U.S. patent application number 10/410882 was filed with the patent office on 2003-12-25 for detection of contaminants using self-contained devices employing target material binding dyes.
This patent application is currently assigned to BioControl Systems, Inc.. Invention is credited to Carpenter, Charles, Clark, Genevieve, Eckenroth, Brian, Ehrenfeld, Elizabeth, Pierson, Mark W., Tornberg, Melanie.
Application Number | 20030235512 10/410882 |
Document ID | / |
Family ID | 22463644 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235512 |
Kind Code |
A1 |
Carpenter, Charles ; et
al. |
December 25, 2003 |
Detection of contaminants using self-contained devices employing
target material binding dyes
Abstract
A self-contained signal generating device and methods for using
and making the same are provided. The device and methods may detect
the presence of a number of different substances, such as proteins,
and utilizes a target material binding dye, which may precipitate a
target material as well as stain it, and/or undergo a detectable
change, e.g., an absorption or emission frequency shift, on binding
of the substance to be detected.
Inventors: |
Carpenter, Charles;
(Scarborough, ME) ; Tornberg, Melanie; (South
Berwick, ME) ; Clark, Genevieve; (Standish, ME)
; Eckenroth, Brian; (Westbrook, ME) ; Pierson,
Mark W.; (Saco, ME) ; Ehrenfeld, Elizabeth;
(Falmouth, ME) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
BioControl Systems, Inc.
Bellevue
WA
|
Family ID: |
22463644 |
Appl. No.: |
10/410882 |
Filed: |
April 9, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10410882 |
Apr 9, 2003 |
|
|
|
09776496 |
Feb 2, 2001 |
|
|
|
6551834 |
|
|
|
|
09776496 |
Feb 2, 2001 |
|
|
|
09134492 |
Aug 14, 1998 |
|
|
|
Current U.S.
Class: |
422/400 ;
436/164; 436/86 |
Current CPC
Class: |
A61B 10/02 20130101;
Y10T 436/13 20150115; Y10T 436/107497 20150115; G01N 33/583
20130101; Y10S 436/80 20130101; G01N 33/68 20130101; A61B 10/0096
20130101 |
Class at
Publication: |
422/56 ; 436/86;
436/164 |
International
Class: |
G01N 021/00 |
Claims
What is claimed is:
1. A self-contained sampling/testing device comprising: a. a
sampler for collecting a target material; b. a sampler washer
comprising a wash solution; and c. a signal generator comprising a
dye which binds to said target material to signal the presence of
said target material.
2. The device of claim 1, further comprising an absorbent material,
wherein said sampler comprises a porous sample collection pad, and
said absorbent material, said sampler, and said sampler washer are
configured and arranged such that said sample collection pad can be
disposed between said absorbent material and said sampler washer so
that said wash solution separates dye bound to said target material
immobilized on said sample collection pad from unbound dye.
3. The device of claim 1, wherein said sampler comprises a porous
sample collection pad for collecting said target material, wherein
said dye and said wash solution are contained in a reagent tray
housing capable of being contacted by said sampler to impart said
dye and said wash solution across said target material contained on
said sampler.
4. The device of claims 2 or 3 wherein said dye and said wash
solution are contained in a plurality of reservoirs, wherein said
reservoirs are serially contacted by said sampler to first expose
said target material to said dye and then to wash unbound dye away
from bound dye.
5. The device of claim 2 or 3, further comprising a wetting agent
for moistening said sample collection pad in advance of collecting
said target material.
6. The device of claim 5 wherein said wetting agent is identical to
said wash solution.
7. The device of claim 2, wherein said sampler is hollow and
wherein said absorbent material is disposed within said sampler to
facilitate transport of said dye and said wash solution across said
target material or said wash solution is disposed within said
sampler.
8. The device of claim 7, further comprising a reagent housing
comprising an additional absorbent material, wherein one of said
absorbent materials is saturated with said wash solution, and the
other of said absorbent materials is unsaturated, wherein unbound
dye from a sample collection surface disposed there between is
washed by the flow of said wash solution from said saturated
absorbent material to said unsaturated absorbent material.
9. The device of claim 8, wherein said dye is transported by said
wash solution to said target material to effect said binding.
10. The device of claim 9, wherein said dye is separated from a
said absorbent material by at least one membrane that is rupturable
during the course of a test.
11. The device of claim 8, wherein said reagent housing also
comprises a cap for protecting said collection pad surface from
contamination when not in use.
12. The device of claim 2 or 3 wherein said dye is a protein
binding dye.
13. The device of claim 12, wherein said dye is Ponceau-S.
14. The device of claim 13, wherein Ponceau-S is used at a final
concentration of about 0.1-1.0% (w/v) in a dilute acetic acid
solution.
15. The device of claim 12, wherein said dye both precipitates and
stains said protein.
16. The device of claim 2 or 3, wherein said dye is dry until
contacted by said sampler.
17. The device of claim 1, further comprising a neutralizing agent
to neutralize any compounds in the sample that might interfere with
the binding of said dye to said target material.
18. The device of claim 17, wherein said neutralizing agent is
selected from the group consisting of sodium thiosulfate,
MgCl.sub.2.
19. The device of claim 1, wherein said dye is a frequency shift
dye.
20. The device of claim 19, wherein said dye is a colloidal
dye.
21. The device of claim 20, wherein said dye is a colloidal
Coomassie.RTM. Brilliant Blue dye.
22. The device of claim 1, further comprising a reaction reading
portion.
23. The device of claim 22, wherein said sampler is contained
within a lower housing providing protection from pre-testing
contamination for said sampler, said device further comprising an
upper housing, wherein said upper housing and said lower housing
sealably engage, and said sampler is attached to said upper
housing.
24. The device of claim 1, wherein said sampler further comprises a
hollow shaft and an absorbent tip; a chamber housing said combined
sample wash signal generator, said chamber further comprising a
breakable shaft which upon breakage exposes an orifice through
which said combined sample wash signal generator may flow, wherein
said chamber is adjoined to a slidably engageable fitting, said
fitting having an inner member and an outer member between which a
lower housing may slidably engage, wherein said combined sample
wash signal generator comprises a frequency shift dye; a lower
housing read portion below said sampler to contain the combined
sample wash signal generator, wherein said read portion comprises
walls allowing detection of a frequency shift of said dye.
25. A method for detecting the presence of a target material or
contaminant, comprising the steps of: a) obtaining on a sampler of
a self-contained sampling/testing device a sample which may contain
a target material that is capable of being bound by a dye to signal
the presence of the target material; and b) contacting said sample
with said dye; c) determining whether a color development or color
change occurs following said contacting, wherein said color
development or color change is indicative of the presence of said
target material in said sample.
26. The method of claim 25, further comprising washing said sample
with a washing solution.
27. The method of claim 26, wherein said dye is a precipitating
dye.
28. The method of claim 25 or 27 wherein said dye is Ponceau-S.
29. The method of claim 28, wherein said Ponceau-S is used at a
final concentration of about 0.1-1.0% weight-volume in a dilute
acetic acid solution.
30. The method of claim 25 or 27, wherein said dye precipitates and
stains protein.
31. The method of claim 25 or 27, further comprising the step of
washing said target material from said sampler so that said target
material may be analyzed in solution in the presence of dye.
32. The method of claim 25, wherein said dye is a frequency shift
dye.
33. The method of claim 32, wherein said dye is a colloidal
dye.
34. The method of claim 33, wherein said colloidal dye is colloidal
Coomassie.RTM. Brilliant Blue dye.
35. The method of claim 26, wherein said wash solution participates
in the collection of said target material by said sampler.
36. A method of making a self-contained sampling/testing device
comprising the steps of: a) providing a device comprising at least
one reservoir, a sampler, and a housing; b) depositing a dye within
a said reservoir; and c) depositing a wash solution with a said
reservoir.
37. The method of claim 36, wherein said dye binds protein.
38. The method of claim 37, wherein said dye is a precipitating
dye.
39. The method of claim 36, wherein said dye is Ponceau-S.
40. The method of claim 39, wherein Ponceau-S is used at a final
concentration of about 0.1-1.0% (w/v) in a dilute acetic acid
solution.
41. The method of claim 36, wherein said dye is a colloidal
dye.
42. The method of claim 41, wherein said colloidal dye is colloidal
Coomassie.RTM. blue dye.
43. The method of claim 36, wherein said dye is frequency shift
dye.
44. A method of using a precipitating dye to fix a target material
in a porous matrix such that said target material is resistant to
migration outside of said matrix on treatment with said dye as
compared to the migration of said target material in the absence of
said dye, comprising the step of: contacting a liquefied sample
containing said target material in said porous matrix with a
precipitating dye, wherein said porous material permits entry of
said target material and binding of said dye to said target
material inhibits egress of said target material from said porous
material.
45. The method of claims 44, wherein said target material is
protein.
46. The method of claim 45, wherein said dye is a sulfonated diazo
dye.
47. The method of claim 46, wherein said sulfonated diazo dye is
Ponceau-S.
48. The method of claim 47, wherein said Ponceau-S is used at a
final concentration of about 0.1-1.0% (w/v) in a dilute acetic acid
solution.
49. A method for detecting a target material in a sample,
comprising the steps of: immobilizing target material/dye complexes
from a liquefied sample in or on a solid matrix, wherein said dye
is a precipitating dye; and washing unbound dye from said solid
matrix with a wash solution, wherein the presence of a greater
amount of dye retained in said porous matrix following said washing
as compared to the amount of dye retained in said porous matrix in
the absence of a sample is indicative of the presence of target
material in said sample.
50. The method of claim 49, wherein said solid matrix is a porous
matrix and said immobilizing is entrapment of said complexes in
said porous matrix.
51. The method of claim 49, wherein said sample is contacted with
said dye in solution and deposited on or in said solid matrix.
52. The method of claim 49, wherein said sample is contacted with
said dye in or on said solid matrix.
53. The method of claim 50, wherein said entrapment results from
binding of said dye to said target material.
54. The method of claims 49, wherein said target material is
protein.
55. The method of claim 54, wherein said dye is a sulfonated diazo
dye.
56. The method of claim 55, wherein said sulfonated diazo dye is
Ponceau-S.
57. The method of claim 56, wherein said Ponceau-S is used at a
final concentration of about 0.1-1.0% (w/v) in a dilute acetic acid
solution.
58. A method for detecting protein on a solid surface, comprising
the step of: contacting said surface with a Ponceau S dye solution;
and determining whether said dye is retained on said surface,
wherein if dye from said solution is retained on said surface it is
indicative of the presence of protein on said surface.
59. The method of claim 58, wherein said surface is washed with a
wash solution following said contacting and before said
determining.
60. The method of claim 58, wherein said Ponceau-S is used at a
final concentration of about 0.1-1.0% (w/v) in a dilute acetic acid
solution
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to the field of contamination
testing, particularly including the field of testing for biological
contaminants.
[0002] This description is provided solely to assist the
understanding of the reader, and does not constitute an admission
that the cited references are prior art to the present
invention.
[0003] In the processing of food materials and in the preparation
of food products, as well as in other fields, it is advantageous to
test for the presence of particular substances, often substances
that would be regarded as contaminants. Materials detected as
indicators of contamination include, for example, viable bacterial
cells and ATP. For many substances, however, the commonly used
detection methods involve a number of different procedural steps,
such as reagent preparation, reagent mixing, sample transfer, and
sample/reagent mixing. As an example, current methods for protein
determination, which can serve as an indicator of a contaminated
surface, often involve on-site reagent preparation due to stability
problems, along with multiple transfer steps, and/or involve highly
subjective color changes which make interpretation difficult,
and/or require the use of complex instrumentation. Examples of
common protein determination methods are described in Stoscheck,
Quantitation of Protein, in METHODS IN ENZYMOLOGY Vol. 182,
pp.50-68, 1990. Among the variants of basic protein detection
methods are methods using colloidal forms of Coomassie.RTM. blue
stain to detect proteins in gels such as polyacrylamide
electrophoresis gels. Such methods are described, for example, in
Neuhoffet al., 1985, Electrophoresis 6:427-488 and Neuhoffet al.,
1988, Electrophoresis 9:255-262.
[0004] In addition, for tests performed in food processing
facilities, contamination of food by assay reagents is a concern
and the individuals performing the tests may lack significant
experience preparing chemical formulations, potentially introducing
significant test error. Therefore, the use of detection methods
which require the preparation or transfers of assay chemicals is
undesirable in many testing environments.
[0005] In addition to the conventional protein assay methods
referenced above, a combination cleaning and protein staining
composition is described in Winicov et al., U.S. Pat. No.
5,424,000, entitled ACID CLEANINGS AND STAINING COMPOSITIONS,
issued Jun. 13, 1995. The solutions preferably include phosphoric,
sulfuric, and nitric acids, and Acid Violet 19 dye.
[0006] A number of different self-contained sampling/testing
devices employing certain assays have been described. Examples of
such assays include sampling for bacterial contaminants in food
processing plants, the sampling for contamination of the
environment by heavy metals such as lead, and the collection of
specimens from a patient to test for microorganism infection.
[0007] Specific examples of self-contained sampling/testing devices
include Nason, U.S. Pat. No. 5,266,266, issued Nov. 30, 1993, and
Nason, U.S. Pat. No. 4,978,504, issued Dec. 18, 1990, both entitled
SPECIMEN TEST UNIT; Nason, U.S. Pat. No. 4,707,450, issued Nov. 17,
1987, entitled SPECIMEN COLLECTION AND TEST UNIT; and Tobin, U.S.
Pat. No. 3,792,699, issued Feb. 19, 1974, all of which are hereby
incorporated by reference in their entireties including
drawings.
SUMMARY OF THE INVENTION
[0008] The need for, and utility of on site, immediate feedback to
cleaning and audit personnel on the presence of residual
contaminating substances in a variety of environments is
well-established. For example, the need for contaminant monitoring
has a well documented role in food safety programs when residual
food residues can result in bacterial contamination and allergic
responses in some individuals. Effective cleaning also reduces the
risk of pathogens contaminating subsequent food products. A variety
of devices and methods have been utilized for contaminant
testing.
[0009] Particularly advantageous devices for the purpose of
evaluating the presence of specific materials require no secondary
reagents or steps, have easily detected changes in the presence of
target material, give immediate results, and allow integrated
collection of sample into the device. The present inventor shows
that such a self-contained sampling/testing device can be
constructed in which the presence of target material in a sample is
detected calorimetrically through use of a dye which binds the
target material. As indicated above, this device is particularly
advantageous for routine sanitation testing procedures.
[0010] In a first aspect, the present invention concerns a
self-contained device having a sampler for collecting a sample
which may contain a target material, a signal generator having a
contactable dye that binds to the collected target material, and a
sampler washer having a wash solution for washing the collected
target material and/or free dye from or on the sampler to
facilitate measurement of a signal produced from the interaction of
dye and target material. The sample collection surface or portion
of the sampler is in communication with or can be placed in
communication with the sampler washer. In preferred embodiments,
the sampler collection surface is also in communication with or can
be placed in communication with an absorbent material able to take
up liquid from a wetting agent and/or dye solution and/or wash
solution. The device may be constructed with any of many possible
structural configurations, depending on the requirements of the
particular application, e.g., depending on the specific type of dye
used and the type of target material to be tested.
[0011] The term "in communication" refers to a contact or channel
or other means that allows fluid contact between the referenced
components. Thus, for example, a sampler washer and an absorbent
material are in communication if fluid transport can occur from the
sampler washer into the absorbent material. The term does not imply
that fluid is actually present, but only that such fluid contact
could occur if fluid were present.
[0012] In preferred embodiments, the device incorporates a target
material precipitating dye, preferably a protein precipitating dye,
for example, Ponceau-S dye. Such a dye binds to and precipitates,
or assists in precipitating or keeping out of solution a target
material. The sample collection surface of the sampler can be
contacted with the dye ( in solution or dry) in a manner such that
a quantity sufficient to dye target material in a sample is taken
up by the sampler. In using such dyes, it is generally advantageous
to separate bound dye from unbound dye to provide convenient
detection of the presence of target material. Thus, preferred
embodiments using such dyes employ an arrangement where the
collected sample (which may contain target material) is or can be
disposed between reservoirs such that wash solution can pass
through or over a solid matrix carrying the collected sample. For
example, the collected sample can be disposed between an absorbent
material able to absorb wash solution and an absorbent material or
other reservoir containing a wash solution. The saturation
differential between these reservoirs provides for a directional
transport of dye and wash solution across the collection pad
surface. Preferably the wash solution is drawn through or over a
matrix bearing a collected sample by capillary action. In
embodiments where the collection surface and dry absorbent material
are in direct contact, the dry absorbent material should have at
least enough capacity to absorb sufficient dye and wash solution to
wash one sample collection surface. In other embodiments, rather
than involving capillary action in an absorbent material to draw
wash solution through a sample-bearing matrix, a wash utilizes
user-applied pressure that pushes wash solution through the sample
bearing matrix.
[0013] The term "matrix" refers to a solid material suitable for
retaining dye/target material complexes. In the context of this
invention, a matrix is preferably, but not necessarily a porous
matrix or porous material, meaning that the matrix is penetrated by
a large number of passages of sufficient size to accept the passage
of fluids such as water, but are preferably not so large that the
matrix is free-draining. Such a porous matrix may be, for example,
a network of interwoven fibers such as paper, cotton swab, or felt.
Thus, the absorbent materials utilized in this invention, for
example, for absorbing fluids to provide a flow through a sample
collection surface are porous matrices or materials.
[0014] In the context of entrapment of complexes of target material
and dye and the removal of unbound dye, the term "wash" or
"washing" refers to a fluid transport of sufficient unbound dye to
enhance the detection of complexes. It is understood that, in many
cases, excess washing of dyed materials can remove bound dye in
addition to unbound dye. Therefore, the washing is not so extensive
that removal of bound dye interferes with the detection of the
presence of target material using detection of the presence of dye
retained in or on a solid support or matrix.
[0015] Preferably the sample collection matrix is an absorbent
material, e.g., an absorbent pad, or the surface of an absorbent
pad. In certain embodiments, the sample collection matrix binds the
target material, in others the sample collection matrix entraps
precipitated target material or the surface of the matrix retains
dye/target material complexes.
[0016] In yet other embodiments, the device is arranged such that
the sample collection matrix of the sampler is washed by wash
solution by diffusion, which may be assisted by physical agitation.
Generally in such embodiments, the sampler would then be removed
from the dye-bearing wash solution. The target material, e.g.,
protein, would be bound to, entrapped by, or otherwise immobilized
on or in a portion of the sampler.
[0017] By "entrapment" or "entrap" is meant a physical association,
which may be chemical, electrostatic or steric in nature, such that
a target material is retained in a matrix even in the presence of
forces that otherwise might have a tendency to remove such target
away from the matrix. This can occur, for example, through
precipitation of target material such that the material becomes
insoluble, e.g., using precipitating dyes such as Ponceau-S. In
this way, washes may be performed to separate small, unreacted or
unbound dye molecules from larger, dye/target material complexes,
thus facilitating testing of samples.
[0018] The term "precipitate" or "precipitation" as used in the
specification and claims includes the usual understanding of
precipitation as a settling or deposit of solid particles out of
solution. Additionally, the term as used herein also includes any
general retention of solid or particulate matter, by any force,
within, or in some cases on, an absorbent collection pad matrix or
sampler or other solid phase surface. Thus, the definition includes
but is not limited to target matter coming out of solution, target
material agglutination, and target material conformational changes
that act to obstruct the exit of these materials out of a matrix by
creating complexes or other physical structures which cannot
readily move through the pores of a porous material. Thus, those
skilled in the art will readily be able to select appropriate
materials and conditions for precipitation or other entrapment of a
particular target material/dye combination, e.g., selection of a
porous material with an appropriate average pore size which will
allow target material to penetrate into the porous material, but
small enough to prevent dye/target material complexes from quickly
being transported out of the porous material.
[0019] In embodiments making use of a target material precipitating
dye, e.g., protein precipitating dye, the dye stains/colors and
immobilizes target material, e.g., a protein (e.g., protein
adsorbed to or precipitated on an absorbent swab or pad). In this
context, "immobilizes" means that the target material is removed
from or prevented from entering the bulk of a solution (e.g., a dye
solution or wash solution), such as by precipitation of the target
material/dye complex, entrapment of the target material and/or
target material/dye complex, or attachment of the target material
to an insoluble or solid material, e.g., a particle, matrix, or
support. In embodiments where target material binds to a matrix or
surface, a precipitating dye need not actually precipitate a target
material as it is immobilized by the binding to the solid matrix or
support.
[0020] Certain of the embodiments described below demonstrate that
the scope of the invention may also contemplate some minimum
manipulation of device components and devices in which the device
does not remain sealed after sample insertion and/or in which
separate manipulation of one or more device components is
needed.
[0021] Thus, the device in one embodiment includes a sampler for
collecting a target material or contaminant, a signal generator for
providing a target material binding dye, a sampler washer for
washing unbound dye away from dye which is bound to target
material, and at least one housing to contain the signal generator
and sampler washer reagents.
[0022] In preferred embodiments, the sampler may take the form of a
wand, stick or any other configuration that is suitable for taking
up a particular type of sample. Such a sampler wand is generally a
on-absorbent stick, preferably flattened, with a sample collection
pad or surface on a terminal portion of the stick. The stick may
also have an absorbent material in communication with the sample
collection pad, e.g., on the other side of the same end of the
stick, with communication through a hole or holes in the stick.
Thus, in these configurations, there is an absorbent pad or
material which is, or is adapted to be, juxtaposed to the
collection surface for drawing dye and/or wash solutions across the
target material, e.g., a protein contaminating a surface, and can
facilitate entrapment of this target material on or within the pad
matrix. The device incorporates a sampler washer to wash unbound
dye from the sampler collection pad, preferably into an absorbent
pad or reservoir, which in some embodiments, such as the wand, is
juxtaposed to or integral with the sampler collection pad, and in
others is an elongated extension of the sampler collection pad or
an abutting absorbent material housed by a sampler stick or housing
and providing for a flow of wash solution across the sample.
Optionally, the device further includes a wetting agent or solution
which can be utilized to wet the sample collection matrix or
surface. Such wetting can assist in sample collection and/or in
picking up a quantity of dry dye. A wetting solution can be the
same or different from the wash solution. Thus, in applications
where a moistened sample collection matrix is desired, the sample
collection surface or matrix can be premoistened or can be
moistened using a wetting solution.
[0023] In certain embodiments such as embodiments including a
sampler wand, the sampler, or a portion of the sampler may be
inserted into a "book" or hinged or flexible housing having a dye,
wetting, and wash reagents, wherein a swabbed sample is subjected,
either successively or at once, to the dye and wash reagents to
effect not only a transfer of dye to the target material, but also
the transfer away or separation of unbound or unreacted dye from
target material. This is accomplished, for example, by using a
saturated dye reservoir or pad or dry dye source, and a washing
solution reservoir or pad contained in a housing, placing the
sample to be tested successively on the dye and washing solution
sources, and opposing the sample from the opposite side with a
nonsaturated absorbent material which, upon proper stimulation, can
receive fluid from the saturated source, in the process "washing"
residual dye away from bound sample. Stimulation in this embodiment
may occur by exerting pressure, for example by squeezing the
sandwiched sample. It is not necessary that the reagent housing be
hinged in book format; it could also be performed using separate
and opposing saturated and nonsaturated absorbent materials.
Alternatively, the sample is not opposed from opposite sides but
rather is placed into a reservoir containing wash solution which
flushes unbound dye from the sample collection pad.
[0024] As indicated above, in particular embodiments, a sampler
wand may be constructed either with a sample collection pad but no
additional absorbent material, or with both a sample collection pad
and an absorbent material for drawing fluids through the sample
collection pad, e.g., with a sample collection pad on one side in
communication with an absorbent pad on the other side. Generally a
sampler wand has a handle, preferably made of a non-porous material
such as various plastics, coated papers, glass, or metal.
Preferably the handle is at least two inches long, and more
preferably 4, 6, or 8 inches long.
[0025] In other embodiments, such as ones including a sampler
stick, there is no sandwich of the type described above. Rather,
the body or housing of the sampler is hollow or integral with an
internal reservoir adjacent or connected to the sample collection
surface for receiving or flushing the dyed sample of unbound dye
with washing solution.
[0026] Thus, the term "sampler stick" refers to an elongated
housing structure which includes a sample collection surface or
pad, and at least one reservoir. For example, a sampler stick may
contain a wash solution, and optionally a wetting agent, along with
a sample collection pad. The wetting agent or wash solution may be
in continuous communication with the collection pad or may be
separated with a separator until communication is desired.
Alternatively, a sampler stick can contain a reservoir with dry
absorbent material for absorbing wash solution in communication
with the sample collection surface. Exemplary sample sticks are
shown in FIGS. 3-5.
[0027] In certain embodiments, the device includes a housing with a
plurality of reservoirs, e.g., three reservoirs containing wetting
agent, dye (dry or in solution), and wash solution. After taking up
a sample and a quantify of dye, the sample collection surface of
the sampler is merely pressed against a wash solution reservoir to
flush unbound dye from the sample, as the sampler itself, unlike
the embodiments above, possesses a complementary receiving
reservoir. The sampler stick formats, exhibited herein, are
illustrative, but not limiting. As an alternative, the reservoir in
the sampler stick can contain wetting/wash solution, and, following
sample and dye uptake, the sample collection surface is pressed
against an absorbent material in a housing to cause a flow of wash
solution across the sample.
[0028] Such sampler sticks can utilize a housing containing
reservoirs in a planar arrangement, e.g., as shown in FIGS. 3 and
4, or can use a housing in the form of a cap (e.g., FIG. 5). For
example, such a cap can be reversible, such that in one orientation
the cap seals and/or protects the sample collection surface. In the
opposite orientation, the cap provides contact with dye and wash
solution. Those skilled in the art will recognize that a variety of
arrangements can be used to provide moistening of the sample
collection surface, dye uptake or transport onto or into the sample
collection surface and matrix, a source of wash solution, and a
complementary absorbent material to receive wash solution as it
washes the sample of unbound dye.
[0029] In some embodiments, certain of the reservoirs and/or
reagents are contiguous or adjacent but separated by rupturable
membranes or separators that, when broken, permit the flow of
reagents across a collected/exposed sample to effectively wash the
sample. FIG. 4 is exemplary but not limiting.
[0030] Some embodiments make use of solid dye which is hydrated and
presented to a sample in response to a physical stimulation such as
a rupturing of a membrane or membranes which maintain the dye in a
dried, segregated state. FIG. 4 is illustrative, although by no
means intended to be limiting. (The combined dye/wash solution
reservoir in FIG. 4 could contain dry dye or a dye solution.) For
example, a moistened sample collection surface can be touched to a
dry dye such that a quantity of dye is transferred to the sample
collection surface. The dye can contact target material directly
and/or by fluid transport through a porous matrix to contact target
material within the porous matrix.
[0031] The foregoing embodiments preferably utilize the properties
of precipitating dyes. As illustrated by those embodiments, the
invention also provides methods of using such dyes to fix or retard
the egress of target materials, e.g., protein, from porous matrices
into which target material has already been introduced, e.g. by
swabbing. Thus, the introduction of target material as contemplated
by the instant invention is not facilitated by or dependent on
movement of particles or molecules in an electric field. Likewise,
the method does not utilize a separation of components of a sample
due to differential migration within the porous matrix. Instead,
the matrix need merely be compatible in size to allow the initial
ingress or association of target with matrix, and the influence of
dye acts to thwart or inhibit the target material from leaving the
matrix. This may be due, for example, to precipitation,
conformational changes, agglutination, or any other result of dye
binding which has the effect of sufficiently immobilizing target
material in the porous matrix that unbound dye can be washed away
and dyed target material visualized in the matrix. Alternatively,
the immobilization can be due to chemical or electrostatic binding
of target material to matrix. Further, matrix constituency is
irrelevant as long as the criteria described above are met and as
long as the dye is otherwise compatible with, or can be made
compatible with, the matrices, e.g. with neutralizing agent.
Illustrative but not limiting of the possible materials that may be
used for the porous matrices are those discussed infra under the
definition of "sampler". The dye should not bind to the matrix
material to such an extent that dye bound to target material in the
matrix cannot be distinguished from dye binding to matrix.
[0032] In accord with the aspects above, the immobilization or
entrapment of target materials with a solid matrix, e.g., in a
porous matrix, provides a method for detecting the presence of
target material in a sample. As previously indicated, the method
involves entrapping or otherwise immobilizing target
material/precipitating dye complexes on or within a solid matrix.
For example, such complexes can be entrapped in a porous matrix by
binding of target material with precipitating dye or by collection
of dye/target material complexes on or in a collection surface or
porous matrix. Generally, the method includes washing away unbound
dye to allow convenient visualization or other detection, e.g.,
detection using an instrument such as a spectrophotometer or
fluorometer. The method can involve various matrix materials,
neutralizing agents, wash solutions, and dyes as described herein
for other aspects.
[0033] For the methods herein involving precipitating dyes, azo
dyes, preferably diazo dyes, which preferably have at least one,
and preferably a plurality of sulfonic acid groups, e.g., 2, 3, or
4 groups (which may be prepared in the corresponding salt form) are
preferred. The red dye, Ponceau-S, Sigma Chemical Co., St. Louis,
Mo., (chemical abstracts service registry number 6226-79-5,
[3-hydroxy-4-[2-sulfo-4-(4-sulfophenyl- azo)
phenylazo]-2,7-naphthalenedisulfonic acid, tetrasodium salt],
HOC.sub.10H.sub.4[N.dbd.NC.sub.6H.sub.3(SO.sub.3Na)(N.dbd.NC.sub.6H.sub.4-
SO.sub.3Na)](SO.sub.3Na).sub.2, F.W. 760.58) is exemplary and most
preferred. Ponceau-S is soluble in water, slightly soluble in
ethanol, and insoluble in vegetable oils. It is stable at room
temperature in acetic acid and in preferred embodiments is used to
stain proteinaceous matter using a dye concentration of about
0.1-1.0% (w/v) in about 1-5% (w/v) acetic acid. The stain may be
quickly removed upon addition of 0.1 N NaOH, or by excess wash
solution. The terms "azo dye" and "diazo dye" have the meanings as
generally accepted in the dye industry. The term "sulfonated" in
connection with the dye compounds refers to the presence of
sulfonic acid substituent groups. Such groups may be present in a
corresponding salt form.
[0034] Advantageously, Ponceau-S binds rapidly to proteins and
precipitates or immobilizes them in addition to staining/coloring
them. Thus, such a precipitating dye is generally used to bind to
and precipitate target material, e.g., protein, in or on a solid
matrix. In such case, unbound dye is generally washed away from
dye/protein complexes, providing visual detection of sample
protein. In such embodiments the dye does not bind to the solid
matrix to such an extent or under such conditions as to prevent or
interfere with detection of dye/protein complex.
[0035] The invention provides a method for detecting protein on a
solid surface. Preferably the method is applied in testing for
contamination on the surface, e.g., food processing residue. The
method involves contacting a solid surface, e.g. a metal surface,
with a Ponceau S dye solution under conditions in which Ponceau S
dye binds to protein. The rapid binding of Ponceau S to protein
allows sufficient dye to bind to protein even on vertical surfaces.
Preferably the method allows immediate visualization of
protein-bound dye on the surface without further processing. If
desired, the method can further include washing the surface with a
wash solution which can wash away unbound dye. Preferably the dye
is used at a concentration of 0.1-1.0% in dilute acetic acid. The
dye solution and/or a wash solution can further contain
neutralizing agent as described above.
[0036] Also in preferred embodiments of the present invention, the
binding of dye to target material is detectable by a color change
of the dye or dye solution, e.g., by a frequency shift of the dye
on binding or a color change of a dye solution by dye depletion.
Preferably a self-contained sampling/testing device incorporates a
frequency shift dye. Frequency shift dyes have their absorbency or
reflection or emission changed on interaction with target material,
thereby differentiating bound from unbound dye. Such dyes can allow
convenient detection of target material even without separation of
bound and unbound dye.
[0037] Therefore, in preferred embodiments, the sample wash is able
to transport sample material, e.g., target material from the
collection portion or surface of the sampler. Device embodiments
wherein liquid-phase analysis is performed typically employ a
reading portion of the device that permits the sample reaction to
be visualized or analyzed, with or without the aid of an instrument
such as a spectrophotometer. The sample material is washed into the
reading portion or alternatively carried into the reading portion
on the sampler.
[0038] In certain embodiments employing frequency shift dyes or
other dyes where a change in dye color is to be detected, the
sampler may be contained within a lower housing that provides
protection for the sampler from pre-testing contamination.
Additionally, an upper housing may sealably engage the lower
housing such that the two housings are in communication during the
test. The sampler is preferably fixed to the upper housing. Within
or comprising such housings may be a chamber or reservoir to hold a
wash solution or a combined sample wash signal generator or
separate reservoirs to hold each of a signal generator and a wash
solution. A chamber may further include a breakable shaft
contiguous with the chamber that, upon breakage, exposes an orifice
through which the contained solution may flow to the sampler and
may further flow to a read portion for evaluation.
[0039] Thus, the dye solution can flow through a hollow shaft in a
swab in the device, flow through the swab tip removing the material
adhering to the swab, and collect in a chamber of the device which
is usable by the user for visual or instrumental detection of the
intended dye reaction. Alternatively, the dye solution can wash a
sample on or in a swab or other sampler, where the target material
is detected on or in the sampler.
[0040] Frequency shift dyes provide convenient detection of bound
dye even in the presence of unbound dye when the frequency shift is
large enough to distinguish the two. In cases where an instrument
is to be used to read the binding results, the frequency shift can
generally be smaller than if a visual reading is to be utilized.
For machine reading, preferably an absorption shift on binding
(expressed as a wavelength shift) is at least 20 nanometers, more
preferably at least 50 nm, still more preferably at least 75 nm,
and most preferably at least 100 nm. For visual reading, preferably
an absorption frequency shift on binding is at least 50 nm, more
preferably at least 75 nm, still more preferably at least 100 nm,
and most preferably at least 120 nm. For example, an absorption
peak of Coomassie blue stain under acidic conditions shifts from
about 465 nm to about 595 nm on binding of the dye to protein. For
a visual reading it is preferable if the absorbance change produces
a color change rather than just a shade change. For example, the
GelCode.RTM. reagent changes from amber to blue on protein binding.
A fluorescent emission shift is preferably at least 20 nm, more
preferably at least 40 nm, still more preferably at least 75 nm,
and most preferably at least 100 nm.
[0041] Gelcode.RTM. includes colloidal Coomassie.RTM. G-250 dye.
Colloidal Coomassie.RTM. blue dyes may also be formed as described
in the art. For example, in Neuhoff, et al., 1985, Electrophoresis
6:427-448 and in Neuhoff, et al., 1988 Electrophoresis 9:255-262.
In general, these solutions utilize Coomassie blue dye.RTM. in an
acidic aqueous solution with ammonium sulphate or ammonium iron
sulphate. In one example, the solution contains 0.1% weight/volume
(w/v) Coomassie blue G-250 in 2% w/v phosphoric acid, and 6% w/v
sulphate. In an alternative solution, the dye contains 10% w/v
ammonium sulphate and 20% w/v methanol. Preferably, the pH of the
solution is between 1 and 2 and the ammonium sulphate or ammonium
iron sulphate concentration is between 2% and 15% more preferably
between 4 and 10%, and most preferably between 5 and 8% w/v. The pH
should not be so low that the dye molecules are rapidly degraded
and the ammonium sulphate concentration should be selected so that
the solution takes on a color characteristic or the colloidal form,
preferably the majority of the dye molecules are present as
colloidal particles rather than being in free solution or
precipitating out of solution.
[0042] As appreciated by one of ordinary skill in the art, certain
device embodiments will accommodate the use of various types of
target material binding dyes, e.g., precipitating dyes or frequency
shift dyes. For example, embodiments which utilize a wash to carry
sample material away from a sampler can be utilized with a
frequency shift dye. Such devices can also be used with a
precipitating dye where there is the capability to wash unbound dye
away from target material bound dye in or on the sampler or on a
porous separator. In accord with certain embodiments described
herein which incorporate a wash solution in a reservoir in a
sampler portion or upper housing, sample can be collected on a
sampler, contacted with dye, e.g., from a reservoir in the sampler
portion or upper housing or by dye contained in the sampler prior
to sample collection, and then washed by a wash solution contained
in a reservoir in the sampler portion or upper housing. Preferably
in such embodiments, the collection surface of the sampler is
pre-moistened. Similarly, a device in which a dye solution is in a
reservoir contactable with a sample collection surface (see e.g.,
FIG. 2 below) can be used with a frequency shift dye or with a
precipitating dye. With either type dye, sample material is
transferred to contact the dye on the sampler. For a frequency
shift dye, dye binding to target material is detected by a color
change of the dye as dye binds target material in the dye solution
or on the sampler, or as dye is depleted from the solution as dye
binds to target material in or on the sampler.
[0043] The terms "sampling/testing device" or "self-contained
sampling/testing device" indicate that the device is constructed so
that all components for a particular assay are provided within a
single device along with a means for introducing a sample into the
device. It may, however, be advantageous for certain embodiments to
utilize separate apparatus for incubation during the assay or for
reading results of the assay.
[0044] By "sampler" is meant a device component (or components)
which allows one to obtain all of or a portion of a sample which
may be present on a surface, in a solution or in an atmosphere to
be tested. For example, the sampler may be an absorbent pad or a
swab with a shaft and an absorbent tip. The shaft of the sampler or
the sampler stick housing may be hollow, and may further include a
vent. As alternatives, the sampler/swab may take the form of a
Q-Tip.RTM. or a simple pad. The swab may include natural or
synthetic materials so long as deposition of a sample thereto may
occur and dye binding to the swab does not interfere with detection
of the target substance so as, to prevent such detection. The
absence or reduction of such interference may be provided, for
example, by selection of material and/or by the physical
interrelationships of device components. The material may be but is
not limited to sponge, mylar, nylon, dacron, rayon, porex, porous
polypropylene, porous polyethylene, glass fibers, paper, or various
other woven or felted fibers such as nitrocellulose, cotton, wool,
cellulose, or combinations thereof. In a preferred embodiment in
which the swab includes a shaft, the swab shaft is preferably
hollow, allowing the sample wash and/or dye solution to flush the
collected sample material from the swab into a reaction and/or read
chamber. Preferably the swab is provided for use in a pre-moistened
form to assist in solubilizing and absorbing sample material into
the sampler, or can be readily moistened from a reservoir within
the device containing a wetting solution, e.g., in a saturated
absorbent matrix. The moistening fluid may be a buffer, water,
acid, or base depending on the type of dye used. Those skilled in
the art understand the selection of a compatible moistening fluid
for the dye and target material involved in a particular type of
test. The sampler may function through capillary action, for
example a capillary tube or tubes. The sampler may comprise a
pipetting means. The sampler may comprise a chamber that captures a
sample of an atmosphere, such as the atmosphere present in an
enclosed work space. The sampler may assume virtually any shape or
combination of shapes, e.g., planar, elongated, circular,
elliptical, cylindrical, spherical, cubical, conical, etc.
Preferably the sampler is designed to enable a user to conveniently
reach into locations in equipment, such as food processing
equipment which are difficult to access. Thus, the sampler is
preferably constructed to provide an extension with a thin
cross-section, e.g., a cross-sectional area of less than 2
in.sup.2, more preferably less than 1 in.sup.2, and in certain
embodiments less than 1/2 in.sup.2. Such extension is preferably at
least 2 inches in length, more preferably at least 4, 6, or 8
inches in length. Such extension may be provided, for example, by a
wand handle, a swab shaft, or an elongated housing, or
combinations.
[0045] The term "sampler portion" refers to a structural assembly
which includes a sampler and also includes additional components
which allow the sampler to be sealed or attached to the remainder
of the device, and may also include one or more reagent spaces,
such as a reservoir for a sample wash solution.
[0046] By "sampler washer" is meant a device component (or
components) which allows the removal of all or a part of a sample
present on the "sampler". For example, in some embodiments an upper
housing or sampler portion or sampler stick comprises a chamber as
a reservoir containing a fluid in which the fluid may be
selectively released as desired, ordinarily to release a sample
that has been obtained or to wash unbound dye away from dye/target
material complexes. In an alternative embodiment, the upper housing
or sampler portion may contain a container such as, but not limited
to, an ampule or a packet. The ampule or packet may contain a fluid
as described above, which may be selectively released. In an
alternative embodiment the upper housing or sampler portion may
contain two containers, both or either comprising, for example but
not limited to, an ampule or packet containing the same or
different fluids or dry substances. In another embodiment a fluid
may be directly contained in the upper housing or sampler portion
and a container or containers containing a fluid or dry substance
(or more than one fluid or dry substance) be contained therein. In
yet other alternatives, the lower portion of the housing or the
lower housing may contain a fluid that is used to wash the sample
from the sampler, for example, by inserting the end of the sampler
into the fluid or otherwise forcing the fluid against or through
the sampler.
[0047] By "wash solution" is meant a solution, e.g., an aqueous
solution, capable of separating unbound dye from dye/target
material complexes and/or carrying sample materials from a sampler
to another location. The solution may also serve the function of a
wetting agent, e.g., for moistening a swab or collection surface in
anticipation of or facilitation of a sample collection. A wash
solution for use with a particular dye does not contain such amount
of agents which tend to disrupt binding of that dye to target
material that determination of target material binding is
prevented. Preferably no such agents are present, but in some cases
it may be desirable to include a low level of such an agent or
agents, for example, to minimize binding of dye to a porous matrix,
thereby enhancing contrast and improving target material detection.
Those skilled in the art will readily be able to select an
appropriate wash solution for a particular dye. In embodiments
utilizing Ponceau S dye, the wash solution and/or wetting agent are
preferably dilute acetic acid solutions, preferably with 0.1 to 10%
acetic acid in water, more preferably 0.5 to 5%, still more
preferably 1.0 to 5% acetic acid.
[0048] By "signal generator" is meant a chemical compound or
physical stimulus or biological agent that provokes a measurable or
discernable response in the presence of a target material; by
chemical compound is meant a chemical dye, an enzyme, or other
organic or inorganic structure capable of inducing such
response.
[0049] The term "target material binding dye" or "dye" refers to a
compound which will preferentially bind to a target material in a
sample as compared to binding to other molecules which are likely
to be present in such samples. Thus, the dye may bind to other
molecules at a level equal or greater than that for binding to the
target material, but such other molecules are ones which are
generally not present in samples to be tested, such as samples to
be tested in evaluating process contamination. The preferential
binding need not occur under all conditions, but at least occurs
under the assay conditions selected for use in the device of the
present invention. The dye compound also is detectable using visual
or spectroscopic means, and preferably absorbs or fluoresces at
visible wavelengths so as to give a characteristic color. Binding
of the dye to target material, e.g., protein, preferably results in
a color change and/or precipitation of the protein that is visible.
Thus, for example, the term "protein-binding dye" refers to a
compound that preferentially binds to protein, polypeptides, or
oligopeptides in preference to other molecules. The dye, while
active in aqueous form may be initially dry and hydrated during the
testing process, e.g., when contacted with wetting/washing
solution.
[0050] By "frequency shift dye" is meant a composition which upon
interaction with the substance to be detected exhibits a
characteristic detectable change in the light emission or
absorption spectrum of the dye molecule. Preferably the alteration
of the light absorption characteristics of the dye molecule is
observed. Changes in absorption or emission spectra can include,
for example, the appearance or increase of absorbance or emission
peaks or bands, the disappearance or reduction of absorbance peaks
or bands and combinations of these. Preferably the frequency shift
dye is a protein binding dye that is colloidal such as
Coomassie.RTM. blue dye, preferably GelCode.RTM. Blue Stain
Reagent, Pierce Chemicals, Rockford, Ill.
[0051] The term "colloidal dye" refers to a dye which is in a
finely divided state in a liquid, such that the solid particles of
dye are in the range of 1 to 1000 nanometers, preferably in the
range of 1-100, and more preferably in the range of 5-100
nanometers. This does not mean that all of the dye present in the
liquid is in the form of such particles, as those skilled in the
art recognize that the colloidal form is generally in thermodynamic
equilibrium with solubilized dye and/or with solid dye particles
larger than colloid size, e.g., larger than 1000 nanometers. Most
useful are colloidal dyes where the amount of dye in colloidal form
is sufficient to alter the color of the dye solution as compared to
solutions containing the same amount of dye but in which the dye is
in solution and/or in non-colloidal particles. In preferred
embodiments, at least 30% of the dye molecules are in colloid size
particles, preferably at least 50%, and more preferably at least
70% or 90%. As recognized by those skilled in the art, a transition
from soluble form to colloidal form of a molecule in liquid
solution can be monitored by an increase in light scattering for
the solution.
[0052] The term "swab" as used in the claims is used as a noun to
denote an absorbent and/or adhesive pad that serves to collect
sample target material in prelude to or concurrent with exposure to
a signal generator, i.e., a dye.
[0053] By "neutralizing agent" is meant a chemical compound or
solution that helps to neutralize potentially interfering compounds
present on the surface being tested or in a wetting agent present
in or on a sampler collection surface, which compounds may
interfere with the dye binding to the target material, e.g.,
protein. Exemplary neutralizing agents include sodium thiosulfate,
MgCl.sub.2, and Triton-X.RTM. (Octoxynols;
.alpha.-[4-(1,1,3,3,-Tetramethylbutyl)phenyl]-w-hydroxypoly(oxy-1,2-ethan-
ediyl). All are available from Sigma, St. Louis, Mo. Triton-X.RTM.
can be used, preferably at an effective concentration of about
0.01-0.5% weight volume. Sodium Thiosulfate may be used, preferably
at an effective concentration of about 0.01-1.0 mg/ml. MgCl.sub.2
is preferably used at a concentration of 0.1-20 mg/ml..These
neutralizing agents may be incorporated into any, all, or a
combination of the wetting, wash, dye, or sample solutions
provided. One of skill in the art will recognize that other
neutralizing agents may be substituted provided they do not
interfere with the signal generator mechanism and measurement, and
will understand what neutralizing agents are appropriate for a
particular application or can determine whether or not a potential
compound is appropriate by simple testing.
[0054] Thus, by "neutralize" is meant to inactivate potentially
interfering compounds present on the sample surface without
disrupting the signal generator's function in combination with the
target material and the rest of the device.
[0055] By "effective concentration" is meant one that supplies, in
whole or in part, the intended or desired effect, e.g., the desired
neutralizing effect.
[0056] The term "participates" as used in the claims denotes an
assistance in the movement and/or gathering of target material onto
the sampler, for instance by pre-moistening of an absorbent
collection swab or pad.
[0057] By "reading portion" is meant a distinct section of the
device housing wherein a reading or measurement or detection may be
taken.
[0058] The term "in succession" connotes a temporal order but does
not preclude the use of wash solution as a wetting agent for a
sampler swab in prelude to exposure to dye. Thus, the wash solution
may be used twice, both before and after the dye.
[0059] The terms "contacted with", "contacted by" or "on contact
with" denotes the direct or indirect touching of one object with
another. Certain embodiments have the contact mediated through a
pierceable membrane, which is rupturable by the sampler to effect
the dyeing and washing of a target material presented on the
sampler.
[0060] The term "segregates" as used herein denotes a separation
and/or containment which may be undone upon proper stimulation, for
example the piercing of a membrane by a sampler to allow mixing of
components from each side of the membrane.
[0061] By "stably packaged" is meant that the dye or other signal
generating component may be stored prior to use for prolonged
periods of time, for example, a year or more if stored at 4.degree.
C., and still provide a signal upon activation. In one embodiment
of the invention the signal generating means, e.g., comprising a
colloidal dye solution, is stably packaged within a sealed glass
ampule. The ampule may be a borosilicate glass, for example
Pyrex.RTM.. It may be an "onionskin" type of glass ampule. In other
embodiments, a signal generating component, e.g., a dye, is sealed
within a chamber by a membrane or membranes.
[0062] As understood by those skilled in the art, the stability of
a dye molecule will depend on the storage conditions, thus, the
storage form can be varied as appropriate for a particular dye. If
the dye is sufficiently stable in the test solution, the dye
solution can be packaged in the device as a single solution.
Alternatively, if the dye is not sufficiently stable in the assay
solution, the stability can be enhanced by packaging the dye within
the device separated from one or more other components of the test
solution until mixing is desired. Thus, for example, the dye and/or
components decreasing dye stability can be separated within the
device by any of a variety of methods, such as by using separate
reservoirs or capsules or ampules or separators or combinations
thereof, such that one or more can be ruptured, broken, or opened
to allow mixing of various components at a desired time or
times.
[0063] By "separator" is meant a device component(s) or structure
for separating two portions of the device, e.g., for separating the
region containing the sampler from the region in which detection is
performed until introduction of the sample into the detection
region (read portion) is desired or for separating a sample on the
sampler from a dye solution until contact between the sample and
the dye is desired. For example, a separator may be a porous
plastic or hydrophobic material filter, however, the porosity is
not such that the sample would filter through without the
application of a force, other than gravity, on the sample. As
further examples, the separator may be a one way valve, or a
puncturable membrane or a breakable or rupturable reservoir or
capsule or ampule.
[0064] A "reservoir" may be a well, ampule, recess, void, or
chamber capable of holding a liquid or solid. Such reservoir may be
encased or contained by a rigid, soft, or flexible housing such as
a plastic. A reservoir may be or include an absorbent pad that is
saturated or capable of absorbing solution or solutions, e.g.,
target material, dye, wash, wetting agent, or combinations thereof.
Such absorbent material is preferably located in a depression,
void, chamber, cavity or the like of a housing.
[0065] Preferably the dye and test conditions are selected such
that a readable result is provided within one hour at room
temperature, more preferably within 30 minutes or 20 minutes, still
more preferably within 10 minutes or 5 minutes, and most preferably
within 2 minutes or 1 minute. Such rapid results are particularly
advantageous for field sanitation testing as retention of the
samples for long periods of time is not required and stability or
consistency of the read of the completed test is enhanced.
[0066] As indicated above, a particularly advantageous embodiment
of the present invention is adapted for protein detection, and
therefore provides for the rapid and convenient testing of surfaces
or solutions for contamination by protein-containing substances.
The presence of protein can be a good indicator of residual food
contamination remaining after cleaning procedures have been
completed, as protein is a component of many food products. For
example, in one application, the device or method will allow for
testing of surfaces in food production plants, supermarkets and
restaurants to ensure that cleanup procedures after food processing
have been effective. Certain preferred embodiments utilize a dye
capable of precipitating as well as staining protein, for example,
Ponceau-S. Ponceau-S is particularly useful due to its speed of
staining as well as its ability to both precipitate and stain
protein. Other embodiments are slower and utilize colloidal protein
binding dyes via a single step, integrated sampling assay device
with visually distinct color changes in the presence of small
amounts of protein material, e.g. colloidal Coomassie.RTM. blue
such as found in GelCode.RTM. Blue Stain Reagent. Colloidal
Coomassie Blue imparts a convenient spectral or color shift in the
presence of protein.
[0067] By "protein" is meant peptide polymers (i.e., polymers of
amino acids) and thus includes oligopeptides, full-length
cellularly-produced polypeptides, degraded cellular polypeptides,
complexes of polypeptides, and polypeptides associated with other
molecules.
[0068] In preferred embodiments, the results of a test using the
device can be read visually. In other embodiments, the result can
be read in an instrument, such as a spectrophotometer or
colorimeter. These devices are most useful for applications
employing frequency shift dyes.
[0069] The device in a preferred embodiment includes a sampler and
a combined sample treatment, sample wash and signal generator
stably packaged, preferably allowing easy visual
interpretation.
[0070] By "a combined sample treatment, sample wash and signal
generator" is meant components or structures to contain a target
material binding dye, which preferably either precipitates and
stains proteins, or else creates a frequency shift on contacting
protein, e.g., colloidal dyes. In either case, the dye solution can
be released at will to wash a sample on or from the sampler thereby
signaling the presence, absence, or quantity of protein present. In
preferred embodiments the solution collects in a reading portion of
the device. The dye solution or wetting agent can also treat the
sample in a desired manner, for example, by solubilizing or
permeabilizing cell walls and/or membranes of microorganisms (e.g.,
bacteria and fungi) or other cells.
[0071] By "fix" is meant that target material in the presence of a
precipitating dye, e.g. Ponceau-S, is relatively slowed or halted
from diffusing from or otherwise exiting the porous matrix in which
it has entered or is contained, as compared with target
material/matrix in the absence of dye.
[0072] Other specific applications of the invention include but are
not limited to the following: the testing of surfaces for other
types of contamination such as carbohydrates, lipids and
microorganisms; the testing of liquid solutions for the presence of
proteins, carbohydrates, lipids and microorganisms; the testing of
air or gas for proteins, carbohydrates, lipids and microorganisms;
and the testing of other materials such as dirt, vegetable
material, manufactured articles, spices, powders, chemicals, debris
and other types of samples familiar to those skilled in the art for
such contaminants as protein, carbohydrates, lipids, nucleic acids,
microorganisms, toxins, poisons, byproducts, adulterants and other
materials recognized by those skilled in the art and capable of
binding to dyes or ligands capable of being contained in colloidal
or other forms which sequester or contain such reagents so that
reaction with specific target materials results in or can be
stimulated to result in rapid, detectable and distinct changes.
[0073] In accord with the provision of sampling/testing devices as
described above, in another aspect, the invention provides a method
of making such a test device by depositing a target material
binding dye, preferably a precipitating-type dye or a frequency
shift dye within a reservoir in a self-contained sampling/testing
device. As indicated above, for device embodiments which include a
sampler portion and a housing, the dye solution may be deposited in
a reservoir in the sampler portion or alternatively in the housing.
In embodiments in which the device includes an upper housing and a
lower housing, the reservoir may be in the upper housing or
alternatively in the lower housing. The dye used is preferably a
precipitating dye, such as Ponceau-S. Alternatively, a colloidal
dye such as colloidal Coomassie.RTM. blue is used which exhibits a
frequency and color shift change on contact with protein.
[0074] In another aspect, the invention provides a method for
detecting the presence of a substance, i.e., a target material, by
using a sampling/testing device as described above. Thus, in
preferred embodiments, the method involves obtaining a sample which
may contain the substance to be detected (i.e., the target
material) on or in the sampler. Depending on the type of sample,
this may, for example, involve swabbing a surface, depositing a
solubilizing or suspension liquid on a surface and then taking up
at least a portion of that liquid, or taking up a sample of liquid
from a bulk liquid, such as by pipetting or in a capillary tube or
by moistening an absorbent material. The sample is contacted with a
target material binding dye, e.g., a precipitating dye or a
frequency shift dye, as described above within the device, and the
presence of the target material in the sample is determined by
detecting the occurrence of a dye color change, e.g., a dye
frequency shift, by observing a visually detectable change in the
color or shade of the dye solution following contact with the
sample or by reading a change or changes in an absorbence or
emission spectrum of the dye in a reading instrument following
contact with the sample or by detecting the presence of bound dye
on or in a matrix which immobilizes dye/target material complexes
but which allows unbound dye to be separated, e.g., by washing
away.
[0075] In particular, a method of sanitation testing is provided in
which a device as described above is used to detect the presence of
contaminants on or from a surface or in solutions, such as
following cleaning procedures on a surface. In particular
embodiments, the method involves contaminant testing of surfaces or
solutions in a food processing facility such as a food production
plant, or a restaurant. In preferred embodiments, the contaminant
to be detected is protein food residue.
[0076] Other and further objects, features and advantages will be
apparent from the following description of the presently preferred
embodiments of the invention and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] FIG. 1 illustrates an embodiment of the present invention
containing a sampler wand with an absorbent sample collection pad,
and a foldable book encompassing reservoir pads for dye, absorbent,
and wash solution.
[0078] FIG. 2 illustrates an embodiment of the invention containing
a sampler wand with a sample collection pad and an absorbent
backing, and a reagent dish with three wells: one with swab wetting
solution, one with dye, and one with washing solution.
[0079] FIG. 3 illustrates an embodiment of the invention containing
a sampler stick and a reagent dish with three wells; one with
sampler wetting solution, one with dye, and one with wash
solution.
[0080] FIG. 4 illustrates another embodiment of the invention
similar to that of FIG. 3 where the reagent tray is simplified to
two wells with the dye and wash reagents present in the same well
separated by an impermeable but rupturable membrane.
[0081] FIG. 5 illustrates an embodiment of the device where the
wash solution is contained in a sampler stick, separated from the
sampling surface by an impermeable membrane. Prior to use, the
sample collection end of the sampler stick sealably engages with a
cavity in a housing. The dye is present in the same cavity and
separated from the wash surface by a permeable membrane. The
opposite end of the housing contains an absorbent material and can
be sealably engaged with the collection end of the sampler stick
following sample collection.
[0082] FIG. 6 illustrates an embodiment of the present invention
containing a swab-type sampler in which the dye solution in the
sampler portion also functions as the sampler wash solution.
[0083] FIG. 7 illustrates an embodiment of a device in which the
housing is divided into an upper housing portion and a lower
housing portion which sealably engage, in which the lower portion
of the lower housing is set off by a separator.
[0084] FIG. 8 shows an embodiment of the device in which the upper
portion of device contains a sampler and a sampler wash solution
which contains no dye. The lower portion of the device (the
housing), protects the sampler when the two portions of the device
are sealed together. The housing also contains a separator which
divides the housing into upper and lower spaces. The lower space
contains a dye composition, preferably a dry dye.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0085] The devices of the present invention generally are
constructed such that a sample to be tested can be obtained on or
in a sampler. The device is constructed so that any remaining steps
involved in detecting the presence of a target material in the
sample can be carried out following placement of the sample-bearing
sampler within the housing without further addition of assay
components. This description generally describes embodiments which
include a precipitating or frequency shift dye, but applies also to
the use of target material binding dyes generally.
[0086] Thus, the device components are arranged so that the sample,
or at least a sufficient portion of the sample to allow detection
of the presence of target material, is contacted with a dye. The
mixture is present in, or is transferred to, a portion of the
device where the results can be read, e.g., visually or in a
spectrophotometer, a fluorometer, or other reading instrument.
Specific embodiments are described below and in the figures with
device elements arranged in particular ways. However, it is clear
that the invention also concerns devices with elements selected and
arranged in other ways to accomplish the above process. Thus, for
example, the dye solution can be located such that it is used as
the wash solution to carry sample from the sampler to the reaction
reading portion, the sample can be directly delivered into the dye
solution (e.g., by pipetting a liquid sample into the dye solution
or by inserting the sample-bearing portion of the sampler into the
dye solution), or a wash solution can carry sample from the sampler
into the dye solution. In view of the description herein, those
skilled in the art will understand how the specifically described
embodiments can be altered to provide each of these and other
formats.
[0087] Embodiments described in FIGS. 1-5 are particularly adapted
for use with precipitating dyes, and embodiments described in FIGS.
6-8 are particularly adapted for use with frequency shift dyes.
[0088] Referring to FIG. 1: the device includes a plastic housing
(3) with three wells. One well contains the target binding dye (5)
and an absorbent dye pad (4). The second well contains only an
absorbent pad (6). The third well, separated from the other two by
a hinge, contains an absorbent pad (1) and wash solution (8). The
housing (3) is covered by a foil seal (9) that is removed prior to
use. A separate sampler wand (1) incorporates a sample collection
pad (2). The sample collection pad (2) is moistened by contacting
it with the wash solution pad (7). The sampler wand (1) is then
used to swab a sample surface, e.g., a food contact sample surface,
removing and absorbing food residue into the sample collection pad
(2). The sampler wand (1) is placed into the device housing (3) and
contacted with the dye pad (4) for a few seconds to transfer dye
(5) to the sample collection pad (2). The sampler wand (1) is then
placed on the absorbent pad (6) and the wash solution pad (7)
pressed against the backside of the sample collection pad (2).
Pressure is maintained for several seconds allowing wash to be
drawn through the sample collection pad (2) into the absorbent pad
(6), removing unbound dye. The sample collection pad (2) is then
observed for the presence of color on its surface. The presence of
colored dye is indicative of the presence of target binding
material. In this embodiment, the sample collection pad is selected
such that target material will remain immobilized on and/or in the
pad matrix during a washing step.
[0089] Referring to FIG. 2: the device includes a sampler wand (1)
and a series of three reservoirs in a housing/reagent tray (17).
One reservoir contains wetting or washing agent/solution (14) used
to moisten the sample wand before swabbing the surface. The second
reservoir contains the dye reagent (15). The third reservoir
contains a wash agent/solution that may or may not be identical to
the wetting/washing agent (16). The reagents are localized in
absorbent pads (18) at the bottom of the individual
well/reservoirs. The reagent tray (17) is covered by a foil seal
(19) that is removed prior to use. The sample wand (10) comprises a
collection surface (11) that abuts an absorbent pad (3) and the two
pieces are held in place by a pad housing (12). The test is
performed by moistening the collection surface (11) of the
sampler/wand (10) in the wetting/washing agent (14). The surface to
be tested is then swabbed and the sample on the collection surface
(11) is then contacted with the dye (15) and then the excess dye is
washed away when the surface is subsequently placed into the wash
well (16). In each case the dye and wash agents are moved
to/through the collection surface by absorption into absorbent
padding or material (13).
[0090] Referring to FIG. 3: the reagent device is essentially the
same as in FIG. 2; however, the sampler (20) is in the form of a
stick instead of a wand, wherein the collection pad (23) surface
may be distinct from but contiguous with an absorbent material (22)
encased by a stick housing (21). Alternatively, the absorbent
collection pad (23) material extends down into the housing (21) and
provides an absorptive draw. Otherwise, this embodiment is
manipulated in the same manner for wetting of the collection
surface, treating the sample with the dye, and using the wash
reagent to wash away excess dye as in FIG. 2.
[0091] The device in FIG. 4 uses the sampler stick as FIG. 3, but
has the reagent housing (34) with 2 instead of 3 wells. The first
well contains the wetting reagent (35) that is used for moistening
the collection pad (33) surface, and the second well has two
compartments vertically arranged, with the dye (36) layered on top
of the wash (37). The dye is present in dry form on top of a
breakable membrane (36a). Following contact with the dye, the
membrane (36a) is pierced with the ampler stick and wash solution
(37) is absorbed into and through the collection pad. (33).
[0092] FIG. 5 is an embodiment that has the reagents in the reagent
tray/housing (46) that is in the shape and function of a cap, as
well as inside the sampler stick (40). The reagent tray/housing
(46) in this embodiment fits onto the end of the sampler stick (40)
as a reversible cap. The collection pad surface (45) is
pre-moistened with wetting agent (51). In the exemplary form, the
collection pad surface (45) is covered by the end of the reagent
tube housing (46) with a breakable membrane (47) protecting the
collection surface (45) from the dye (48) in an absorbent pad (49).
The sample stick (40) is removed from the reagent tube housing (46)
and used to collect the sample. The reagent tube housing (46) is
then put back on the sampler stick (40) after being rotated 180
degrees and the same side of the cap is placed on the sampler stick
employing the alignment guide (42). The collection pad surface (46)
pierces an initial barrier (47), thereby coming into contact with
dye (48) and taking a quantity of that dye on the collection
surface (45). Then the sampler stick (40) is put onto the other end
of the cap at which point the breakable seal (43) in the sampler
stick housing (41) is broken, allowing the wash reagent (44) in the
sampler stick (40) to migrate through the collection pad (45)
surface and into the absorbent material (50) in the cap/reagent
tray housing (46). This effectively washes away excess dye, so that
only the dye remaining on the collection surface is dye which has
been immobilized due to binding to target material, e.g.,
protein.
[0093] Referring to FIG. 6: in one embodiment the device (60)
includes a sampler portion or upper housing (61) a dye reservoir
(62) containing the target binding dye (70); an orifice (64)
communicating with the hollow swab shaft (66), exposed by breaking
off the snap plug (68); a housing (74); an absorbent swab tip (72);
and a lower read chamber or read portion (76).
[0094] Referring to FIG. 7, in a another embodiment the device
includes an upper housing (80), an upper barrier means (81) between
the upper housing (80) and the upper section (82) of a lower
housing (87). The upper housing (80) and upper barrier means (81)
define a chamber (88). A sampler (83) is attached to the upper
housing. The lower portion of the lower housing (87) forms a read
portion (85).
[0095] Referring to FIG. 8, in another embodiment. This embodiment
is as in FIG. 6, except that the dye reservoir (62), contains a
wash solution (71) which does not contain a dye. The housing
contains a foil barrier (78) (i.e., a separator) dividing the
housing into an upper section (73) and a lower section (75). The
lower section contains a dry dye (79), and forms a sealing,
slidable junction with the upper section (73). In this embodiment,
other types of barriers can be used to prevent the wash solution
from washing the sampler before such washing is desired. Similarly,
other types of separators can be used to divide the housing into
upper and lower sections. Also, the dye in the lower section can be
a dye solution or suspension rather than a dry dye. The slidable
junction between the upper and lower sections of the housing may
include a threaded surface(s) such that the upper and lower
sections may be screwed together, thereby piercing the separator
with the sampler.
[0096] In addition to the embodiments described in the figures,
additional embodiments can be constructed with various combinations
and arrangements of elements which also accomplish contacting a
sample with a target material binding dye, e.g., a precipitating or
frequency shift dye, within the self-contained sampling testing
device. Exemplary selections and arrangements are described. In
accord with the embodiments described above, a device may be
constructed to include a sampler portion which sealably attaches to
a housing, or may be constructed as an upper and a lower housing in
which the sampler is attached to the upper housing and the upper
and lower housing sealably engage. -Other variants can also be
constructed.
[0097] As previously indicated, in the various embodiments
different types of samplers can be utilized. These include, for
example, swabs, pipettes and capillaries. For embodiments in which
the sampler is a swab or other wiping device, a sample washer is
provided. In preferred embodiments, the sample washer includes a
reservoir containing a wash solution that can be used to wash the
sample from the sampler. Delivery of the wash solution to the
sampler can be accomplished in a variety of ways including, for
example, rupture of a membrane to allow wash solution to pass
through a hollow sampler shaft, or breaking the tip or plug to
expose an orifice communicating with a hollow sampler shaft
allowing the fluid to flow down the shaft, or rupture of a packet
or ampule thereby releasing a fluid that can then flow down a
sampler shaft to wash the sample.
[0098] The wash solution may also be constituted and packaged in a
variety of different ways as appropriate for various configurations
and dye selections. For example, as described above, the wash
solution may include the dye. However, in certain embodiments it
may be preferable to package the dye separately from the wash
solution. For example, the dye and other wash solution components
may be separated in the upper reservoir until mixing is desired. As
an example, a concentrated dye solution may be provided in a
breakable ampule or rupturable packet within a reservoir chamber
containing other wash solution components. Alternatively the dye
and other wash solution components may be in separate chambers
separated by a separator. Breakage of the dye container or
combining the contents of separate chambers, then results in mixing
and thus provides a combined dye wash solution. Such an arrangement
may be desirable, for example, where the dye molecules would not
have long-term stability in the presence of one or more other wash
solution components. Alternatively, the wash solution and dye may
be separated by providing the wash solution only in the upper
reservoir and providing the dye in a reservoir or ampule or packet
or chamber in a lower portion of the device, e.g., in a lower
portion of the housing or lower housing.
[0099] In embodiments where the sampler is a pipette or a capillary
the sample can be removed from the sampler in a variety of ways,
such as by expelling the liquid sample with air, or by washing the
sample from the pipette or capillary with a wash solution. In
general, to remove the sample, the upper portion of the device will
be deformable to allow a creation of pressure to push the liquid
sample from the pipette or capillary.
[0100] Similar to the embodiment described above in which the dye
is separated from other solution components in the sampler portion
of upper housing, in embodiments where the dye is contained in a
lower portion of the device the dye can be separated from other
solution components by placing either or both of the dye or other
components into separate chambers, ampules, packets, or other
structures such that the components can be mixed at a desired
time.
[0101] In yet another embodiment, the sampler is directly inserted
into a solution in a lower portion of the device. For example, in
certain embodiments the upper portion of the device does not
contain a reservoir with a wash solution. Instead, the wash
solution with or without dye is contained in a lower portion of the
device and the sampler is inserted into the wash solution following
sample collection. In such embodiments the wash solution can be
separated from upper portions of the device by a barrier, for
example, a rupturable membrane or one-way valve or deformable
constriction through which the sampler can be inserted. Also, in
such embodiments, as noted, the dye may be packaged separately from
the wash solution or may be incorporated in the wash solution. As
described before, such separation may be accomplished by the use of
separate chambers, rupturable packets, breakable ampules,
rupturable membranes, semi-porous filters, and other such
structures.
[0102] The method of using one embodiment of the device to test for
the presence of protein will be briefly described. This embodiment
of the device has the structure of the device illustrated in FIG.
6, and utilizes a dye solution to detect the presence of protein on
a surface.
[0103] The device is opened by removing the sampler (61) from the
housing (74). An area to be tested for protein is swabbed with a
pre-moistened swab (72), allowing a portion of the protein material
to be absorbed into the swab. The sampler (61) is then sealably
engaged onto the housing (74). The dye (70) is released by bending
a bulb defining a dye reservoir (62) containing the dye (70),
thereby breaking off the snap plug (68), exposing the orifice (64)
communicating with the upper end of the hollow swab shaft (66) and
allowing the fluid to be flushed down the hollow interior of the
swab. The fluid flow can be accelerated by squeezing the bulb to
force out the dye solution. The dye solution washes the
protein-containing residues from the swab (72) into the bottom of
the device, which forms a read portion (76). The walls of the read
portion containing the expelled liquid are translucent or
transparent allowing direct visualization of the color changes
resulting from the reaction between the dye and protein.
[0104] The device embodiments described herein are constructed from
any of a variety of materials or material combinations, including
but not limited to plastics. Injection mold castings or any other
means for generating suitable device housings may be employed. In
appropriate devices, well/reservoirs may be machine-drilled or
injection molded or formed by other methods suitable for forming
such cavities in the particular materials. Those skilled in the art
are familiar and can select suitable materials and construction
techniques. Also where appropriate, as in embodiments such as the
book of FIG. 1, separate housings and pieces may be joined by
hinges, snaps, latches, Velcro.RTM., or any other connector that
does not impede the ability of the reagents to function. The
absorbent swabs and collection surface materials, already
described, are comprised of any of the following illustrative
materials or functional equivalents thereof, sponge, mylar, nylon,
dacron, rayon, porex, porous polypropylene, porous polyethylene,
glass fibers, paper, or various other woven or felted fibers such
as nitrocellulose, cotton, wool, cellulose, or combinations
thereof. These may in turn be attached to housings where
appropriate, such as in the embodiments of FIGS. 1, 2, or 3, by
glue, adhesive, or any other means which does not interfere with
target material collection, staining or, in the case of
precipitating dye use, the precipitation or other immobilization of
target material.
[0105] Those skilled in the art will recognize that this and other
embodiments of the present invention can be used in a variety of
ways, including the following:
[0106] (1) Testing of liquid samples to determine if they contain
contaminating material. The procedure utilized to test for material
in a liquid sample would be similar to the procedure used to test a
surface, with the difference being that the sample tested is a
liquid.
[0107] (2) Testing of any sample for contamination and using an
instrument read instead of a visual read.
EXAMPLES
Example 1
[0108] Exemplary devices were constructed as generally described in
FIG. 2 and used with a precipitating dye (Ponceau S) and a
frequency shift dye (a colloidal Coomassie Blue dye, Gelcode.RTM.)
and used to test food surfaces soiled with milk, cheese, roast
beef, turkey, or tomato. The surfaces were also tested with an
industry-accepted means of measuring surface contamination based on
ATP detection (LIGHTNING.RTM., produced by IDEXX Laboratories,
Westbrook, Me.)(used according to manufacturer's instructions) as
well as the protein detection devices described for this invention.
As indicated, two different embodiments of the present invention
were used. One with Ponceau-S as the protein-binding dye, and one
with Gelcode.RTM.--a colloidal Coomassie blue dye.
[0109] Stainless steel surfaces were smeared with the indicated
food materials. For each test, a sample was obtained from the
surface by swabbing with the moistened sampler collection surface
of a sampler from the particular device. "Dirty" indicates that the
surface was tested following application of the food residue to the
surface; "wiped clean" indicates that the surface was wiped free of
visible food residue with a dry paper towel; and "scrubbed clean"
indicates that the surface was wet cleaned with a brush and
detergent type cleaning solution in a manner commonly used for
cleaning in the food processing industry.
[0110] For the Ponceau S device, the absorbent pad of the sampler
was moistened with the wetting agent, a sample was swabbed from the
surface, then the absorbent pad of the sampler was touched briefly
(a few seconds) against the dye. The absorbent pad of the sampler
was then dipped in the wash solution to wash away unbound dye.
[0111] The Gelcode.RTM. device was used similarly except that the
color change of the dye was observable both in the dye solution and
on the sampler pad.
[0112] The results are shown in Table 1. The data indicate that the
device is able to distinguish the three different states of the
surfaces (dirty, wiped clean, and the more thorough, scrubbed
clean) for each food type. Both dyes gave results that allow the
test operator to distinguish between dirty, minimally cleaned
(wiped) and thoroughly cleaned (scrubbed) surfaces.
[0113] Results for the LIGHTNING.RTM. device range from 0-7.5. Dye
results are read by eye and assigned a numberic value from 0-5. In
both cases the higher the number, the greater the indicated level
of contamination.
1TABLE 1 Comparison of Bioluminescence assay (Lightning) to protein
detection devices. Lightning results in zones (0-7.5). Dye results
are read by eye and assigned a numeric value from 0-5. roast- milk
cheese beef turkey tomato Bioluminescence Lightning dirty 3.3 2.55
4.9 5 6.05 wiped clean 2.4 2.1 3.45 3 4.85 scrubbed clean 2.05 1.65
1.65 2.05 2.35 Protein detection devices Ponceau S dirty 3.5 4.5 4
3 1.5 wiped clean 1.25 1.25 0.75 0.25 0.25 scrubbed clean 0 0 0 0 0
Gelcode dirty 4.5 4 5 4 3 wiped clean 3.5 2.5 2.25 1.5 0.75
scrubbed clean 0.25 0 0 0.25 0.5
Example 2
[0114] An exemplary device constructed as generally described in
FIG. 6 and containing 2 ml Pierce Gelcode.RTM. dye was used in a
test to determine detection sensitivity of the device. Presence of
protein was detected using qualitative visual reading and by
reading the optical density (OD) at 595 nm, with the reported OD
being the mean of two readings.
[0115] Bovine serum albumin (BSA) at various concentrations was
dried on clean 4".times.4" stainless steel coupons. For each sample
tested, the pre-moistened swab portion of a device was swiped over
the coupon surface with firm pressure to collect the sample. The
swab was inserted into the housing, and the dye reservoir bulb
snapped to the side to deliver the dye into the lower read chamber.
A visual interpretation is then made, followed by transfer from the
read chamber to a disposable cuvette for reading at 595 nm. The
results are shown in Table 2.
2 TABLE 2 Sample O.D. Visual Interpretation Negative control (PBS)
0.0006 Negative BSA, 5 mg/test 3.3800 ++++ BSA, 50 pg/test 1.3570
+++ BSA, 10 pg/test 0.4 130 ++ BSA, 5 pg/test 0.1930 + BSA, 2.5
pg/test 0.0900 +
[0116] BSA refers to Bovine Serum Albumin.
[0117] The results demonstrate that this embodiment of the device
has a detection sensitivity of about 2.5 .mu.g protein/test.
Example 3
[0118] An exemplary device as in Example 2 was used in a comparison
test of biological contamination with the Konica Hygiene Monitoring
Kit. The Konica kit was utilized according to manufacturer's
instructions with reading after 10 minutes at room temperature. The
exemplary device was utilized as follows.
[0119] Various different sources of protein were dried upon clean
4".times.4" stainless steel coupons, which had been marked to
divide each coupon into two equal parts. The exemplary device was
used to collect the sample from the left side of the coupon
surface. Following the Konica kit procedure, the corresponding
right side of the coupon was sampled with the Konica swab. Visual
interpretation for the exemplary device was made immediately upon
activation. The Konica test was read at 10 minutes according to kit
instructions. The stainless steel coupons were then washed with a
mild detergent (Palmolive.RTM.) and water, and after drying, each
side of the coupon was retested to detect any remaining
contamination on the surface.
[0120] The results of the comparison test are shown in Table 3.
Cleanliness levels for the Konica kit are shown according to a
cleanliness standard where:
[0121] Level 1 (Clean)
[0122] Level 2 (Less Clean)
[0123] Level 3 (Slightly Dirty)
[0124] Level 4 (Dirty)
3TABLE 3 Sample Konica kit Exemplary device Milk on coupon Level
3-3.5 (slightly dirty) ++++ 1 wash Level 1-1.5 (clean) +++ 2 washes
Level 1 (clean) Negative 5 .mu.g/test BSA standard Level 1 (clean)
+/- 1 wash Level 1 (clean) Negative 1:400 Plasma Level 1 (clean) ++
1 wash Level 1 (clean) Negative
[0125] The results indicate that the device is more sensitive than
the Konica test system, in addition to the advantages of being
faster and more convenient to use.
[0126] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. All references cited in this
disclosure are incorporated by reference to the same extent as if
each reference had been incorporated by reference in its entirety
individually.
[0127] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The solutions, dyes, and methods described herein as
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the claims.
[0128] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. For example, those skilled in the art will
recognize that the invention may be practiced using a variety of
different dyes, and pH buffers, as well as additional reaction
components.
[0129] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0130] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
[0131] Thus, additional embodiments are within the scope of the
invention and within the following claims.
* * * * *