini Mojito X X X Old Fashioned Long Island Iced Tea
X X X White Russian Pina Colada Jose Cuervo Ready to Drink Classic
Margarita Screwdriver Cosmopolitan Tequila Sunrise Margarita X X X
Daiquiri X X X Irish Coffee X X X Bloody Mary Liquor Smirnoff Vodka
Captain Morgan Spiced Rum Jack Daniel's Whiskey Jagermeister
Tanqueray Gin X X X Bacardi Rum Crown Royal Whisky X X X Jim Beam
Bourbon Jose Cuervo Tequila Fireball Cinnamon Whisky Dekuyper
Peachtree Malibu Coconut Rum Mixers Cranberry Juice Lemonade X X
Hawaiian Punch Half and Half Coffee (hot) X X X Orange Juice Rose's
Mojito Mix X X X Tonic Water Pineapple Juice X X X Coke V8 Club
Soda Lime Juice X X X
TABLE-US-00002 TABLE 2 With potassium carbonate pre-treatment of
sample pad/area. Valium Xanax Rohypnol (diazepam) (alprazolam)
(flunitrazepam) Beer/ Sam Adams Boston Lager Other Guinness Blue
Moon Big Boss Bad Penny Lonerider Shotgun Betty Hefeweizen
Foothills People's Porter Duck-Rabbit Amber Sweetwater IPA Sierra
Nevada Pale Ale Bell's Oberon Mike's Hard Lemonade Angry Orchard
Cider White Yellowtail Pinot Grigio Wine Barefoot Moscato Gallo
Chardonnay Mondavi Woodbridge Sauvignon Blanc Barefoot Riesling
Rose Gallo White Merlot Wine Sutter Home Pink Moscato Yellowtail
Pink Moscato Barefoot Red Moscato Mondavi Woodbridge White
Zinfandel Red Yellowtail Merlot Wine Sutter Home Pinot Noir
Barefoot Shiraz Mondavi Woodbridge Zinfandel Gallo Cabernet
Sauvignon Mixed Rum and Coke Drinks Martini Mojito Old Fashioned
Long Island Iced Tea White Russian Pina Colada Jose Cuervo Ready to
Drink Classic Margarita Screwdriver Cosmopolitan Tequila Sunrise
Margarita Daiquiri Irish Coffee Bloody Mary Liquor Smirnoff Vodka
Captain Morgan Spiced Rum Jack Daniel's Whiskey Jagermeister
Tanqueray Gin Bacardi Rum Crown Royal Whisky Jim Beam Bourbon Jose
Cuervo Tequila Fireball Cinnamon Whisky Dekuyper Peachtree Malibu
Coconut Rum Mixers Cranberry Juice Lemonade Hawaiian Punch Half and
Half Coffee (hot) X X X Orange Juice Rose's Mojito Mix Tonic Water
Pineapple Juice Coke V8 Club Soda Lime Juice
Example 5: Faster Development of Test Results in Inventive Assays
Vs. Comparative Assays
[0205] Lateral flow assays were prepared by the process of Example
3 and were compared to commercial lateral flow assays (DBZ-114
distributed by Innovacon, San Diego, Calif.) 30 seconds after
exposure to a test fluid. The inventive assay results are fully
developed by 30 seconds, whereas the comparative assays had not
fully developed at 30 seconds.
Procedure:
[0206] 1.) Prepare assays according to the procedure described in
Example 3. To prepare linear assays use a rectangular Ahlstrom 319
wick. To prepare miniaturized assays use the U-shaped Ahlstrom 319
wick. 2.) Arrange the miniaturized and linear assays on the testing
sheet. 3.) Deposit 20 .mu.L of blank Corona beer on the sample area
of the linear assays marked blank. 4.) Deposit 20 .mu.L of Corona
beer spiked with 1000 ng/mL Flunitrazepam on the sample area of the
linear assays. 5.) Deposit 20 .mu.L of blank Corona beer on the
sample pad of the U-wick assays marked blank. 6.) Deposit 20 .mu.L
of Corona beer spiked with 1000 ng/mL Flunitrazepam on the sample
pad of the U-wick assays. 7.) Take picture at 30 seconds
[0207] Results are shown in FIG. 3 with the lateral flow assays
prepared by Example 3 shown on the left and the commercial lateral
flow assays shown on the right.
Example 6: Beverage Components Cause False Negative Results in
Comparative Assays, but not in Inventive Assays
[0208] Comparative commercial assays as used in Example 5 fail (due
to false negatives) in whiskey and moscato after 5 minutes of
development due to specifications of the commercial assay. The
commercial assay completely fails to run in daiquiri, and no
results are visible after 5 minutes. The inventive assays (prepared
as in Example 3) perform successfully in all cases, with no false
negative results.
Procedure:
[0209] 1.) Prepare miniature assays according to the procedure
described in Example 3. 2.) Arrange the miniaturized and commercial
assays on the testing sheet. 3.) Deposit 20 .mu.L of the designated
blank beverage on the sample area of the miniature assays marked
blank. 4.) Deposit 20 .mu.L of designated beverage spiked with 1000
ng/mL Flunitrazepam on the sample area of the miniature assays. 5.)
Deposit 100 .mu.L of designated blank beverage on the sample pad of
the commercial assays marked blank. 6.) Deposit 100 .mu.L of
designated beverage with 1000 ng/mL Flunitrazepam on the sample pad
of the commercial assays. 7.) Take picture at 5 minutes to allow
time for the commercial assays to fully develop.
[0210] Results are shown in FIG. 4 for daiquiri, whisky, and water,
and in FIG. 5 for Corona, orange juice, and moscato, with the six
commercial assays on top and the six inventive assays on the bottom
in both Figures.
Example 7: U-Shaped Wick Shortens Assay Length without Affecting
Performance
[0211] Inventive assays (prepared as in Example 3) with a U-shaped
wick (shown on right), where the fluid path is longer than the
assay length, perform just as well as inventive assays (prepared as
in Example 3) with a linear wick (shown on left), where the fluid
path equals the assay length.
Procedure:
[0212] 1.) Prepare assays according to the procedure described in
Example 3. To prepare linear assays use a rectangular Ahlstrom 319
wick. To prepare miniaturized assays use the U-shaped Ahlstrom 319
wick. 2.) Arrange the miniaturized and linear assays on the testing
sheet. 3.) Deposit 20 .mu.L of blank Corona on the sample area of
the linear assays marked blank. 4.) Deposit 20 .mu.L of Corona
spiked with 1000 ng/mL Flunitrazepam on the sample area of the
linear assays. 5.) Deposit 20 .mu.L of blank Corona on the sample
pad of the U-wick assays marked blank. 6.) Deposit 20 .mu.L of
Corona spiked with 1000 ng/mL Flunitrazepam on the sample pad of
the U-wick assays. 7.) Take picture at 30 seconds
[0213] Results are shown in FIG. 6 with the U-shaped wick assays on
the right and the linear wick assays on the left.
* * * * *