U.S. patent application number 16/090045 was filed with the patent office on 2019-05-16 for detecting microbial infections in wounds.
The applicant listed for this patent is ConvaTec Technologies Inc., QUALIZYME DIAGNOSTICS GMBH AND CO KG, SYNOVO GMBH. Invention is credited to Lucy BALLAMY, Philip BOWLER, Michael BURNET, Clemens GAMERITH, Andrea HEINZLE, Daniel LUSCHNIG, Daniel Gary METCALF, David PARSONS, Eva SIGL, Jade STEVEN, Sarah WROE.
Application Number | 20190142642 16/090045 |
Document ID | / |
Family ID | 59965180 |
Filed Date | 2019-05-16 |
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United States Patent
Application |
20190142642 |
Kind Code |
A1 |
BURNET; Michael ; et
al. |
May 16, 2019 |
DETECTING MICROBIAL INFECTIONS IN WOUNDS
Abstract
Provided herein are microbial infection indicator devices,
including dressing with indicators, standalone indicator inserts or
disks that can be freely placed at a wound site or dressing, and
applications thereof for displaying a visible or detectable signal
to a user upon detection of an analyte or biomarker indicative of
an infection, such as a color change.
Inventors: |
BURNET; Michael; (Tubingen,
DE) ; BOWLER; Philip; (Appleton, GB) ; WROE;
Sarah; (Manchester, GB) ; STEVEN; Jade;
(Ellesmere Port, GB) ; METCALF; Daniel Gary;
(Sale, GB) ; PARSONS; David; (West Kirby, GB)
; BALLAMY; Lucy; (Llangollen, GB) ; HEINZLE;
Andrea; (Gratwein-Strassengel, AT) ; SIGL; Eva;
(Sankt Barbara im Murztal, AT) ; LUSCHNIG; Daniel;
(Graz, AT) ; GAMERITH; Clemens; (Graz,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ConvaTec Technologies Inc.
SYNOVO GMBH
QUALIZYME DIAGNOSTICS GMBH AND CO KG |
Las Vegas
Tubingen
Graz |
NY |
US
DE
AT |
|
|
Family ID: |
59965180 |
Appl. No.: |
16/090045 |
Filed: |
March 30, 2017 |
PCT Filed: |
March 30, 2017 |
PCT NO: |
PCT/US17/24991 |
371 Date: |
September 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62315565 |
Mar 30, 2016 |
|
|
|
Current U.S.
Class: |
600/362 |
Current CPC
Class: |
A61B 10/0045 20130101;
A61B 5/445 20130101; A61F 2013/00429 20130101; A61F 13/00051
20130101; A61F 2013/8473 20130101; C12Q 1/37 20130101; G01N 33/00
20130101; A61L 15/56 20130101; A61L 15/38 20130101 |
International
Class: |
A61F 13/00 20060101
A61F013/00; A61L 15/56 20060101 A61L015/56; A61L 15/38 20060101
A61L015/38 |
Claims
1. A wound dressing comprising a) a wound contacting layer; b) a
reagent layer comprising one or more testing regions, wherein the
reagent layer is in fluid communication with the wound contacting
layer; and c) an outer layer that overlays the reagent layer.
2. The wound dressing of claim 1, wherein the wound contacting
layer comprises gel-forming polymers.
3. The wound dressing of claim 1, wherein each of the one or more
testing regions comprises one or more of each of a back-flow trap,
a reagent pad, a filter pad, an indicator trap, and an absorbent
area, and wherein one or more viewing windows are located either
above the reagent pad or the indicator trap.
4. The wound dressing of claim 3, wherein: a) the reagent pad is in
fluid communication with the filter pad; b) the filter pad is in
fluid communication with the indicator trap; and c) the indicator
trap is in fluid communication with the absorbent area.
5. The wound dressing of claim 1, wherein each of the one or more
testing regions comprises one or more reagents selected from the
group consisting of enzyme-reactive indicators, reagents that are
sources of peroxide, enzymes that produce colored products, pH
indicators, protein responsive reagents, and moisture-detecting
reagents.
6. The wound dressing of claim 5, wherein enzyme-reactive
indicators are protein-indicator conjugates.
7. The wound dressing of claim 6, wherein the protein-indicator
conjugates are deposited in or on the reagent pad.
8. The wound dressing of claim 6, wherein the protein-indicator
conjugate has the structure of Formula (I): A-B Formula (I)
wherein: A is an anchor region or moiety that helps to bind an
enzyme-reactive region to the reagent pad; and B is the
enzyme-reactive region.
9. The wound dressing of claim 8, wherein the enzyme-reactive
region comprises a peptide.
10. The wound dressing of claim 8, wherein the enzyme-reactive
region comprises an indicator region having an enzyme-reaction
indicator.
11. The wound dressing of claim 8, wherein B further comprises an
indicator region.
12. The wound dressing of claim 11, wherein the indicator region,
after having been cleaved by the target enzyme is transformed into
a colored species by accessory enzymes selected from the group
consisting of lipase, esterase, hexosaminidase, peroxidase,
oxidase, glycosidase, glucosidase, laccase, and a combination of
two or more thereof.
13. The wound dressing of claim 10, wherein the enzyme-reactive
indicators interact with one or more enzymes selected from the
group consisting of elastase, lysozyme, cathepsin G,
myeloperoxidase, and any combination thereof.
14. The wound dressing of claim 10, wherein the enzyme-reactive
indicators comprise a moiety capable of producing a visible color
or a detectable electronic change upon interaction of the
enzyme-labile or enzyme-reactive region with one or more enzymes,
wherein the moiety is selected from the group consisting of a
peroxidase substrate, arylamine, an amino phenol, a neutral dye, a
charged dye, a nanoparticle, a colloidal gold particle, and an
analog thereof.
15. The wound dressing of claim 8, wherein the anchor region is
covalently attached to the reagent pad.
16. The wound dressing of claim 8, wherein the anchor region is
non-covalently attached to the reagent pad.
17. The wound dressing of claim 16, wherein the anchor region is
ionically attached to the reagent pad.
18. The wound dressing of claim 1, further comprising one or more
lines of wicking stitching or wicking tufting throughout all layers
of the wound dressing except the outer layer, and wherein the
wicking stitching or wicking tufting provides fluid communication
between the reagent layer and the wound contacting layer.
19. The wound dressing of claim 18, wherein one or more lines of
wicking stitching or wicking tufting comprise fibers that are
wettable and exhibit capillary action.
20. The wound dressing of claim 19, wherein the fibers comprise
cotton, rayon, viscose, wool, silk, polyester, polyamide, CMC,
polypropylene, or any combination thereof.
21. The wound dressing of claim 1, wherein one or more testing
regions comprise a leach-back trap in fluid communication with a
reagent pad and one or more lines of wicking stitching or wicking
tufting crossing through one or more testing regions only at the
leach-back trap.
22. The wound dressing of claim 1, further comprising a foam layer
between the wound contacting layer and the reagent layer.
23. The wound dressing of claim 22, further comprising one or more
perforations in the wound contacting layer.
24. The wound dressing of claim 22, further comprising one or more
perforations in the foam layer and the wound contacting layer.
25. The wound dressing of claim 21, wherein each of the one or more
testing regions further comprises a leach-back trap in fluid
communication with the reagent pad and one or more perforations
aligned with the leach-back trap.
26. The wound dressing of claim 1, wherein each of the one or more
testing regions comprises a multichannel testing region, wherein
each channel is separated from an adjacent channel by one or more
impermeable separators or borders.
27. The wound dressing of claim 26, comprising a plurality of
testing regions.
28. The wound dressing of claims 26, wherein the testing regions
are arranged in a linear configuration.
29. The wound dressing of claim 26, wherein the testing regions are
arranged in a radial configuration.
30. The wound dressing of claim 1, wherein the outer layer has one
or more windows that permit visualization of a signal from the
reagent layer.
31. The wound dressing of claim 30, wherein the signal is a color
change.
32. The wound dressing of claim 30, wherein the signal is a
fluorescent signal, a luminescent signal, or a signal mediated by
physical means, such as an electrical change.
33. A method of detecting the level of one or more enzymes in a
mammalian wound, the method comprising: a) contacting the mammalian
wound with a wound dressing of claim 1; b) observing one or more
signals in the reagent layer, wherein the signal is a color change,
a fluorescent signal, a luminescent signal, or an electrical
change; and c) comparing the signal to a reference or a control to
determine the level of an enzyme.
34. A method of detecting the presence of one or more enzymes in a
mammalian wound, the method comprising: a) contacting the mammalian
wound with a wound dressing of claim 1; and b) observing one or
more signals in the reagent layer, wherein the signal is a color
change, a fluorescent signal, a luminescent signal, or an
electrical change.
35. A method of detecting an infection in a mammalian wound, the
method comprising: a) contacting the wound with a wound dressing of
claim 1; and b) observing one or more signals in the reagent layer,
wherein the signal is a color change, a fluorescent signal, a
luminescent signal, or an electrical change.
36. A method of treating an infection in a wound of a mammal, the
method comprising: a) contacting the wound with a wound dressing of
claim 1; b) observing one or more signals in the reagent layer
indicative of an infection, wherein the signal is a color change, a
fluorescent signal, a luminescent signal, or an electrical change;
and c) administering a medical treatment to the mammal.
37. A device for detecting an infection in a wound, comprising: a)
a wound contacting layer; b) a reaction layer comprising one or
more reagents that can indicate the presence of one or more
analytes associated with an infection, wherein the reagents are
affixed to a solid phase and produce a detectable signal in a
reporter area; c) a cover on top of the reaction layer, wherein the
cover comprises one or more windows or clear areas to allow
visualization of the detectable signal; and d) fluid communication
between the wound contacting layer and the reaction layer.
38. The device of claim 37, wherein the reagents comprise
enzyme-reactive regions that interact with one or more target
enzymes selected from the group consisting of lysozyme, MPO,
cathepsin G, elastase, catalase, lipase, esterase, and any
combination thereof.
39. The device of claim 37, wherein one or more reagents produce a
visible color upon a change in the pH, at an acidic pH, or at a
basic pH, and wherein the pH-sensitive reagent comprises
bromothymol blue, phenol red, bromophenol red, chlorophenol red,
thymol blue, bromocresol green, bromocresol purple; nitrazine
yellow; or other sulfophthalein dyes.
40. The device of claim 38, wherein the enzyme-reactive regions
comprise a moiety capable of producing a visible color or
detectable electronic change upon interaction with one or more
target enzymes, wherein the moiety is a peroxidase substrate,
arylamine, an amino phenol, an indoxyl, a neutral dye, a charged
dye, a nanoparticle, a colloidal gold particle, or an analog
thereof.
41. The device of claim 40, wherein the reagent interacts with a
target enzyme to produce a colored species or to produce an
intermediate product that interacts with an accessory enzyme
selected from the group consisting of a lipase, esterase,
hexosaminidase, peroxidase, oxidase, glycosidase, glucosidase, and
laccase.
42. The device of claim 37, wherein the fluid communication
comprises wicking stitching or wicking tufting of an absorbent
material that allows fluid communication between the wound
contacting layer and the reaction layer.
43. The device of claim 37, wherein the reagents are printed,
sprayed, or deposited on the solid phase.
44. The device of claim 37, wherein the solid phase is selected
from the group consisting of paper, viscose, regenerated cellulose,
glass fiber, and any combination thereof.
45. The device of claim 37, wherein the detectable signal is a
color change, a fluorescent signal, a luminescent signal, or an
electrical change.
46. The device of claim 37, wherein the device is a wound
dressing.
47. A device for detection of infection associated enzymes that is
provided as an independent entity and can be placed in any dressing
system, comprising a sample inlet in fluid communication with
reagent cells, wherein reagent cells comprise indicators for sample
delivery and/or pH, and one or more indicators for biomarkers of an
infection selected from the group consisting of lysozyme, MPO,
cathepsin G, elastase, catalase, lipase, and esterase.
48. The device of claim 47, wherein the indicators comprise
enzyme-reactive indicators, reagents that are sources of peroxide,
enzymes that produce colored products, pH indicators, protein
responsive reagents, or moisture-detecting reagents.
49. The device of claim 47, wherein the indicators are
enzyme-reactive indicators or protein-indicator conjugates.
50. The device of claim 47, wherein the protein-indicator
conjugates are deposited in or on the reagent pad.
51. The device of claim 47, wherein the protein-indicator conjugate
has the structure of Formula (I): A-B, wherein A is an anchor
region or moiety that helps to bind an enzyme-reactive region to
the reagent pad; B is the enzyme-reactive region; and wherein the
enzyme-reactive region comprises a peptide or an indicator
region.
52. The device of claim 51, wherein the indicator region interacts
with an enzyme to produce a colored species or wherein an
intermediate species interacts with an accessory enzyme selected
from a group consisting of a lipase, esterase, hexosaminidase,
peroxidase, oxidase, glycosidase, glucosidase, and laccase to
produce a colored species.
53. The device of claim 47, wherein the fluid communication
comprises one to ten indicator channels separated by impermeable
lanes, borders, or separators, wherein each indicator channel
comprises a different reagent or control.
54. The device of claim 47, wherein the fluid communication
comprises a plurality of separate indicator channels, and wherein
each indicator channel comprises a different reagent or
control.
55. The device of claim 47, wherein the reagents are printed,
sprayed, or deposited on a solid phase in a radial configuration to
form a disk.
56. The device of claim 47, wherein the reagents are printed,
sprayed, or deposited on a solid phase in a linear configuration to
form a testing strip or dipstick-type device.
57. The device of claim 55, wherein the disk comprises reagents
printed, sprayed, or deposited on the top surface of the disk with
a trap material and a substrate material on the bottom surface,
wherein the substrate can be digested by one or more enzymes in the
sample to release one or more products that migrate toward the
trap.
58. The device of claim 57, wherein one or more products are
colored or produce a color change through interaction with an
accessory enzyme, and wherein the color change can be visualized on
the top surface of the disk.
59. A diagnostic disk for detecting an infection in a wound,
comprising: a) a reaction layer comprising one or more reagents
that interact with a target enzyme indicative of an infection,
wherein the reagents are affixed to a solid phase; b) each reagent
is sprayed, printed, or deposited in a reagent area in a lane
separated from adjacent lanes by impermeable separators; c) each
lane comprises a reporter area wherein a color, color change, or
other detectable signal is observed; and d) a cover comprising a
window for visualizing the signal in the reporter area.
60. The diagnostic disk of claim 59, wherein one or more reagents
produce a visible color upon a change in the pH, at an acidic pH,
or at a basic pH, wherein the pH-sensitive reagent comprises
bromothymol blue, phenol red, bromophenol red, chlorophenol red,
thymol blue, bromocresol green, bromocresol purple; nitrazine
yellow; or other sulfophthalein dyes.
61. The diagnostic disk of claim 59, wherein multiple lanes are
arranged in a linear or radial configuration about a cut access,
perforation, or wicking material that allows fluid communication
between a wound contacting area to the reagents in the reaction
layer.
62. The diagnostic disk of claim 59, wherein one or more reagents
produce a color signal or other detectable signal upon interaction
with an enzyme selected from the group consisting of lysozyme, MPO,
cathepsin G, elastase, catalase, lipase, and esterase.
63. The diagnostic disk of claim 59, wherein the reagents comprise
a moiety selected from the group consisting of peroxidase
substrate, arylamine, an amino phenol, an indoxyl, a neutral dye, a
charged dye, a nanoparticle, and a colloidal gold particle, and an
analog thereof.
64. The diagnostic disk of claim 59, wherein the reagents comprise
accessory enzymes that produce a colored species or color signal,
and wherein the accessory enzyme is a lipase, esterase,
hexosaminidase, peroxidase, oxidase, glycosidase, glucosidase,
laccase, or a combination of one or more thereof.
65. The diagnostic disk of claim 59, wherein the solid phase is
selected from the group consisting of paper, viscose, regenerated
cellulose, glass fiber, and similar material.
66. The diagnostic disk of claim 59, wherein there are a plurality
of lanes, each with a different reagent or control.
67. The diagnostic disk of claim 59, wherein the detectable signal
comprises a color change, appearance or disappearance of a color, a
fluorescent signal, a luminescent signal, or an electrical change
or signal.
68. A lateral flow or dipstick device for detecting an infection in
a wound, comprising: one or more reagent disks arranged in a linear
configuration, wherein each reagent disk is impregnated with a
reagent that interacts with an enzyme to produce a color change
and/or is pH-sensitive, comprising bromothymol blue, phenol red,
bromophenol red, chlorophenol red, thymol blue, bromocresol green,
bromocresol purple; nitrazine yellow; or other sulfophthalein dyes,
and wherein the disks are affixed to a solid phase.
69. The device in claim 68, wherein the reagents produce a color
signal upon interaction with an enzyme selected from the group
consisting of lysozyme, MPO, cathepsin G, elastase, catalase,
lipase, and esterase.
70. The device in claim 68, wherein the solid phase is selected
from the group consisting of paper, viscose, regenerated cellulose,
glass fiber, and any combination thereof.
71. A device for detecting an infection in a wound or a sample,
comprising a housing, wherein the housing comprises: a) a sampling
component for collecting the sample; b) a sample preparation
chamber in fluid communication with a reaction chamber, wherein the
sample preparation chamber receives the sample; c) the reaction
chamber comprising one or more reaction cells containing reagents
that interact with one or more enzymes in the sample to indicate
the presence of an infection and/or pH of the sample; and d) a
window or a clear area for visualizing a detectable signal, wherein
the signal is a color change.
72. The device in claim 71, wherein one or more reagents produce a
color signal upon interaction with an enzyme selected from a group
consisting of lysozyme, MPO, cathepsin G, elastase, catalase,
lipase, and esterase.
73. The device in claim 71, wherein one of the reagents produces a
color change in response to a change in pH, a basic pH, or an
acidic pH.
74. The device in claim 71, wherein the reagents perform in a
primarily liquid medium.
75. The device in claim 71, wherein the reagents are provided in
tablet form for use in the reaction cells.
76. The device in claim 71, wherein the reagents are printed,
sprayed, or deposited in separate reagent fields on a support
material to form a panel of tests for use in the reaction
chamber.
77. The device in 71, wherein the support material is selected from
the group consisting of paper, viscose, regenerated cellulose, and
glass fiber, arrayed in a line along a carrier strip.
78. The device in claim 76, wherein the reagent fields are arrayed
in a line along a carrier strip capable of absorbing the sample in
the reaction chamber.
79. The device in claim 71, wherein the sampling component is a
swab device.
80. The device in claim 71, wherein the sampling component is a
hook or needle device adapted to removing a thread from a wound
dressing without disturbing the wound dressing.
81. A kit for detecting an infection in a sample, comprising: a) a
sampling component for collecting the sample; b) a test device
comprising a housing surrounding a tube to define an opening in the
housing for receiving the sampling component, the housing
comprising: c) a diluent chamber that holds a liquid diluent; d) a
reaction well in liquid communication with the tube, wherein the
reaction well holds one or more reagents that interact with one or
more analytes to produce a color change or a detectable signal; e)
a viewing window or reporter area wherein the color change or
detectable signal can be observed; and f) wherein the liquid
diluent flows from the sample component into the reaction well to
mix the sample with the reagents in the reaction well.
82. The kit of claim 81, wherein the reagents comprise one or more
enzyme-reactive indicators and/or pH indicator.
83. The kit of claim 81, wherein one or more reagents produce a
color signal upon interaction with an enzyme selected from a group
consisting of lysozyme, MPO, cathepsin G, elastase, catalase,
lipase, and esterase.
84. The kit of claim 81, wherein the reagents comprise a moiety
selected from the group consisting of peroxidase substrate,
arylamine, an amino phenol, an indoxyl, a neutral dye, a charged
dye, a nanoparticle, a colloidal gold particle, and an analog
thereof.
85. The kit of claim 81, wherein the detectable signal comprises a
color signal or color change, a fluorescent signal, a luminescent
signal, or an electrical signal.
86. The kit of claim 81, wherein at least one reagent produces a
color signal in response to a basic pH, an acidic pH, or a change
in pH, wherein the pH-sensitive reagent is bromothymol blue, phenol
red, bromophenol red, chlorophenol red, thymol blue, bromocresol
green, bromocresol purple; nitrazine yellow; or other
sulfophthalein dyes.
87. The kit of claim 81, wherein the sample is obtained from a
wound, a wound dressing, or a surgical site.
88. The kit of claim 81, wherein the sampling component is a swab
device or a hook or needle device.
89. The kit of claim 81, wherein the reagents are deposited in
separate fields on a testing strip to form a panel of tests.
90. The kit of claim 81, wherein there are a plurality of reaction
wells, wherein each reaction well comprises a different reagent or
control.
91. The kit of claim 90, wherein the reaction wells are arranged in
a linear configuration.
92. The kit of claim 90, wherein the reaction wells are arranged in
a radial configuration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States
Provisional Application No. 62/315,565, filed Mar. 30, 2016, the
disclosure in which is incorporated herein by reference in its
entirety and made a part hereof.
TECHNICAL FIELD
[0002] Embodiments described herein generally relate to wound
healing, and in particular to compositions, apparatuses and methods
for the detection and treatment of wounds.
BACKGROUND
[0003] In mammals, dermal injury triggers an organized complex
cascade of cellular and biochemical events that result in a healed
wound. Wound healing is a complex dynamic process that results in
the restoration of anatomic continuity and function: an ideally
healed wound is one that has returned to normal anatomic structure,
function, and appearance. A typical wound heals via a model
consisting of four stages--`exudative` phase, proliferative phase,
reparative phase and epithelial maturation (Hatz et al., Wound
Healing and Wound Management, Springer-Verlag, Munich, 1994) or
hemostatic, inflammatory, proliferative and remodeling phase
(Nwomeh et al., Clin. Plast. Surg. 1998, 25, 341). The inflammatory
phase is particularly important to the wound healing process,
wherein biochemical reactions at the wound situs facilitate healing
but also cause tissue breakdown due to production of excess
proteases.
[0004] Infection of the wound results in either a slower, or an
arrested healing process. For example, pathogens in a wound can
produce toxins (e.g., Clostridium species), generate noxious
metabolites like ammonia that raise pH (e.g., Proteus species),
activate or produce tissue lytic enzymes like proteases, or promote
tissue invasion, thereby leading to an increase in the size or
seriousness of the wound. In a worst case, pathogens can leave the
wound and cause sepsis.
[0005] In order to keep the chronicity of wounds in check, a
variety of assessment techniques and/or tools are employed in the
clinical and veterinary setting. Current methods of assessing an
infected wound are based primarily on assaying for a variety of
parameters associated with the wound. For instance, a wound may be
assessed visually, length and depth measurements may be taken,
digital photography may be used where available to track the visual
condition and size of a wound (Krasner et al., supra). In clinical
practice, diagnosis of infection is based on measurement of
secondary parameters, such as, odor, presence of local pain, heat,
swelling, discharge, and redness. Many of these clinical
indicators, such as inflammation and discharge have a low
predictive value of infection in wounds. In other instances, the
number(s) and type(s) of pathogenic flora at the wound situs may be
determined using laboratory and/or clinical diagnostic procedures.
Swabbing of a wound followed by microbiology testing in the
hospital laboratory is an option for confirmation of bacterial
colonization and identification of the strains associated with
infection, thus allowing for the prescription of correct antibiotic
course. However, this process is time consuming and labor
intensive. Delay in diagnosis of infection can delay the
administration of antibiotics and may increase the risk of
developing sepsis.
[0006] One of the biggest drawbacks associated with existing
clinical diagnostics is a lag associated with the onset of
infection and the timing of detection. For instance, positive
identification of infection using swabbing procedures often depends
on attainment of a "critical mass" of microorganisms at the wound
site and so early detection cannot be made until a detectable level
is reached. Also, the swabs may be contaminated with the flora of
the surrounding tissue, thereby complicating the diagnostic
procedure. Other drawbacks include, e.g., sampling errors, delays
in transport of the swabs, errors in analytical procedures, and/or
errors in reporting. See, the review by Bowler et al., Clin
Microbiol Rev. 14(2): 244-269, 2001.
[0007] There is therefore an imminent but unmet need for diagnostic
reagents and methods that enable early diagnosis of clinical
infection, preferably, which permit clinical diagnosis prior to
manifestation of clinical symptoms of infection. There is also a
need for compositions and methods that would assist in predicting
clinical infection of a wound prior to the manifestation of
clinical symptoms. Such a prognostic aid would allow early
intervention with suitable treatment (e.g., antimicrobial
treatment) before the wound is exacerbated and surgery or other
drastic intervention is required to prevent further infection.
Additionally, if clinicians could respond to wound infection as
early as possible, the infection could also be treated with minimal
antibiotic usage. This would reduce the need for hospitalization
and would reduce the risk of secondary infections, e.g., as a
result of contact with other diseased subjects.
SUMMARY
[0008] The technology disclosed herein provides for compositions
and methods of detecting infected and/or chronic wounds. The
disclosed technology improves upon exiting assays by: increasing
the sensitivity, precision and specificity of detection of infected
wounds; providing for the ability of qualitative and quantitative
measurements; and, increasing the speed of detection of infected
wounds in situ and in real-time. The assays and methods described
herein are partly based on the use of specific reagents that detect
biomarkers and/or probes which are present in infected or chronic
wounds. The detection process may involve use of reagents that are
specific to the markers present in infected wounds but not
non-infected or non-chronic wounds and the detection step may
involve qualitative or quantitative measurements of the signal(s)
that are generated when the probe is acted upon by the marker. In
embodiments wherein the detection method involves detection of
enzymes present in wounds, the probes comprise modified enzyme
substrates that are specific to the enzyme, which generate signals
that may be optionally amplified. This greatly improves efficiency
and specificity of detection. Moreover, a plurality of detection
probes, each specific for one or more targets, e.g., enzymes that
are specific to the wounds, may be employed. This greatly helps to
maximize both efficiency and accuracy of diagnostic assays while
minimizing the incidence of false positives (e.g., due non-specific
interactions and/or target redundancy). Furthermore, the
experimental results disclosed herein confirm that the novel probes
and the assay techniques based thereon are capable of detecting and
characterizing various types of wounds. Finally, the reagents of
the disclosed technology may be used together with therapeutic
molecules such as antibiotics, antifungal agents, etc. to monitor
and evaluate treatment and management of chronic wounds.
[0009] Embodiments described herein are based, in part, on the
discovery that cells of the immune system, including enzymes
generated thereby, may serve as markers in the early diagnosis of
wounds. These cells, e.g., neutrophils, are recruited at the wound
situs to combat infection, do so by engulfing bacteria (and other
pathogens) and/or neutralizing them with enzymes. Some enzymes are
specific towards proteins (e.g., elastase, cathepsin G), others are
specific towards cell wall components (e.g., lysozyme) and yet
others mediate protein denaturation (e.g., NADPH oxidase, xanthine
oxidase, myeloperoxidase (MPO) and other peroxidases). These cells,
e.g., neutrophils, are generally only short-lived and when they
lyse in the area of the infection, they release the contents of
their lysosomes including the enzymes, which can then be detected
to provide a reliable measurement of the status of the wound.
[0010] Accordingly, various embodiments described herein utilize
the detection of enzyme markers, which are indicative of the
presence of myeloid cells, and neutrophils in particular, in a
biological sample of interest, for example, wound tissue. Increased
level or activity of such enzymes in the wound fluid, therefore,
corresponds to a heightened bacterial challenge and a manifestation
of disturbed host/bacteria equilibrium in favor of the invasive
bacteria.
[0011] Provided herein are embodiments of a wound dressing,
devices, and methods for detecting an infection in a wound or a
sample. One embodiment is a wound dressing comprising a wound
contacting layer, a reagent layer comprising one or more testing
regions, wherein the reagent layer is in fluid communication with
the wound contacting layer, and an outer layer that overlays the
reagent layer. In some embodiments, the wound contacting layer
comprises gel-forming polymers. In further embodiments, each of the
one or more testing regions comprises one or more of each of:
back-flow trap, reagent pad, filter pad, indicator trap, and
absorbent area, wherein one or more viewing windows are located
either above the reagent pad or the indicator trap. In further
embodiments, the reagent pad is in fluid communication with the
filter pad; the filter pad is in fluid communication with the
indicator trap; and the indicator trap is in fluid communication
with the absorbent area.
[0012] In other embodiments, one or more testing regions comprises
one or more reagents selected from the group consisting of
enzyme-reactive indicators, reagents that are sources of peroxide,
enzymes that produce colored products, pH indicators, protein
responsive reagents, and moisture-detecting reagents. The
enzyme-reactive indicators include protein-indicator conjugates
printed, sprayed, or otherwise deposited in or on the reagent pad.
In some embodiments, the protein-indicator conjugate has the
structure of Formula (I): A-B, wherein A is an anchor region or
moiety that helps to bind an enzyme-reactive region to the reagent
pad, and B is the enzyme-reactive region.
[0013] In some embodiments, the enzyme-reactive region comprises a
peptide and/or an indicator region. In further embodiments, the
wound dressing comprises an indicator region that after having been
cleaved by the target enzyme in a sample is further transformed
into a colored species by accessory enzymes selected from a lipase,
esterase, hexosaminidase, peroxidase, oxidase, galactosidase,
glycosidase, glucosidase, and laccase, or a combination of two or
more thereof. In some embodiments, the enzyme-reactive indicators
interact with elastase, lysozyme, cathepsin G, myeloperoxidase, or
any combination thereof. In further embodiments, the
enzyme-reactive indicators comprise a moiety capable of producing a
visible color or a detectable electronic change upon interaction of
the enzyme-labile or enzyme-reactive region with one or more
enzymes, wherein the moiety is selected from the group consisting
of a peroxidase substrate, arylamine, an amino phenol, a neutral
dye, a charged dye, a nanoparticle, a colloidal gold particle, or
an analog thereof. The anchor region can be attached to the reagent
pad covalently, non-covalently, or ionically. In some embodiments,
pH-sensitive reagents produce a visible color comprise bromothymol
blue, phenol red, bromophenol red, chlorophenol red, thymol blue,
bromocresol green, bromocresol purple; nitrazine yellow; or other
sulfophthalein dyes.
[0014] In some embodiments, the wound dressing also comprises one
or more lines of wicking stitching or wicking tufting throughout
all layers of the wound dressing except the outer layer, wherein
the wicking stitching or wicking tufting provides fluid
communication between the reagent layer and the wound contacting
layer. Fibers that are wettable and exhibit capillary action may be
used for wicking stitching or wicking tufting to form fluid
communication between a sample or a wound and the reagents. In some
embodiments, the wicking fibers are solid or hollow. Examples of
wicking fibers include, but are not limited to, cotton, rayon,
viscose, wool, silk, polyester, polyamide, CMC, and
polypropylene.
[0015] In further embodiments, the wound dressing comprises one or
more testing regions, comprising a leach-back trap in fluid
communication with the reagent pad and one or more lines of wicking
stitching or wicking tufting crossing through one or more testing
regions only at the leach-back trap. In some embodiments, a foam
layer is added between the wound contacting layer and the reagent
layer. One or more perforations can be added in the wound
contacting layer or in the foam layer and the wound contacting
layer. In further embodiments, each of the one or more testing
regions further comprises a leach-back trap in fluid communication
with the reagent pad and one or more perforations aligned with the
leach-back trap.
[0016] In some embodiments, the testing regions comprise a
multichannel testing region, wherein each channel within the
multichannel testing region is separated from an adjacent channel
by one or more impermeable separators or borders. Such multichannel
testing regions can comprise 1 to 10 testing regions, preferably 3,
4, or 5 testing regions, wherein the testing regions are arranged
in a linear or a radial configuration. Arrays of multichannel
testing regions can be combined to cover a broader area of a wound
or wound dressing. In further embodiments, the outer layer of the
wound dressing comprises one or more windows that permit
visualization of a signal from the reagent layer, wherein the
signal is a color change.
[0017] Such wound dressing or device provides a method of detecting
the level of one or more enzymes in a mammalian wound, comprising
contacting the mammalian wound with the wound dressing; observing
one or more signals in the reagent layer, wherein the signal is a
color change; and comparing the signal to a reference or control to
determine the level of an enzyme. In another embodiment, the wound
dressing can be used to detect the presence of one or more enzymes
and/or pH in a mammalian wound, comprising contacting the mammalian
wound with the wound dressing and observing one or more signals in
the reagent layer, wherein the signal is a color change. In another
embodiment, the wound dressing can be used to treat an infection in
a wound of a mammal or to determine when such treatment is
necessary, comprising contacting the wound with a wound dressing
described herein, observing one or more signals in the reagent
layer, wherein the signal is a color change and indicates the
presence of an infection, and administering a medical treatment to
the mammal.
[0018] In some embodiments, a device for detecting an infection in
a wound comprises a wound contacting layer, a reaction layer
comprising one or more reagents that can indicate the presence of
one or more analytes associated with an infection, wherein the
reagents are affixed to a solid phase and produce a detectable
signal in a reporter area, a cover on top of the reaction layer,
wherein the cover comprises one or more windows or clear areas to
allow visualization of the detectable signal, such as a color
change, and fluid communication between the wound contacting layer
and the reaction layer. Reagents include enzyme-reactive indicators
that interact with one or more enzymes selected from the group
consisting of lysozyme, MPO, cathepsin G, elastase, catalase,
lipase, esterase, and any combination thereof, at least one
indicator for pH or a change in pH, wherein the indicators may be
printed, sprayed, or deposited on a solid phase or support
material, including paper, viscose, regenerated cellulose, glass
fiber, or similar materials. In further embodiments, the
enzyme-reactive indicators comprise a moiety capable of producing a
visible color or a detectable electronic change upon interaction of
the enzyme-labile or enzyme-reactive region with one or more
enzymes, wherein the moiety is selected from the group consisting
of a peroxidase substrate, arylamine, an amino phenol, a neutral
dye, a charged dye, a nanoparticle, a colloidal gold particle, and
an analog thereof. In further embodiments, the device comprises
wicking stitching or wicking tufting of an absorbent material to
form fluid communication between the wound contacting layer and the
reaction layer.
[0019] A device for detection of infection associated enzymes that
is provided as an independent entity and can be placed into any
dressing or bandage system, comprising a sample inlet in fluid
communication with reagent cells, wherein reagent cells comprise
indicators for sample delivery and/or pH change, which can be one
and the same, and one or more indicators for biomarkers of an
infection, including lysozyme, MPO, cathepsin G, elastase,
catalase, lipase, esterase, and any combination thereof. The fluid
communication comprises at least one indicator channel, lane, or
arm, such as one to ten indicator channels, or one, two, three,
four, five, six, seven, eight, nine, or ten separate indicator
channels, wherein the indicators are printed, sprayed, or deposited
in a reaction area or field on a carrier material or solid phase
and arranged in a radial configuration to form a disk, and wherein
the reaction areas or fields are separated by impermeable
separators or lanes. The carrier material may comprise a non-woven
material. In some embodiments, the disk comprises reagents printed,
sprayed, or deposited on the top surface of the disk with a trap
material and a substrate material on the bottom surface, wherein
the substrate can be digested by one or more enzymes in the sample
to release one or more products that migrate towards the trap. In
further embodiments, one or more products are colored or produce a
color change capable of being visualized on the top surface of the
disk.
[0020] In additional embodiments, a diagnostic disk for detecting
an infection in a wound comprises a reaction layer comprising one
or more reagents that interact with an enzyme indicative of an
infection, wherein the reagents are affixed to a solid phase; each
reagent is sprayed, printed, or deposited in a reagent area
separated by impermeable separators; each lane comprises a reporter
area wherein a color change can be observed; and a cover comprising
a window for visualizing the color change in the reported area. The
diagnostic disk may further comprise at least one reagent that
produces a color change in response to a change in pH. Multiple
lanes in the diagnostic disk, wherein each lane contains a
different indicator/reagent, can be arranged in a linear or radial
configuration about a cut access, perforation, or wicking material
that allows fluid communication between a sample or wound contact
material and the reagents in the reaction layer. The reagents
include indicators as described above, namely, reagents that
interact with lysozyme, MPO, cathepsin G, elastase, catalase,
lipase, or esterase. In some embodiments, the diagnostic disk
comprises a solid phase material selected from the group consisting
of paper, viscose, regenerated cellulose, glass fiber, and similar
material. In further embodiments, the disk is attached to a
non-woven carrier in a wound dressing, wherein means for such
attachment include, but are not limited to, a continuous adhesive,
ring or annular adhesive, welding with UV printed border, and
welding with a polyethylene component or the non-woven carrier.
[0021] In further embodiments, the reagents describes herein may be
applied to form a lateral flow or dipstick device for detecting an
infection in a wound, comprising one or more reagent disks arranged
in a linear configuration, wherein each reagent disk is impregnated
with a reagent that interacts with an enzyme to produce a color
change or a similar detectable signal, wherein one of the disks
produces a color change based on pH, and wherein the disks are
affixed to a solid phase comprising paper, viscose, regenerated
cellulose, glass fiber, or similar materials. Reagents include
enzyme-reactive indicators that produce a color signal in the
presence of lysozyme, MPO, cathepsin G, elastase, catalase, lipase,
or esterase. In one embodiment, each disk is separated by an
impermeable border or lane.
[0022] In a further embodiment, a standalone device for detecting
an infection in a wound or a sample comprises a housing,
comprising: a sampling component for collecting the sample; a
sample preparation chamber in fluid communication with a reaction
chamber, wherein the sample preparation chamber receives the
sample; the reaction chamber comprising one or more reaction cells
containing reagents that interact with one or more enzymes in the
sample to indicate the presence of an infection and/or pH of the
sample; and a window or a clear area for visualizing a detectable
signal, wherein the signal is a color change or an electronic
output. One or more reagents interact with an enzyme selected from
the group consisting of lysozyme, MPO, cathepsin G, elastase,
catalase, lipase, and esterase to produce a detectable signal,
wherein the signal is a color change. One or more regents produce a
color change in response to a change in pH, a basic pH, or an
acidic pH. In further embodiments, the reagents perform the
reactions in a primarily liquid medium, wherein the reagents may be
provided in tablet form for use in the reaction cells. In some
embodiments, the reagents may be printed, sprayed, or deposited in
separate reagent fields on a support material to form a panel of
tests, such as a testing strip, for use in the reaction chamber.
Support materials include paper, viscose, regenerated cellulose,
and glass fiber. Reagent fields can be arrayed in a line along a
plastic or paper carrier strip, which is capable of absorbing the
sample in the reaction chamber, allowing the sample to interact
with the reagents in the reaction chamber. In some embodiments, the
sampling component comprises a swab device, or a hook or needle
device adapted to removing a sampling thread from a wound dressing
to sample the wound fluid without disturbing the dressing.
[0023] In further embodiments, a kit for detecting an infection in
a sample comprises a sampling component for collecting the sample;
a test device comprising a housing surrounding a tube to define an
opening in the housing for receiving the sampling component, the
housing comprising: a diluent chamber that holds a liquid diluent;
a reaction well in liquid communication with the tube or the
sample, the reaction well holding one or more reagents that
interacts with one or more analytes to produce a color change or
similar detectable signal; a viewing window or reporter area
wherein the color change or similar detectable signal can be
observed; and wherein the liquid diluent flows from the sample
component into the reaction well to mix the sample with the
reagents in the reaction well. The reagents comprise one or more
enzyme-reactive indicators and/or pH indicator, as described above.
The sample may be obtained from a wound, a wound dressing, or a
surgical site. In some embodiments, the sampling component is a
swab device or a hook or needle device. The reagents can be
provided in tablet form, which are dissolved upon contacting the
liquid diluent and the sample. The reagents can also be deposited
in separate fields on a testing strip to form a panel of tests,
which can be applied in the reaction wells.
[0024] In another embodiment, the reagents are provided in liquid
form for use in the reaction wells. The number of reaction wells is
based upon the number of analytes to be analyzed, ranging from one
to ten, including indicators that produce a detectable signal in
response to pH or the presence of one of the following enzymes:
lysozyme, MPO, cathepsin G, elastase, catalase, lipase, and
esterase. The reaction wells can be arranged in various
configurations, including a linear or a radial configuration.
[0025] In another embodiment, a wound dressing is disclosed
comprising: a wound contacting layer; a reagent layer comprising
one or more testing regions, wherein the reagent layer is in fluid
communication with the wound contacting layer; and an outer layer
that overlays the reagent layer.
[0026] In another embodiment, a wound dressing is disclosed wherein
each of the one or more testing regions comprises one or more of
each of a back-flow trap, a reagent pad, a filter pad, an indicator
trap, and an absorbent area, and wherein one or more viewing
windows are located either above the reagent pad or the indicator
trap.
[0027] In another embodiment, a method of detecting the level of
one or more enzymes in a mammalian wound is disclosed, the method
comprising: contacting the mammalian wound with a wound dressing;
observing one or more signals in the reagent layer, wherein the
signal is a color change, a fluorescent signal, a luminescent
signal, or an electrical change; and comparing the signal to a
reference or a control to determine the level of an enzyme.
[0028] In another embodiment, a method of detecting the presence of
one or more enzymes in a mammalian wound is disclosed, the method
comprising: contacting the mammalian wound with a wound dressing;
and observing one or more signals in the reagent layer, wherein the
signal is a color change, a fluorescent signal, a luminescent
signal, or an electrical change.
[0029] In another embodiment, a method of detecting an infection in
a mammalian wound is disclosed, the method comprising: contacting
the wound with a wound dressing; and, observing one or more signals
in the reagent layer, wherein the signal is a color change, a
fluorescent signal, a luminescent signal, or an electrical
change.
[0030] In another embodiment, a device for detecting an infection
in a wound is disclosed, comprising: a wound contacting layer; a
reaction layer comprising one or more reagents that can indicate
the presence of one or more analytes associated with an infection,
wherein the reagents are affixed to a solid phase and produce a
detectable signal in a reporter area; a cover on top of the
reaction layer, wherein the cover comprises one or more windows or
clear areas to allow visualization of the detectable signal; and,
fluid communication between the wound contacting layer and the
reaction layer.
[0031] In another embodiment, a wound dressing is disclosed wherein
the reagent pad is in fluid communication with the filter pad; the
filter pad is in fluid communication with the indicator trap; and
the indicator trap is in fluid communication with the absorbent
area.
[0032] In another embodiment, a diagnostic disk for detecting an
infection in a wound is disclosed, comprising: a reaction layer
comprising one or more reagents that interact with a target enzyme
indicative of an infection, wherein the reagents are affixed to a
solid phase; each reagent is sprayed, printed, or deposited in a
reagent area in a lane separated from adjacent lanes by impermeable
separators; each lane comprises a reporter area wherein a color,
color change, or other detectable signal is observed; and a cover
comprising a window for visualizing the signal in the reporter
area.
[0033] In another embodiment, a lateral flow or dipstick device for
detecting an infection in a wound is disclosed, comprising: one or
more reagent disks arranged in a linear configuration, wherein each
reagent disk is impregnated with a reagent that interacts with an
enzyme to produce a color change and/or is pH-sensitive, comprising
bromothymol blue, phenol red, bromophenol red, chlorophenol red,
thymol blue, bromocresol green, bromocresol purple; nitrazine
yellow; or other sulfophthalein dyes, and wherein the disks are
affixed to a solid phase.
[0034] In another embodiment, a device for detecting an infection
in a wound or a sample is disclosed, comprising a housing, wherein
the housing comprises: a sampling component for collecting the
sample; a sample preparation chamber in fluid communication with a
reaction chamber, wherein the sample preparation chamber receives
the sample; the reaction chamber comprising one or more reaction
cells containing reagents that interact with one or more enzymes in
the sample to indicate the presence of an infection and/or pH of
the sample; and a window or a clear area for visualizing a
detectable signal, wherein the signal is a color change.
[0035] In another embodiment, a kit for detecting an infection in a
sample is disclosed, comprising: a sampling component for
collecting the sample; a test device comprising a housing
surrounding a tube to define an opening in the housing for
receiving the sampling component, the housing comprising: a diluent
chamber that holds a liquid diluent; a reaction well in liquid
communication with the tube, wherein the reaction well holds one or
more reagents that interact with one or more analytes to produce a
color change or a detectable signal; a viewing window or reporter
area wherein the color change or detectable signal can be observed;
and wherein the liquid diluent flows from the sample component into
the reaction well to mix the sample with the reagents in the
reaction well.
[0036] It is understood that other embodiments and configurations
of the subject technology will become readily apparent to those
skilled in the art from the following detailed description, wherein
various configurations of the subject technology are shown and
described by way of example or illustration. As will be realized,
the subject technology is capable of other and different
configurations and its several details are capable of modification
in various other respects, all without departing from the scope of
the subject technology. Accordingly, the figures and detailed
description are to be regarded as illustrative in nature and not as
restrictive.
INCORPORATION BY REFERENCE
[0037] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0038] To understand the present disclosure, it will now be
described by way of example, with reference to the accompanying
figures in which embodiments and examples of the disclosures are
illustrated and, together with the descriptions below, serve to
explain the principles of the disclosure.
[0039] FIG. 1: Examples of engineered three-dimensional fabric
structures, such as corrugations.
[0040] FIG. 2: Example of a dressing with AQUACEL showing different
layers of a dressing and stitching that draws fluid from a wound to
the reaction layer of the dressing.
[0041] FIG. 3: Schematic of reaction cells showing different
components of a reaction cell with stitching (21) in (A) and cut
access (27) in (B). In some embodiments, each reaction cell can be
a different reporter or dye system.
[0042] FIG. 4: Movement of indicators in reaction cells upon
exposure to fluid, which flows from cut access and reagents (22)
toward absorbent or evaporation area (25). Over time, the reaction
products diffuse and migrate toward an absorbent or evaporation
area. Movement of indicators arranged in a radial manner is shown
in (B). In some embodiments, each lane or reaction cell can be a
different reporter or color system. Multiple reaction cells can be
used as shown in (C). Multiple reaction cells can be used in arrays
or combinations to provide indicator function over an area. Leach
back traps may be used to prevent backflow.
[0043] FIG. 5: Indicators can be arranged in a circular or radial
manner to form indicator disks (A). In some embodiments, each lane
or reaction cell (45-48) can be a different reporter or color
system, such as bromothymol blue, phenol red, bromophenol red,
chlorophenol red, thymol blue, bromocresol green, bromocresol
purple; nitrazine yellow; or other sulfophthalein dyes. (B) shows
views of a radial indicator disk from above and from below.
[0044] FIG. 6: Dressing printed for MPO detection. In one
embodiment, a wound contact material is sprayed or printed with
amylase, starch, and glucose oxidase, followed by printing of a
substrate for MPO printed in the centers of each sprayed area.
[0045] FIG. 7: In-place color development of MPO and elastase
substrates on testing strips are shown. These test strips represent
prototypes of visualization methods for detecting the presence of
MPO and elastase in a sample, wherein color (e.g., blue) increases
in intensity with greater substrate concentration.
[0046] FIG. 8: Examples of substrates, including MPO substrate
(Fast Blue derivative), elastase substrate, and oxidation of
indoxyl to blue colored indigo are shown.
[0047] FIG. 9: In-place color development of different indicators
in radial arrangement. (A) and (B) represent prototypes of
indicators for detecting certain analytes, including pH change,
MPO, lysozyme, and elastase. In one embodiment, pH change can be
reported as a color change from yellow to green; MPO reported as an
appearance of a blue color; lysozyme reported as an appearance of
pink or red color; elastase reported as an appearance of green or
blue color; and liquid control reported as an appearance of a blue
or purple color.
[0048] FIG. 10: Schematics of a radial indicator insert or
disk.
[0049] FIG. 11: Schematics of a radial indicator insert or disk
with a window for detection.
[0050] FIG. 12: Schematics of another embodiment of a radial
indicator insert or disk with a window for detection.
[0051] FIG. 13: Transport of Remazol Brilliant Blue, showing
migration of indicators to reporter area after liquid
transport.
[0052] FIG. 14: Example of a pH indicator. In one embodiment, the
color can change from green to blue with increase in pH.
[0053] FIG. 15: Schematic of a lysozyme test strip. Fluid flow
causing stained peptidoglycan particles to move upwards to trap
layer.
[0054] FIG. 16: Examples of indicator substrates and reactions.
[0055] FIG. 17: Example of indicator disk freely placed in a
dressing.
[0056] FIG. 18: Embodiments of diagnostic disks in non-woven layer
in dressing.
[0057] FIG. 19: Embodiments of diagnostic disks in non-woven layer
in dressing.
[0058] FIG. 20: Example of manufacturing diagnostic disks in
sheets.
[0059] FIG. 21: Embodiments of printed and applied paper disks. In
some embodiments, each disk can be a different reporter or color
system.
[0060] FIG. 22: Methods of attaching or applying diagnostic disks
to non-woven layer in dressing.
[0061] FIG. 23: Dipstick devices with indicator inserts or disks
arranged in different arrays and combinations are shown. In some
embodiments, each insert, disk, or lane can be a different reporter
or color system.
[0062] FIG. 24: Sampling thread and use in dressing. Sampling
thread can be incorporated in a wound dressing or at a surgical
site, wherein the thread can be pulled out without disturbing the
dressing to test for the presence of microbial infection or
condition of the surgical site or wound in a diagnostic device.
[0063] FIG. 25: Assembly for manufacturing indicator inserts.
[0064] FIG. 26: Cross section of a standalone device kit
[0065] FIG. 27: Sampling tip inserted in the housing of standalone
device kit
[0066] FIG. 28: A plan view of the standalone device kit
[0067] FIG. 29: Another view of the standalone device kit
[0068] FIG. 30: A plan view of the standalone device kit with
housing slid apart
[0069] FIG. 31: Diluent chamber, tube and reaction chamber in
standalone device kit
[0070] FIG. 32: Distribution of test solution to each reaction
chamber in standalone device kit
[0071] FIG. 33: Diagnostic swab device with housing, wherein
reaction with indicator disks or inserts can be observed from a
viewing window in the housing.
[0072] FIG. 34: Thread hook diagnostic device, suitable for pulling
out a sampling thread from a dressing for analysis.
[0073] FIG. 35: Swab diagnostic device, wherein a swab is used to
obtain a sample for testing with a diagnostic device, further
comprising a diluent chamber, gas outlet, and a plunger.
[0074] FIG. 36: Diluent chamber for sample preparation. A diluent
chamber comprising a diluent is adapted for use with a swab device,
a thread hook device, and similar sample preparation devices,
comprising a resealable top and a seal or film at the bottom,
wherein breaking the seal or film (402) allows the sample to mix
with the diluent solution, which flows out of the diluent chamber
and into a testing device comprising reaction chambers or
wells.
[0075] FIG. 37: Embodiment of diagnostic device with sampling
chamber and reaction wells. One embodiment of a diagnostic device
with reaction chambers (502) adapted to being connected to sampling
chamber or diluent chamber (202) for receiving a sample from a
sample preparation device (300), such as the swab device.
[0076] FIG. 38: Embodiment of diagnostic device or transfer system,
wherein the sample chamber or diluent chamber uses a Luer-lock
connector to attach to reaction chambers for testing a sample
fluid. In one embodiment, the plunger or piston comprises a gas
outlet, hook for holding a sample, and membrane that lets out gas
as the plunger is depressed into the diluent chamber.
[0077] FIG. 39: Further embodiments of an analytic or diagnostic
system, wherein reaction chambers are arranged in a radial
arrangement.
DETAILED DESCRIPTION
[0078] Various aspects of the disclosed technology will be
described more fully hereinafter. Such aspects may, however, be
embodied in many different forms and should not be construed as
limited to the embodiments set forth herein; rather, these
embodiments are provided so that this disclosure will be thorough
and complete, and will fully convey its scope to those skilled in
the art.
[0079] Throughout this disclosure, various patents, patent
applications and publications are referenced. The disclosures of
these patents, patent applications and publications in their
entireties are incorporated into this disclosure by reference in
order to more fully describe the state of the art as known to those
skilled therein as of the date of this disclosure. This disclosure
will govern in the instance that there is any inconsistency between
the patents, patent applications and publications cited and this
disclosure.
[0080] Provided herein are means to detect infections in wounds. In
some embodiments these are wound dressings able to detect infection
in one or more body fluids before such infection is otherwise
apparent. In some embodiments, the wound dressing reacts with wound
exudate or wound fluid to detect infection in a wound through a
visible or otherwise detectable change in the dressing. In some
embodiments, wound exudate or wound fluid is drawn up through the
wound dressing to a reagent layer for assessment of possible
infection without the need to remove the dressing. In some
embodiments, wound exudate or wound fluid reacts with the reagent
layer to give rise to a color or other visible or observable
marker. In some embodiments, the color is easily distinguishable
from those colors that are common in wounds or body fluids. In some
embodiments, the reaction between the wound exudate or wound fluid
and the reagent layer of the wound dressing occurs at ambient
temperature and within a period of time short enough to allow
timely response, such as a decision to make a dressing change after
cleaning the wound and examining the test result and/or to
administer antiseptics or local or systemic antibiotics. In some
embodiments, the color or other visible or observable marker and/or
the location of the color or other visible or observable marker
indicates one or more areas of the wound that deserve closer
attention and/or antisepsis. In some embodiments, the color change
function is embedded in parts of the dressing that are only visible
on dressing change. In further embodiments, the reagent layer that
gives rise to a color change or other visible or observable marker
is a standalone device, disk, or insert, capable of application
with any wound dressing, at a surgical or wound site, or by itself
as a dipstick-type of device. In further embodiments, indicator
reagents are applied in a "swab sample preparation device" or a
stand-alone device into which wound fluids are injected. In some
embodiments, indicator reagents are printed directly on support
materials, such as the various layers within a wound dressing.
[0081] In some embodiments disclosed herein, a wound dressing
comprises a wound contacting layer; a reagent layer comprising one
or more testing regions or indicator reagents; and an outer layer
that overlays the reagent layer. The wound dressing may comprise
one or more testing regions, which further comprise one or more of
a back-flow trap, reagent pad, a filter pad, an indicator trap, and
an absorbent area, wherein the viewing window is located either
above the reagent pad or the indicator trap and the reagent pad is
in fluid communication with a filter pad; the filter pad is in
fluid communication with the indicator trap; and the indicator trap
is in fluid communication with the absorbent area.
[0082] In some embodiments, testing regions comprise one or more
components selected from the group consisting of enzyme-reactive
indicators, reagents that are sources of peroxide, enzymes that are
able to transform color reactions, pH indicators, protein
responsive reagents, and moisture-detecting reagents.
Enzyme-reactive indicators may comprise protein-indicator
conjugates.
[0083] In some embodiments, protein-indicator conjugates are
deposited in or on the reagent pad. In some embodiments,
protein-indicator conjugate has the structure of Formula (I): A-B,
wherein: A is an anchor region for attachment to the testing
region; and B is an enzyme-reactive region. In further embodiments,
the enzyme-reactive region comprises a peptide or an indicator
region. The anchor region may be covalently or non-covalently
attached to the reagent pad.
[0084] In further embodiments, the wound dressing comprises one or
more lines of wicking stitching or wicking tufting throughout all
layers of the wound dressing except the outer layer. One or more
testing regions further comprises a leach-back trap in fluid
communication with the reagent pad, the one or more lines of
wicking stitching or wicking tufting crossing through each of the
one or more testing regions only at the leach-back trap. In further
embodiments, the wound dressing comprises a foam layer between the
wound contacting layer and the reagent layer. In some embodiments,
the wound dressing further comprises one or more perforations of
the wound contacting layer.
[0085] In some embodiments, enzyme-labile or enzyme-reactive
regions contained therein may interact with target enzymes
including elastase, lysozyme, cathepsin G, and myeloperoxidase. In
further embodiments, the enzyme-labile or enzyme-reactive region
comprises a moiety capable of producing a visible color or
detectable electronic change upon interaction of the enzyme-labile
or enzyme-reactive region with one or more target enzymes, the
moiety being selected from a peroxidase substrate, arylamine, an
amino phenol, an indoxyl, a neutral dye, a charged dye, a
nanoparticle, and a colloidal gold particle, and an analog thereof.
In some embodiments, after the target enzyme has cleaved the
indicator from the substrate it is further reacted by an accessory
enzyme selected from a lipase, esterase, hexosaminidase,
peroxidase, oxidase, glycosidase, glucuronidase, glucosidase, and
laccase, or a combination of one or more thereof.
[0086] Applications of the reactive regions may include a device
for detection of infection associated enzymes, on a solid phase
such as paper, viscose, regenerated cellulose, glass fiber,
mixtures of same or similar material, or arrayed in a line along a
plastic or paper carrier strip.
[0087] In some embodiments, reagent or indicator inserts or disks
for detection of infection associated with certain enzymes may be
provided as an independent entity and placed into any dressing
system comprising a sample inlet, diffusion channels toward
different areas containing reagents, an indicator for sample
delivery and or an indicator of pH which may be one in the same,
and one or more indicators for the following markers selected from
lysozyme, MPO, cathepsin G, elastase, catalase, lipase,
esterase.
[0088] In some embodiments, the enzyme labile region is labile to a
protease and the polymer binding domains are selected from
cellulose binding domains or are hydrophobic binding domains.
[0089] In some embodiments, the enzyme labile region is labile to
cathepsin or elastase.
[0090] In some embodiments, the chemical entity is selected from a
small molecule entity, a modified oligomer, and a modified
polymer.
[0091] In another aspect, provided herein is a chemical entity for
the detection of infection in a wound, the chemical entity
comprising an indicator region comprising a pH-sensitive moiety
that presents a visible color change.
[0092] In some embodiments, the chemical entity further comprises
an anchor region wherein the anchor region enables binding of the
chemical entity to a support material.
[0093] In some embodiments, the pH-sensitive moiety that presents a
visible color change at alkaline pH. In some embodiments, the
pH-sensitive moiety that presents a visible color change at neutral
pH. In some embodiments, the pH-sensitive moiety that presents a
visible color change at acidic pH.
[0094] In some instances, the pH of a wound can influence many
factors of wound healing, such as angiogenesis, protease activity,
oxygen release, and bacterial toxicity. Chronic non-healing wounds
may have an elevated alkaline environment. As the wound progresses
towards healing, the pH of the wound moves to neutral and then
becomes acidic. Monitoring of the pH of the wound may provide a
method to assess the condition of the wound (e.g., infection or no
infection) and aid in determining a wound's response to
treatment.
[0095] Accordingly, in some aspect of the disclosed technology, the
chemical entity for the detection of infection in a wound comprises
an indicator region comprising a pH-sensitive moiety that presents
a visible color change. In some embodiments, the chemical entity
further comprises an anchor region wherein the anchor region
enables binding of the chemical entity to a support material. In
some embodiments, the pH-sensitive moiety presents a visible color
change at alkaline pH. In some embodiments, the pH-sensitive moiety
presents a visible color change at pH=7.2-9.5. In some embodiments,
the pH-sensitive moiety presents a visible color change at
pH=7.2-9.0. In some embodiments, the pH-sensitive moiety presents a
visible color change at pH=7.2-8.5. In some embodiments, the
pH-sensitive moiety presents a visible color change at pH=7.2-8.0.
In some embodiments, the pH-sensitive moiety presents a visible
color change at pH=7.5-8.5. In some embodiments, the pH-sensitive
moiety presents a visible color change at pH=7.5-9.0. In some
embodiments, the pH-sensitive moiety presents a visible color
change at pH=8.0-9.0. In some embodiments, the pH-sensitive moiety
presents a visible color change at pH=7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,
9.1, 9.2, 9.3, 9.4, or 9.5, or increments thereof.
[0096] In some embodiments, the pH-sensitive moiety presents a
visible color change at neutral pH. In some embodiments, the
pH-sensitive moiety presents a visible color change at pH =6.9,
7.0, or 7.1, or increments thereof.
[0097] In some embodiments, the pH-sensitive moiety presents a
visible color change at acidic pH. In some embodiments, the
pH-sensitive moiety presents a visible color change at pH=4.5-6.8.
In some embodiments, the pH-sensitive moiety presents a visible
color change at pH=4.5-6.5. In some embodiments, the pH-sensitive
moiety presents a visible color change at pH=5.0-6.8. In some
embodiments, the pH-sensitive moiety presents a visible color
change at pH=5.4-6.8. In some embodiments, the pH-sensitive moiety
presents a visible color change at pH=5.4-6.5. In some embodiments,
the pH-sensitive moiety presents a visible color change at pH=4.5,
4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,
5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or
increments thereof.
[0098] In some embodiments, the pH-sensitive moiety is bromothymol
blue, phenol red, bromophenol red, chlorophenol red, thymol blue,
bromocresol green, bromocresol purple; nitrazine yellow; or other
sulfophthalein dyes.
[0099] Other embodiments include reagents printed on dressing or
solid support materials, dipstick devices with indicator disks
arranged in various arrays, and devices with separate sample
preparation chamber that transfer a sample of a bodily fluid or
wound fluid to a standalone diagnostic device that uses reagent
pills, solutions, or disks in reaction chambers for detecting
biomarkers associated with microbial detection. In further
embodiments, indicator reagents are printed, sprayed, or overlayed
on support materials, such as dressing, wound dressing, bandage,
filter paper, and test strips.
[0100] Generally, when a pathogen encounters the human body
interior, cells react through innate receptor systems, either to
injury, toxins, or to the bacterial cell wall. All of these
recognition events result in the recruitment of innate immune
cells. These cells are stimulated by pathogens like bacteria to
activate bacterial killing systems that are normally present in
polymorphonuclear leukoctyes (PMNs) and are mainly enzymatic in
character. The cells engulf bacteria and lyse them with enzymes
that hydrolyze proteins (e.g., protease, elastase, cathepsin G) and
cell walls (lysozyme), or mediate protein denaturation (NADPH
oxidase, xanthine oxidase, myeloperoxidase (MPO)). These PMNs are
generally only short lived and will themselves lyse in the area of
the infection. When they lyse, they release the contents of their
lysosomes including the enzymes.
[0101] These enzymes are, therefore, biomarkers for the presence of
myeloid cells, and PMNs in particular. A rising level of these
enzymes in the wound fluid, therefore, corresponds to a heightened
bacterial challenge and one that is not being adequately met by the
innate defense. The association of these enzyme levels with
clinical infection has been validated using a clinical trial
approach (Blokhuis-Arkes et al., 2015).
[0102] In addition, the pH of a wound can influence many factors of
wound healing, such as angiogenesis, protease activity, oxygen
release, and bacterial toxicity. Chronic non-healing wounds, and
those that are infected or at risk of infection, typically have an
elevated alkaline environment. As the wound progresses towards
healing, the pH of the wound moves to neutral and then becomes
acidic. Monitoring of the pH of the wound may provide a method to
assess the condition of the wound (e.g., infection or no infection)
and aid in determining a wound's response to treatment.
[0103] A typical lateral flow device utilizes the concept of
lateral liquid flow in order to transport a given sample to the
test. The benefits of lateral flow tests include rapid results,
long-term stability and low cost to manufacture. These features
make lateral flow tests well-suited for applications involving drug
testing in urine, in particular with rapid point of care testing in
hospitals and doctor's offices being an advantage. A test strip can
be dipped directly in the sample which is taken in a liquid form.
The sample travels up the lateral flow strip and binds to available
antibodies, which causes a reaction that can be visually detected
on the strip. Applying this technology to samples other than urine
or blood has however been problematic.
[0104] Early detection of markers for infection in wounds has
advantages in that treatment of infection can be commenced before
the infection becomes established and other signs of infection
become apparent, for example, discharge from the wound, redness,
pain and unpleasant odor. A difficulty in testing for markers in
wound fluid is that wound fluid differs greatly in its consistency
and quantity. For instance it can be scant but viscous making the
use of a lateral flow test difficult.
[0105] Thus it would be desirable to have a single kit for
collecting and testing a sample of fluid taken from a wound that is
easy to operate and not limited by the type or quantity of exudate
from the wound. One embodiment of the standalone device kit
described herein mitigates the above problems in a kit which
comprises a sampling component and a test device where the test
device does not rely on a lateral flow strip to move the sample
through the device and achieve a diagnosis.
[0106] Wound Dressing
[0107] In some embodiments, the wound dressing comprises a wound
contacting layer; a reagent layer comprising one or more testing
regions; and an outer layer that overlays the reagent layer. In
some embodiments, the wound dressing further comprises a protective
cushioning layer (for example a foam or a nonwoven layer) between
the wound contacting layer and the reagent layer. In some
embodiments, the wound dressing further comprises one or more lines
of wicking stitching or wicking tufting throughout all layers of
the wound dressing except the outer layer. In some embodiments, the
wound dressing comprises perforation through the wound contacting
layer, the protective cushioning layer, or a combination of both.
In some embodiments, such perforation allows for wound fluid
transfer from the wound to the reagent layer.
[0108] Wound Contacting Layer
[0109] When in use, the wound contacting layer of the wound
dressing absorbs wound exudate and/or wound fluid. In some
embodiments, the wound contacting layer comprises gel-forming
polymers or hydrofiber. Gel-forming polymers include, but are not
limited to cellulose, carboxymethylcellulose (CMC),
carboxyethylcellulose, oxidized cellulose (or a derivative
thereof), cellulose ethyl sulfonate, other chemically modified
cellulose, pectin, alginate, chitosan, modified chitosan,
hyaluronic acid, polysaccharide, or gum-derived polymer, or any
combination thereof. In some embodiments, the wound contacting
layer may comprise polyvinylpyrrolidone, polyvinyl alcohols,
polyvinyl ethers, polyurethanes, polyacrylates, polyacrylamides,
collagen, gelatin or mixtures thereof. In some embodiments, the
wound contacting layer comprises fibers of gel-forming polymers. In
some embodiments, the wound contacting layer comprises a nonwoven
layer of gel-forming fibers.
[0110] In some embodiments, the wound contacting layer further
comprises non-gel-forming polymers. In some embodiments, the wound
contacting layer comprises cellulose (for example, Lyocell),
modified cellulose (for example, viscose or rayon), Polyester,
silk, wool, Nylon, Polypropylene, Elastane or mixtures thereof.
[0111] In one embodiment, the thickness of the wound contact layer
is from 0.1 to 10 mm, in a preferred embodiment it is from 0.1 to 5
mm and in a still more preferred embodiment it is from 0.3 to 3.5
mm.
[0112] Protective Cushioning Layer
[0113] In some embodiments, the protective cushioning layer
provides mechanical protection of the wound and also assists in the
management of excess exudate by acting as a large surface area for
evaporation. In some embodiments, the protective cushioning layer
may also serve as the material that accepts fluid exiting reagent
layer or device and may add functionality by pulling or directing
fluid through the reagent layer or device. Suitable materials
include foams, (non-gelling) fiber fleeces, (non-gelling) nonwoven
fabrics, and engineered three-dimensional fabric structures, such
as corrugations. Examples of engineered three-dimensional fabric
structures are shown at FIG. 1. Preferably, materials used for the
protective cushioning layer possess mechanical cushioning
properties that are unaffected or are minimally affected by contact
with wound exudate. In some embodiments, the protective cushioning
layer comprises plastics based on olefins or olefin derived
polymers, such as polyethylene, polypropylene, nylon, polyurethane,
polystyrene and polyvinyl chloride. In some embodiments, these
materials may further comprise agents such as surfactants or
absorbents that improve their wettability.
[0114] In some embodiments, hydrophilic polyurethane foam is 2.5 mm
(+/-0.5 mm) thick, with a density of 90 kg/m.sup.3 to 150
kg/m.sup.3, absorption of .gtoreq.12 g/g.
[0115] Wicking Stitching and/or Wicking Tufting
[0116] In some embodiments, the transfer of wound fluid to the
reagent layer is optimized by fiber tufts from the wound contact
layer to the reagent layer. In some embodiments, gel forming
polymers from the wound contact layer can be used as the transport
mechanism of fluid from the wound to reagent layer. In some
embodiments, the increased hydrophilic nature of gel forming
polymers in comparison to materials within alternate layers of the
dressing allows enhanced wicking action to the reagent layer.
[0117] In some embodiments, yarns can be used to provide capillary
action of fluid from the wound contact layer to the reagent layer.
This can be achieved using stitching of one or more layers of the
dressing or using tufting of yarn through one or more dressing
layers.
[0118] In some embodiments, the wicking stitching and/or wicking
tufting is selected from various fibers that are wettable and
exhibit capillary action. Such fibers include, but are not limited
to, cotton, rayon, viscose, wool, silk, polyester, polyamide, and
CMC fibers, solid and hollow fibers. In some embodiments, the
wicking stitching comprises cotton, polyester, polyamide,
polypropylene, or a combination thereof. In some embodiments, using
increased number of plies or multifilament yarn, increased linear
density of yarn, and/or decreased linear density of fiber may
enhance capillary action of yarn. In some embodiments, the wicking
stitching comprises cotton. In some embodiments, the wicking
stitching comprises polyester. In some embodiments, the wicking
stitching comprises polyamide. In some embodiments, the wicking
tufting comprises CMC fibers. In some embodiments, the wicking
occurs across all areas of the dressing layers. In some
embodiments, the wicking is concentrated immediately beneath or
adjacent to the reagent layer to provide focused, enhanced wicking
action and/or reaction with the reagent layer.
[0119] In some embodiments, stitching of yarn through hydrofiber
and/or foam layer using hydrophilic yarn provides wicking capacity.
The wound fluid can be wicked up by yarns in a more direct route to
the printed substrate or reaction layer. Increase in yarn linear
density may allow more of a decrease in wicking time and/or amount
of fluid required.
[0120] In some embodiments, needling of hydrofiber-foam laminate in
wound dressing creates tufts of hydrofiber on the foam side of the
dressing. Variable parameters of needling include punch density and
penetration depth, such as 10-100 punches/cm.sup.2 at 1-10 mm
penetration, 20-90 punches/cm.sup.2 at 2-9 mm penetration, 30-80
punches/cm.sup.2 at 3-8 mm penetration, 40-80 punches/cm.sup.2 at
4-8 mm penetration, 50-80 punches/cm.sup.2 at 5-8 mm penetration,
60-80 punches/cm.sup.2 at 6-8 mm penetration, 70 punches/cm.sup.2
at 6 mm penetration. Channels of hydrofiber are created through the
foam, leading to vertical wicking of fluid. Hydrofiber tufts may
enable quicker fluid and enzyme transfer. Type of needles used for
tufting include felting (crown), felting (regular), and fork. In
some embodiments, use of felting needles allowed gelling fiber
tufts to be created through the foam layer without causing a
detrimental effect on the foam or gelling fiber. Penetration depth
may be 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm,
11 mm or 12 mm, or at least 6 mm, or less than 7 mm, less than 8
mm, less than 9 mm or less than 10 mm. Preferably, penetration
depth is 6 mm, which enabled an 18% decrease in vertical wicking
time at 70 p/cm.sup.2 punch density. As punch density increases,
more hydrofiber tufts are created on the foam layer. Enhanced fluid
transfer was seen in all punch densities at 6 mm penetration
depth.
[0121] In some embodiments, stitching of yarn through hydrofiber
and/or foam layer using hydrophilic yarn provides wicking capacity.
Stitches may be about 1 mm, about 2 mm, about 3 mm, about 4 mm,
about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about
10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15
mm, at least about 5 mm, less than about 6 mm, less than about 7
mm, less than about 8 mm, less than about 9 mm or less than about
10 mm. The wound fluid can be wicked up by yarns in a more direct
route to the printed substrate or reaction layer. Increase in yarn
linear density allows more of a decrease in wicking time and/or
amount of fluid required. Short stitches (less than 3.5 mm) do not
reduce wicking time/volume required to wick through the foam layer.
Stiches may be 5 mm to allow a reduction in wicking time by about
45%. In some embodiments, hydrofiber-foam laminate material with
combined thickness of 4.3 mm was tested for stitching with two
types of yarn: high wicking polyester (continuous filament) and
standard polyester thread. Three stitch lengths were tested,
including 2.5 mm, 3.5 mm, and 5.0 mm. Incorporating stiches
enhances fluid transfer, while increasing stitch lengths reduced
vertical wicking time.
[0122] Perforation
[0123] In some embodiments, the wicking action of the various
layers of the dressing, such as the gel-forming wound contacting
layer and the foam, is adequate as it is with the factory porosity
and no further treatment. In other embodiments, the wicking action
can be enhanced by fine needling to create channels that have
capillary action. In some embodiments, the needling can occur
across all areas of the dressing layers to provide generally
enhanced capillary action. In some embodiments, the needling is
concentrated immediately beneath or adjacent to the common entrance
to the reagent layer to provide focused, enhanced capillary action.
In some embodiments, the perforation occurs through all layers of
the dressing. In further embodiments, the perforation occurs in the
one or more layers between the wound contact layer and the reagent
layer. In some embodiments, capillary action can be enhanced by
increasing the punch density of the needling to produce higher
number of perforations per unit area.
[0124] Perforations allow direct fluid transfer through hydrofiber
and/or foam layers to the printed substrate layer. The larger the
hole, the more fluid may be transferred, reducing the wicking
time/volume required for the fluid to interact with the printed
substrate layer. However, if the hole is too large, fluid handling
capacity of the dressing may be affected. Gelling fibers swell upon
hydration and may obstruct the perforation channel of the gelling
fabric. Perforations may be formed using a hypodermic needle. At a
higher density, the vertical wicking time can be reduced by about
28%. In some embodiments, the vertical wicking time is reduced by
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45% or about 50%.
[0125] Reactive or Reagent Layer
[0126] In some embodiments, the wound contact layer, or the layer
supporting it contains a material that reacts to wound exudates to
indicate potential infection, or a reactive layer. A reactive layer
may comprise one or more dyes and/or the reagents necessary to
support these reactions. In one embodiment, these dyes comprise
amino acids, peptides, or proteins conjugated to dyes with strong
ionic functions, strong contrasting colors, or the ability to form
colors, such as indoxyl/indigo. In a preferred embodiment,
addressing includes a layer within the dressing printed with an
immobile trapping material to which said dyes bind. This layer is
optionally in the outer part of the dressing or at various levels
within the dressing such that it may be observed without dressing
change, or at dressing change.
[0127] In another preferred embodiment, the reactive layer is
comprised of an MPO substrate, glucose oxidase and an energy
source, such as glucose or starch, and gamma-amylase. In another
embodiment, the dressing contains particles comprised of chitosan
or a derivative that releases dyes on hydrolysis by lysozyme. These
dyes may be highly charged or otherwise functional to allow their
accumulation at sites of signal interpretation. In yet other
embodiments, the reactive layer comprises compounds such as
p-aminophenol, ABTS (2,2inophenol, ABTS (strate. In some
embodiments, acid) diammonium salt), 3,3'-diaminobenzidine, 3,4
diaminobenzoic acid, DCPIP, N,N-dimethyl-p-phenylenediamine,
o-dianisidine, p-phenylenediamine, 4-chloro-1-naphthol,
o-phenylenediamine N-(4-aminobutyl)-N-ethylisoluminol,
3-amino-9-ethylcarbazole, 4-aminophthalhydrazide, 5-aminosalicylic
acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),
indoxyl, indigo, Fast Blue RR, 4-chloro-7-nitrobenzofurazan. In
some embodiments, the reactive layer comprises an arylamine. In
some embodiments, the reactive layer comprises an amino phenol. In
some embodiments, the reactive layer comprises an amino phenol an
aminophenol ether. In some embodiments, the reactive layer
comprises an indoxyl. In some embodiments, the reactive layer
comprises an a neutral dye. In some embodiments, the reactive layer
comprises a charged dye, e.g., a dye selected from remazole
brilliant blue, toluidine blue, reactive black 5, remazol brilliant
blue, reactive violet 5, and reactive orange 16, or a hydrolytic or
ammonolytic derivatives thereof, toluidine blue, reactive black 5,
or ahydrolytic or ammonolytic derivatives thereof; reactive violet
5, or hydrolytic or ammonolytic derivatives thereof; reactive
orange 16, or hydrolytic or ammonolytic derivatives thereof; a
dichlorotriazine-based reactive dye such as reactive blue 4,
reactive red 120, reactive blue 2, reactive green 19 and reactive
brown 10. In some embodiments, the dichlorotriazine-based reactive
dye appears black.
[0128] In particular embodiments, the reactive layer comprises
compounds such as a reactive dye containing a
sulfonylethyl-hydrogensulphate-reactive-group. In some embodiments,
the reactive dye is reactive black 5, remazol brilliant blue,
reactive violet 5 or reactive orange 16. In some embodiments, the
reactive dye is reactive black 5. In some embodiments, the reactive
dye is remazol brilliant blue. In some embodiments, the reactive
dye is reactive violet 5. In some embodiments, the reactive dye is
reactive orange 16. In some embodiments, the reactive dye is
reactive black 5, remazol brilliant blue, or reactive violet 5. In
some embodiments, the reactive dye is reactive black 5 or remazol
brilliant blue.
[0129] In some embodiments, the reactive layer comprises a
nanoparticle. In some embodiments, the reactive layer comprises a
colloidal gold particle. In some embodiments, the reactive layer
comprises a charged dye, an indole derivative, or a luminol
derivative. Especially, the reactive layer comprises a dye
containing a sulfonylethyl-hydrogensulphate-reactive-group, e.g.,
reactive black 5, remazol brilliant blue, reactive violet 5 or
reactive orange 16, or a combination thereof, or a dye containing a
dichlortriazine reactive-group, e.g., reactive blue 4, reactive red
120, reactive blue 2, reactive green 19 and reactive brown 10, or a
combination thereof.
[0130] FIG. 3 shows two embodiments of a reaction cell, comprising
indicator units or testing regions. In (A) of FIG. 3, stitching
(21) using wicking fibers helps to draw wound or bodily fluid from
a wound toward a reagent pad (22), then through testing regions (23
and 24) and toward absorbent or evaporation area (25). In (B) of
FIG. 3, a perforation or cut access (27) is made, such as in the
reagent pad (22) to allow the flow of wound fluid from the wound to
the reagent pad via capillary action. The reagent pad (22) may
comprise reagents that react with microbial biomarkers in the wound
fluid, such as substrates that react with MPO (29), elastase (30),
and lysozyme (31) in the wound fluid. In some embodiments, one or
more testing regions may comprise a sulfonic acid filter pad (23)
and a quaternary amine trap (24). In some embodiments, one or more
testing regions comprise a leach-back trap (28) and an amine back
flow trap or filter (29). Some embodiments contain pH indicators
(32) and protein indicators (33) that allow a user to detect a
visible signal resulting from reactions between microbial
biomarkers in the wound fluid and the reagents in the reagent pad
(22). Absorbent or evaporation area (25) helps to draw the flow of
the fluid from the reagent pad (22) toward (25). In a preferred
embodiment, impermeable separators (26) keep adjacent testing
regions separate.
[0131] In some embodiments, the indicator trap catches reaction
products between the wound fluid and the one or more components
selected from the group consisting of enzyme-reactive indicators,
reagents that are sources of peroxide, enzymes that are able to
transform color reactions, pH indicators, and moisture-detecting
reagents. In some embodiments, the indicator trap comprises a
positively charged or negatively charged trap for reaction
products. In some embodiments, the positively charged trap
comprises a quaternary amine polymer, a mixture of secondary and
tertiary amines, other amine-containing polymers, or a combination
thereof. In some embodiments, the positively charged trap comprises
polyDADMAC, or an analog thereof. In some embodiments, the
negatively charged trap comprises polymers or reagents containing
carboxy, sulfate, sulfonate, or other acidic chemical groups. In
some embodiments, the negatively charged trap comprises styrene
sulfonate. In some embodiments, the indicator trap comprises a
total protein indicator which is eluted by wound fluid to indicate
overall flow and capacity of the testing region. In some
embodiments, the control region contains a substrate for a
ubiquitous enzyme such as esterase or carbonic anhydrase, or an
indicator for a ubiquitous metabolite like lactate, glucose,
ammonia or lipid. In some embodiments, one or more testing regions
comprise a sulfonic acid filter pad and a quaternary amine trap. In
some embodiments, one or more testing regions comprise a leach-back
trap, a sulfonic acid filter pad and a quaternary amine trap. In
some embodiments, each of the one or testing regions is used to
evaluate the presence of one or more analytes and one or more
positive or negative control indicators. In further embodiments,
the one or more analytes is associated with enzyme activity. In
some embodiments, the enzyme is selected from one or more of the
group consisting of elastase, lysozyme, cathepsin G,
myeloperoxidase, and leukocyte peroxidase. In some embodiments, the
enzyme is elastase. In some embodiments, the enzyme is lysozyme. In
some embodiments, the enzyme is cathepsin G. In some embodiments,
the enzyme is myeloperoxidase. In some embodiments, the enzyme is
leukocyte peroxidase.
[0132] In some embodiments, the wound dressing comprises a reagent
layer comprising one or more testing regions. In some embodiments,
the reagent layer comprises a support material. In some
embodiments, the support material comprises a woven or non-woven
material that is capable of being wet by a wound fluid and which
displays capillary action. In a preferred embodiment, the capillary
action is uniform in the plane of the material. In a preferred
embodiment, the test regions are arranged in a circle so that
diffusion occurs radially when a liquid is applied. Support
material includes, but is not limited to, paper, cellulose,
cellulose derivatives, viscose, polyamide, polyester, polyacrylate,
and other similar polymers that are useful as fibers, and any
combination thereof. In some embodiments, the support material is
cellulose-based, such as refined papers, or non-woven material
containing bonded cellulose fibers. In some embodiments, the
support material is polyamide. In some embodiments, the support
material is polyester. In some embodiments, the support material is
polyacrylate. In some embodiments, the role of the solid support is
to adhere substrates and provide a field in which analyte enzymes
can travel to and interact with the detector. In some embodiments,
cellulose content aids adherence of the enzyme substrates, and a
significant cellulose or cellulose like content is preferred.
[0133] In some embodiments, each of the one or more testing regions
is printed on or in the support material. In some embodiments, each
of the one or more testing regions comprises an inlet for wound
fluid, an area for the wound fluid to react with reagents (e.g., a
reagent pad), an area to observe each product of one or more
reactions, and an area for the accumulation of excess wound fluid
(e.g., an absorbent area), which is then evaporated from an area
sufficiently large as to not block due to accumulated solutes. In
some embodiments, the evaporation zone helps to drive pull-through
of more wound fluid.
[0134] FIG. 4 shows multiple embodiments of the movement of
indicators in various reaction cells. When testing regions in the
embodiment of (A) of FIG. 4 are exposed to wound fluid, wound fluid
flows from the reagent pad (22) to absorbent or evaporation area
(25), as shown in the right panel of FIG. 4(A). The embodiment of
(B) of FIG. 4 shows an embodiment of reaction cells wherein
indicators are arranged in a radial arrangement, and wherein fluid
flows outward from the center upon encountering the reagent pad.
The embodiments of (C) of FIG. 4 illustrates how multiple reaction
cells can be used to cover a broader area, with trap leach-back
(41) preventing backflow. In some embodiments, each reagent cell or
lane of reagent pad (22) may be a different reporter or color
system, such as bromothymol blue, phenol red, bromophenol red,
chlorophenol red, thymol blue, bromocresol green, bromocresol
purple; nitrazine yellow; or other sulfophthalein dyes. In the
presence of wound fluid, in one embodiment reagents interact with
analytes in the wound fluid and migrate or diffuse toward the
absorbent or evaporation area (25).
[0135] In some embodiments, reagents are used that require trapping
of the reaction product, and, to this end, each of the one or more
testing regions comprises a reagent pad or a reagent cell (22), a
filter pad (23), an indicator trap (24), and an
absorbent/evaporation area (25). In embodiments comprising a color
change reagent, each of the one or more testing regions comprises a
reagent pad that is also under a viewing window and an
absorbent/evaporation area. In some further embodiments, each of
the one or more testing regions comprises a leach-back trap which
is a trap field that contains an absorbent that absorbs the
reagents and prevents their back flow to the dressing below. In
some embodiments, an outer layer overlays the reagent layer in
order to modulate evaporation of wound fluid, the outer layer
containing one or more windows to visualize the underlying
indicator trap and/or reagent pad from one or more testing
regions.
[0136] In some embodiments, each of the one or more testing regions
detects at least one biomarker. In some embodiments, each of the
one or more testing regions comprises one or more impermeable
separators, wherein each of the one or more testing regions detects
more than one biomarker. In some embodiments, the one or more
impermeable separators are printed strips of hydrophobic
non-permeable material. In some embodiments, the one or more
impermeable separators are arranged in parallel lanes. In some
embodiments, the one or more impermeable separators are arranged in
a radial pattern. In some embodiments, each of the one or more
testing regions detects two biomarkers. In some embodiments, each
of the one or more testing regions detects three biomarkers. In
some embodiments, each of the one or more testing regions detects
four biomarkers. In some embodiments, each of the one or more
testing regions detects five biomarkers. In some embodiments, each
of the one or more testing regions detects six biomarkers. In some
embodiments, each of the one or more testing regions detects seven
biomarkers. In some embodiments, each of the one or more testing
regions detects eight biomarkers. In some embodiments, each of the
one or more testing regions detects nine biomarkers. In some
embodiments, each of the one or more testing regions detects ten
biomarkers. In some embodiment, each of the one or more testing
regions detects one or more biomarkers.
[0137] FIG. 5 shows a radial arrangement of indicators or a radial
indicator patch. As shown in (A) of FIG. 5, testing regions or
reagents may be arranged in a circular or radial orientation. The
indicator includes reagents (22), a quaternary amine trap (24), and
an absorbent or evaporation area (25). A hole or cut access (27) in
the middle of the indicator helps to draw fluid from a wound into
the indicator. The fluid typically will flow from the access (27)
outward to the evaporation area (25). When reagents (22) are
exposed to wound fluid and react to microbial biomarkers, the
resulting products migrate to amine trap (24), allowing detection
by a user. The indicator may also have impermeable separators or
lanes (26). As shown in (B) of FIG. 5, a top or "above" view is
provided and a bottom or "below" view is provided for a radial
indicator patch. In one embodiment, substrates may be printed as
dots to allow for greater freedom of printing. Moisture impermeable
film with adhesive on both sides allows the radial indicator patch
to attach to foam or other support material. In some embodiments,
each reaction cell or lane (45-48) can be a different reporter or
color system, allowing analysis of multiple analytes on one
indicator patch.
[0138] In some embodiments, each of the one or more testing regions
comprises one or more components selected from the group consisting
of enzyme-reactive indicators, reagents that are sources of
peroxide, enzymes that are able to transform color reactions, pH
indicators, total protein-detecting reagents, and
moisture-detecting reagents. In some embodiments, the reagents that
are sources of peroxide are selected from peroxy acids, sodium
percarbonate, and peroxide-generating oxidases, such as glucose
oxidase or lactate oxidase. In some embodiments, the enzymes that
are able to assist the transformation of color reactions are
selected from peroxidases and laccases. In some embodiments, one or
more components are immobilized within the one or more testing
regions. In some embodiments, one or more components are mobilized
by wound fluid within the one or more testing regions. In some
embodiments, one or more components bind to the one or more testing
regions due to interaction with wound fluid. In further
embodiments, each of the one or more testing regions further
comprises one or more of the group consisting of buffers, binders,
and solubility enhancers. In some embodiments, one or more buffers,
binders, and/or solubility enhancers improves printing or
stability.
[0139] In some embodiments, each of the one or more testing regions
comprises an enzyme-reactive indicator, further comprising an
enzyme-labile or enzyme-reactive moiety, an immobilizing moiety
that holds the reactive indicator in place, and a moiety that gives
rise to a visible change upon interaction of the reactive indicator
with a target enzyme. In some embodiments, each moiety is
distinctly different from the other. In some embodiments, one
moiety incorporates another moiety either partially or entirely. In
some embodiments, the reagent pad comprises one or more
enzyme-reactive indicators.
[0140] In some embodiments, the enzyme-reactive indicator is a
protein-indicator conjugate such as a protease substrate comprising
both protein and dye materials. In a preferred embodiment, the
protein-indicator conjugate is a protein with a binding function to
a solid phase, such as a cellulose binding domain conjugated with a
protease recognition site and dyes that are released upon
proteolysis.
[0141] In some embodiments, the pH indicator presents a visible
color change at alkaline pH. In some embodiments, the pH indicator
presents a visible color change at pH=7.2-9.5. In some embodiments,
the pH indicator presents a visible color change at pH=7.2-9.0. In
some embodiments, the pH indicator presents a visible color change
at pH=7.2-8.5. In some embodiments, the pH indicator presents a
visible color change at pH=7.2-8.0. In some embodiments, the pH
indicator presents a visible color change at pH=7.5-8.5. In some
embodiments, the pH indicator presents a visible color change at
pH=7.5-9.0. In some embodiments, the pH indicator presents a
visible color change at pH=8.0-9.0. In some embodiments, the pH
indicator presents a visible color change at pH=7.2, 7.3, 7.4, 7.5,
7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,
8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5, or increments thereof.
[0142] In some embodiments, the pH indicator presents a visible
color change at neutral pH. In some embodiments, the pH indicator
presents a visible color change at pH=6.9, 7.0, or 7.1, or
increments thereof.
[0143] In some embodiments, the pH indicator presents a visible
color change at acidic pH. In some embodiments, the pH indicator
presents a visible color change at pH=4.5-6.8. In some embodiments,
the pH indicator presents a visible color change at pH=4.5-6.5. In
some embodiments, the pH indicator presents a visible color change
at pH=5.0-6.8. In some embodiments, the pH indicator presents a
visible color change at pH=5.4-6.8. In some embodiments, the pH
indicator presents a visible color change at pH=5.4-6.5. In some
embodiments, the pH indicator presents a visible color change at
pH=4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,
5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or 6.9, or
increments thereof.
[0144] In some embodiments, the pH indicator is nitrazine yellow,
bromocresol purple or bromothymol blue or an analog thereof.
[0145] In some embodiments, the filter pad removes unwanted
components of wound fluid, such as fibrinogen, albumins or
globulins, and cellular components or non-cellular debris, i.e.,
dressing components, medicaments, metabolites, microbes, microbial
debris, microbial metabolites, etc. In some embodiments, the
leach-back trap prevents backflow of reagents in the reagent pad or
reagent cell from entering the inlet for wound fluid in the testing
region. In some embodiments, the filter pad and/or leach-back trap
comprises a quaternary amine polymer, a mixture of secondary and
tertiary amines, other amine-containing polymers, or a combination
thereof. In some embodiments, the filter pad and/or leach-back trap
comprises a quaternary amine polymer. In some embodiments, the
filter pad and/or leach-back trap comprises a mixture of secondary
and tertiary amines. In some embodiments, the quaternary amine
polymer is polydiallyldimethylammonium chloride (polyDADMAC or
polyDDA). In some embodiments, the mixture of secondary and
tertiary amines is polyethylenimine (PEI). In some embodiments, the
filter pad and/or leach-back trap is held in place by cross-linking
with bifunctional reagents, such as epichlorhydrin,
diglycidylethers, di-epoxides or arylazideisothiocyanates. In some
embodiments, such reagents when mixed with a reactive
amine-containing polymer link different polymer chains and trap the
longer polyDADMAC chains within a matrix. In some embodiments, the
trap is composed of choline acrylate derivatives polymerized in
situ using a radical initiator such as benzphenone. In some
embodiments, the filter pad and/or leach-back trap comprises
polymers or reagents containing carboxy, sulfate, sulfonate, or
other acidic chemical groups. In some embodiments, the filter pad
and/or leach-back trap comprises styrene sulfonate.
[0146] In some embodiments, the indicator trap catches reaction
products between the wound fluid and the one or more components
selected from the group consisting of enzyme-reactive indicators,
reagents that are sources of peroxide, enzymes that are able to
transform color reactions, pH indicators, and moisture-detecting
reagents. In some embodiments, the indicator trap comprises a
positively charged or negatively charged trap for reaction
products. In some embodiments, the positively charged trap
comprises a quaternary amine polymer, a mixture of secondary and
tertiary amines, other amine-containing polymers, or a combination
thereof. In some embodiments, the positively charged trap comprises
polyDADMAC, or an analog thereof. In some embodiments, the
negatively charged trap comprises polymers or reagents containing
carboxy, sulfate, sulfonate, or other acidic chemical groups. In
some embodiments, the negatively charged trap comprises styrene
sulfonate. In some embodiments, styrene sulfonate is diluted to
0.02 to 0.8% in water and printed in this form to the support
material. In yet other embodiments, styrene sulfonate is diluted to
between about 0.01% to 2.0%, about 0.01% to 1.5%, about 0.01% to
1%, about 0.05% to 1%, about 0.1% to 1% or about 0.5% to 1%.
[0147] In some embodiments, the indicator trap comprises a total
protein indicator which is eluted by wound fluid to indicate
overall flow and capacity of the testing region. This region is
distinct from the moisture indicator. In one embodiment, a blue
polysulfonate dye, such as Evans or Trypan blue, is weakly bound to
a tertiary amine trap. On arrival of protein, the dye is displaced
and re-trapped as a Protein complex on a quaternary amine trap. In
another embodiment, Coomassie Blue G250 is weakly bound to a
Styrene sulfonate field and is displaced by protein to be
re-trapped on a quaternary amine trap. The dye undergoes a mild
color change from the sulfonic acid environment to the amine
environment increasing the effect. In another embodiment, the
visualization field is pre-printed with the Ponceau S complex of
the quaternary amine trap such that it is red indicating
non-function. The conversion of the trap to the Blue form indicates
the progress of protein elution.
[0148] In an embodiment of an indicator of the arrival of fluid in
the system, Brilliant Black or a similar dark tetra sulfonate is
printed into a reagent pad as a free reagent without any polymer
complexing. Being water soluble, it is readily mobilized by the
wound fluid and migrates to the window where it is avidly trapped
by a quaternary amine trap. The high polysulfonation increases the
avidity for the amine and resists further elution by proteins.
Under conditions of high secretion, the eventual removal of the dye
from the trap may also serve to indicate exhaustion of the device
or a need to change it.
[0149] In some embodiments, one or more testing regions comprise a
sulfonic acid filter pad and a quaternary amine trap. In some
embodiments, one or more testing regions comprise a leach-back
trap, a sulfonic acid filter pad and a quaternary amine trap.
[0150] In some embodiments, each of the one or testing regions is
used to evaluate the presence of one or more analytes and one or
more positive or negative control indicators. In some embodiments,
the one or more analytes is associated with enzyme activity. In
some embodiments, the enzyme is selected from one or more of the
group consisting of elastase, lysozyme, cathepsin G,
myeloperoxidase, and leukocyte peroxidase. In some embodiments, the
enzyme is elastase. In some embodiments, the enzyme is lysozyme. In
some embodiments, the enzyme is cathepsin G. In some embodiments,
the enzyme is myeloperoxidase. In some embodiments, the enzyme is
leukocyte peroxidase.
[0151] In some embodiments, a positive result (e.g., indication of
infection) from the one or more testing regions is in the form of a
visible change. In some embodiments, the visible change is a color.
In some embodiments, the color is selected from dark blue, dark
green, and black. It is clear to those skilled in the art that the
signal effect of the color change depends on context and practical
consideration of interfering colors from the wound itself. Thus,
red is a useful signal to indicate a problem, or to indicate stop
or not ready, but it is readily confused with colors associated
with wound fluids. Thus, colors that are not likely to emerge from
a wound offer potentially less source of error. In some
embodiments, the visible change is fluorescent, luminescent, or
mediated by physical means such as electrical, refraction, gas
evolution or polymer state change. Some fluorescent systems have
the drawback that they require a source of light and potentially a
darkened room or chamber for viewing, however, other fluorescent
systems do not have such drawbacks. Conventional colors are visible
under normal treatment conditions. Given that a color may be
diluted or covered by fluids such as blood, there remains an
embodiment in which a dual indicator is used in which a fluorescent
indicator is mixed with a conventional color indicator. Thus, if a
field is covered by blood, the result may be optionally
interrogated with a black-light to determine whether a signal is
present.
[0152] Outer Layer
[0153] In some embodiments, the outer layer comprises a polymer
that is not easily penetrated by wound fluid. Such polymers
include, but are not limited to, a polyolefin, a polypropylene, a
polyethylene, polyurethane, polyamides, ethylene-vinyl alcohol
(EVOH), acrylonitrile (PAN), polyvinyl choride (PVC),
polyvinylidene chloride (PVDC), polyacrylates (e.g.,
(1-methyl-1,2-ethandiyl)bis[oxy(methyl-2,1-ethandiyl) diacrylate)
or other similar hydrophobic impermeable polymers that, in some
embodiments, are laid down as films by printing, spraying or film
blowing. In some embodiments, the outer layer is water vapor
permeable. In some embodiments, the outer layer prevents moisture
loss in specific areas (e.g., where a visible change indicating
infection is observed) and promotes moisture loss in other specific
areas (e.g., where excess wound fluid accumulates).
[0154] In some embodiments, the reaction layer is protected by two
layers: a top layer and a bottom layer. The bottom layer typically
has an opening that allows fluid sample inflow. The top layer
generally prevents premature evaporation of the sample and may
force it to migrate through the device to the evaporation zone. The
top layer may also contain one or more windows that allow the
response of the reagents to be seen or detected.
[0155] Devices
[0156] In yet other embodiments, the disclosure herein provides a
device comprising a sampling component and a test device
comprising:
[0157] (a) a housing surrounding a tube to define an opening in the
housing to receive the sampling component, the housing also having
disposed within it:
[0158] (b) a sealed diluent chamber connected to the tube and
holding a liquid diluent for removing the sample from the sampling
tip to form a liquid test sample;
[0159] (c) a reaction well in liquid communication with the tube,
the reaction well holding a reagent capable of indicating the
presence of the analyte within the test liquid; and
[0160] (d) a forcing mechanism capable of moving the diluent
through the device from the chamber, over the sample tip and into
the reaction well.
[0161] In some embodiments, the device operates by driving the
diluent over the sample and into a reaction well, and a test
solution is made by the flow of the diluent over the sample.
Preferably, it is not necessary to first mix the sample with the
diluent to make a test solution and then move that solution via a
lateral flow strip to the reaction well. The moving of the diluent
past the sample and to the reaction well means that the kit can be
used with a minimum number of steps, for instance taking the
sample, inserting the sampling component into the housing and
activating the moving or driving mechanism. This procedure
minimizes user error and thus minimizes false-negative results and
misdiagnoses.
[0162] In some embodiments, the diluent is forced through the
device in a one-step or multi-step process. For instance, in a
one-step process, the diluent is forced through the device which
creates a test liquid, which is forced into the reaction well. In a
multi-step process, such as a two-step process, the diluent could
first be forced through the device to a mixing chamber where a test
liquid is prepared. That liquid could then be forced from the
mixing chamber to the reaction well in a further step.
[0163] In another embodiment, the means of mixing and loading the
sample may be achieved in a separate step to its analysis. In one
embodiment, a sample swab is first inserted into a recipient fluid
container, and then a coaxial plunger is pushed over the swab to
eject diluted sample into the analysis device. In a preferred
embodiment, gas is removed, such as by using Goretex membranes
which are gas and vapor permeable but not permeable to liquid
water. Said membranes can be used to degas both the sample as it is
injected and to vent the fluid chambers where the assay takes
place.
[0164] In one embodiment, preferably the diluted sample is
distributed to each analysis chamber equally through microchannels.
However, when each exit from a chamber contains a Goretex membrane,
back pressure ensures that each chamber is only filled once. In a
more preferred embodiment, the loss of liquid sample from the
assembly is prevented by an absorbent between the last outlet and
the exterior of the device.
[0165] In still other embodiments, the disclosure herein provides a
kit for detecting an analyte or biological marker or target in a
sample comprising:
[0166] (i) a sampling component comprising a sampling tip for
collecting the sample and
[0167] (ii) a test device comprising: a housing surrounding a tube
to define an opening in the housing to receive the sampling
component, the housing also having disposed within it: a sealed
diluent chamber connected to the tube and holding a liquid diluent
for removing the sample from the sampling tip to form a test
liquid; a reaction well in liquid communication with the tube, the
reaction well holding a reagent capable of indicating the presence
of the analyte within the test liquid; and a forcing mechanism
capable of moving the diluent through the device from the chamber,
over the sample tip and into the reaction well.
[0168] The sealed diluent chamber may contain a specified volume of
diluent so that an expected volume of test solution reaches the
reaction well or wells. In addition the pathway between the diluent
chamber and the reaction well is preferably vented at the reaction
well end so that trapped air does not affect the flow of test
solution through the device or prevent the test solution from
reaching the reaction well or prevent the test liquid from
correctly filling the reaction well.
[0169] The housing preferably has two parts which are capable of
moving with respect to each other while remaining connected to one
another. The action of moving the parts may provide the forcing
mechanism by which diluent is moved through the device. The diluent
may be driven through the device by compression of the diluent
chamber which forces the diluent past the sample tip and to the
reaction well or wells emptying the compression chamber. The
compression of the diluent chamber can occur when the parts of the
housing are moved with respect to one another such as by sliding
one part past another. Alternatively the diluent can be pulled
through the device again for example by moving parts of the housing
with respect to one another.
[0170] The sampling component preferably comprises a handle and a
sampling tip, the handle preferably comprising a seal which engages
with the opening in the housing to seal the tube when the sampling
component is fully inserted in the tube. The seal prevents escape
of the sample and diluent from the device reducing the chance of
cross contamination from the wound fluid. Preferably the seal and
tube engage to lock the sampling component in the device and
prevent removal of the sampling component once it has been used.
This further reduces the chance of cross-contamination from the
sampling component. The sampling component preferably activates
release of the diluent from the diluent chamber.
[0171] The housing may comprise a locking mechanism which locks the
housing in position once the driving mechanism has been activated
and prevents reuse of the device. In this way it is immediately
apparent that the device has been used and cannot be used again.
This minimizes false results from, for instance, a device that has
been mistakenly activated in transit or from reuse of a device
whose reagents have been spent.
[0172] Preferably insertion of the sampling component in the device
releases the seal on the diluent chamber. Preferably the seal is a
ball valve or can be a film or membrane seal or a duck bill valve
or other non-return valve known in the art which is activated when
the sampling component is inserted in the device. The sampling
component preferably bursts, punctures or displaces the seal on the
diluent chamber.
[0173] Preferably the tube is the same or similar size to the
sampling tip of the sampling component so that the act of inserting
the sampling tip into the tube causes it to be scraped along the
walls of the tube aiding the dispersion of the sample in the
diluent once it is released from the diluent chamber and is flushed
through the device. The diluent can be flushed along the whole
length of the tube or only part thereof. The sizing of the sampling
tip to match the tube also forces the diluent to be flushed through
the tip when the diluent is driven from the diluent chamber.
Preferably the tube is wider at its mouth to aid insertion.
[0174] Preferably the diluent chamber is shaped like a bellows to
assist in the compression of the chamber alternatively the chamber
can be a combination of a plunger and tube similar to that found in
a syringe, or sample preparation device, or can be a filled
flexible sachet which is compressed by hand by the user or a
balloon which contracts when the seal is released.
[0175] Methods of Use
[0176] In one aspect, provided herein are methods to diagnose and
indicate need for treatment of chronic wounds using a wound
dressing described herein.
[0177] In some embodiments, the methods and devices disclosed
herein detect biological markers or targets from body fluid. In
some embodiments, the body fluid is blood, plasma, serum,
cerebrospinal fluid, sputum, urine or wound exudate. In preferred
embodiments, the body fluid is wound exudate.
[0178] In another aspect, provided herein are methods to diagnose
chronic wounds using a wound dressing described herein.
[0179] In another aspect, provided herein are methods to indicate
need for treatment of chronic wounds using a wound dressing
described herein.
[0180] In another aspect, provided herein are methods to indicate
need for treatment of surgical or acute wounds using a wound
dressing described herein.
[0181] In another aspect, provided herein are methods of detecting
biomarkers of infection in wounds using a wound dressing described
herein.
[0182] In another aspect, provided herein are methods of detecting
the pH and/or the presence of biomarkers of infection in wounds
using a wound dressing described herein. In some embodiments, the
biomarkers of infection are leukocyte enzymes. In some embodiments,
alkaline pH in the wound indicates infection in the wound.
[0183] In another aspect, provided herein are methods of detecting
protease activity in wounds using a wound dressing described
herein.
[0184] In another aspect, provided herein are methods of monitoring
the condition of a wound or surgical site and its healing process
or status.
EXAMPLES
Example 1: Wound Dressing
[0185] One example of a construction of a wound dressing
incorporating the device is shown in FIG. 2. The wound contact
layer in this example is carboxymethylcellulose marketed as
"AQUACEL", and the AQUACEL is backed by a polyurethane foam. In the
infection-indicating area of the device is an impermeable area
beneath the reagent layer. Connecting to this area is a material
such as a polyester thread, methylcellulose fibers, or a similar
wicking, hydrophilic, capillary or similar material, or capillary
channels. This fluid connection brings wound exudate or fluid into
contact with the reagent layer, where it may react with and
mobilize indicator reagents into visible products that are either
visible in place or trapped in window visible from the outside of
the dressing. This example also demonstrates the use of
AQUACEL.
[0186] In one embodiment of a wound dressing is shown in
cross-section in FIG. 2. In that wound dressing the wound contact
layer (4) comprises carboxymethylcellulose, marketed as AQUACEL. In
FIG. 2, the wound contact layer (4) is backed by a polyurethane
foam (3). In the infection-indicating area of the dressing is an
impermeable area beneath the reagent layer (2) and above the
polyurethane foam (3). Accordingly, in this embodiment the
infection-indicating area is provided between the reagent layer (2)
and the polyurethane foam (3). Connecting to the
infection-indicating area is a fluid connection (1) component, such
as a material such as a polyester thread, methylcellulose fibers,
or a similar wicking, hydrophilic, capillary or similar material,
or capillary channels (1). This fluid connection component (1)
brings wound fluid into contact with the reagent layer (2), where
it may react with and mobilize indicator reagents into visible
products that are either visible in place or trapped in window (6)
visible from the outside of the dressing as shown in the top view
of the wound dressing shwon in (C) of FIG. 2. As explained above,
views (A) and (B) of FIG. 2 show side views of the wound dressing
(7). View (B) of FIG. 2 shows the flow of wound fluid (5) from the
wound contacting layer (4) at the bottom upward via capillary
channels (1), which may be formed by stitching using wicking
fibers. The wound fluid reacts with reagents in reagent layer (2),
which may contain windows (6), allowing users to observe a visible
signal resulting from reactions between wound fluid and reagents in
the wound dressing. View (C) of FIG. 2 shows a top view of a wound
dressing (7), wherein an opaque film on top of the reagent layer
(2) contains windows or clear areas (6) that allow the observation
of indicators or changes associated with reagent interaction with
an analyte. In some embodiments, a visible signal may be a color
change indicative of a microbial infection in the wound.
Example 2: A Dressing Material Printed with a Patterned Reactive
Ink to Report MPO Activity
[0187] A dressing wound contact layer has an upper and lower
surface in which the lower surface is the wound contact layer.
Reagents can be sprayed or printed on a wound dressing material.
One embodiment of such dressing is shown in FIG. 6, wherein (A)
depicts a view of the surface of the wound dressing material and
illustrates the topside of wound contact material; (B) represents
the wound material sprayed with amylase, starch, and glucose
oxidase; and (C) represents substrate-printed in the centers of the
sprayed area.
[0188] In alternate embodiments, onto the upper surface are printed
multiple layers, such as three layers, to report MPO activity. In
one embodiment the first layer is the substrate which is printed on
the upper surface of the wound dressing material, such as at a
concentration of 30 mg/mL in ethanol/heptane using a line width of
0.8 mm and a print density of 1 .mu.L/cm. Alternatively, the fast
blue substrate is printed a grid of circles each 3 mm in diameter
(FIG. 6). In one embodiment the next layer is a spray application
of a solution of gamma-amylase and glucose oxidase immobilized on
hydoxypropyl cellulose. The material may be sprayed in a water
buffer solution such that approximately 3 .mu.g of glucose oxidase
is deposited per cm2, in parallel, 0.5 .mu.g/cm2 of gamma amylase
is applied as the conjugate. Once dried, a starch suspension may be
sprayed at a density of 150 .mu.g per cm2. Once printed, the wound
contact layer is preferably bonded to an upper protecting layer.
The same printing regime can be printed on the upper side an upper
protecting layer. When exposed to artificial wound fluid containing
enzymes, the grid becomes blue colored over time.
Example 3: An Absorbent Material Printed with a Patterned Reactive
Ink to Report Elastase Activity
[0189] In this example a dressing has an absorbent and protective
layer which has an upper and lower surface in which the lower
surface contacts the wound contact layer. Onto the upper surface a
grid pattern is printed with 1 cm grid spacing. In one embodiment,
as shown in FIG. 8, the print is performed with a solution of the
AAPV-indoxyl ester 30 mg/mL in heptane/butanol using a line width
of 1 mm and a print density of 1.3 .mu.L/cm. FIG. 7 illustrates
embodiments of in-place color development of MPO and elastase
substrates.
Example 4: A Multi-Biomarker Device Insert
[0190] The visualization methods are preferably either a color
change of an immobile enzyme substrate, directly printed in the
window of the reporter area, or of the appearance of an
immobilization of the substrate caused by hydrophobic properties of
the substance and non-covalent chemical interactions with the
carrier material. The amount of applied substrate and possible
impregnation mixtures for color improvement were tested in this
example as described below
[0191] Optimization of the reporter area and color signal: Circles
(diameter 5 mm) were punched out of carrier material, in this case
filter paper. Circles were impregnated with different mixtures of
buffers (see specific reagents: Artificial wound fluid 2% bovine
serum albumin in phosphate buffered saline containing potassium
chloride, urea pH 7.2). See FIG. 8 for examples of substrates in a
water solution followed by a drying step. After drying, varying
amounts of substrate, usually in an organic solution, were pipetted
on the test circles.
[0192] The reactivity to wound fluid was tested as follows: 10
.mu.L test liquid (buffer or artificial wound fluid 2% albumin)
with or without enzyme were pipetted on the dried test disks. Disks
were incubated either in open air or in a closed system. Color
development was evaluated visually at various times after
initiation. All observations were at room temperature to simulate
the condition expected outside the dressing.
[0193] After optimization of the two visualization methods,
prototypes were prepared in lab scale to test the interaction of
the different enzyme substrates/their color development. Prototypes
were designed and assembled as described in FIGS. 7 and 9.
[0194] FIGS. 7 and 9 show embodiments of in-place color development
of different indicators. FIG. 9 shows a prototype with the reporter
areas for lysozyme, elastase and MPO detection, a pH indicator and
the liquid control was constructed. On the left portion in (A) the
diagnostic material is shown. On the right portion in (A) a
magnification of the reporter area is shown. (B) shows the
diagnostic area after liquid application (artificial wound fluid
0.5% albumin, 1 U/mL elastase, 10 .mu.g/mL MPO, 30000 U/mL
lysozyme). The experiment was run over 2 h with a flow rate of 100
.mu.L/min for the first 10 min, followed by 10 .mu.L/min. The
experiment was repeated in n=10.
[0195] Embodiments of diagnostic inserts or disks are shown in
FIGS. 10, 11, and 12. FIG. 10(A) shows the top view of a diagnostic
insert, comprising a reporter area (60), reaction area (61), and
evaporation area (62). FIG. 10(B) shows the bottom layer,
comprising an impermeable layer of plastic film, either white or
transparent, with a diameter of about 40 mm. The hole in the middle
allows for liquid transport and has a diameter of about 4 mm. The
bottom layer is covered with adhesive and in the same shape
underneath for an exact fixation on a dressing. FIG. 10 shows
embodiments of the reaction material comprising an adhesive layer
(C) and a reaction layer (D), wherein each arm has a different
substrate/indicator and/or pH system. FIG. 10(E) shows the cover,
which comprises an impermeable white plastic foil with a diameter
of 20 mm. The outer ring may have an insider diameter of 25 mm and
an outer diameter of 31 mm. The top layer may be covered with
adhesive underneath for an exact fixation on the reaction
material.
[0196] Top view of the assembled completed diagnostic insert. See
FIG. 10(A). The reporter area is designed as a window surrounded by
an off-white layer to achieve a maximum contrast to the color
signals. In this embodiment, there are five radial arms, each of
which contains a different reporter and color system. In one
embodiment, three are for enzymes and two are for controls.
[0197] The evaporation area ensures a continuous liquid transport
through the diagnostic material, necessary for the enzyme reaction
and color development in the reporter area.
[0198] Bottom layer as liquid barrier between the dressing and the
diagnostic material. Liquid will preferably pass only through the
hole in the middle of the layer which leads to a directed radial
distribution into the arms of the reaction material (diagnostic
material).
[0199] Diagnostic material was designed with four or five radial
"arms" depending on the favored number of enzyme-substrates and
controls to be included. The reaction material is fixed on the
bottom layer with medical adhesive. Alternatively, the reaction
arms are printed or coated with the less permeable bottom layer in
place of the adhesive (one material can serve both purposes).
[0200] In some embodiments, the device insert comprises at least
one arm or fewer than ten arms. The number of arms may depend on
the number of analytes to be determined in a sample and control(s),
as applicable. In further embodiments, the device insert comprises
one, two, three, four, five, six, seven, eight, nine, or ten
arms.
[0201] The reaction material is prepared with impregnation mixtures
and substrates in accordance to the optimized conditions described
above before assembling the detection material.
[0202] As shown in FIG. 10(E), the cover has several functions.
Firstly, it preferably maintains the reaction zone moist by
preferably preventing premature drying. Fluids should pass through
the reaction area into the reporter area where there is a
transparent window that allows color changes to be seen. The second
function is preferably to avoid a stop of liquid flow and to cover
the chemistry area so that colored reagents are not seen before
they are transported to the window. The cover is water impermeable
and includes the windows for signal visualization.
[0203] The detection material is preferably fixed with a medical
adhesive to the foam backing layer of a hydrofiber dressing.
[0204] Optimization of the first visualization method (accumulation
and trapping) established the following conditions:
[0205] Trapping mixture: Volume of 1.5 .mu.L per 10 mm2, thickener
Methylcellulose (Methocel A4C) max. 1.25%. Drying at room
temperature for at least 1 h.
[0206] Transport of Remazol Brilliant Blue (FIG. 13) and
visualization in the trap coated reporter area containing the
amino-trap (triplicates), test liquid was artificial wound fluid 2%
albumin. This visualization method was used for the
Lysozyme-substrate (results obtained by QZY); released and trapped
dye after enzyme cleavage: Remazol Brilliant Black) and the liquid
control (dye: Brilliant Black BN).
[0207] FIG. 13 shows visualization of dye in reporter area (D)
after exposure of reaction area (C) to artificial wound fluid. The
direction of the fluid flow was from reaction area (C) to reported
area (D), further comprising amino trap. The experiment was done in
triplicates.
[0208] Optimization of the second visualization method (in-place
color change) led to clearly colored signals for the MPO-substrate,
the elastase substrate and a pH Indicator.
[0209] MPO-substrate: The MPO substrate in this example is a Fast
Blue derivative. The substrate is soluble in 50.degree. C. ethanol.
After pipetting of 1.5 .mu.L of a saturated solution at the
reporter area followed by a drying step (20 min, room temperature)
the substrate cannot be mobilized by artificial wound fluid 2%
albumin. The slightly beige MPO substrate is converted by MPO under
development to a deep blue to black color in the reporter area. As
the MPO reaction is H.sub.2O.sub.2 dependent, a glucose/glucose
oxidase based H.sub.2O.sub.2 generating system is printed in the
reaction area.
[0210] Optimized conditions led to the results shown in FIG. 7.
Test circles contain 1.5 MPO substrate as described above, 10 .mu.g
glucose and 1 .mu.L of 0.1% glucose oxidase (1 .mu.g) in water.
After drying of the test circles 5 .mu.L test liquid (artificial
wound fluid 2% albumin, pH 7, without/with MPO) were applied. The
picture of FIG. 7 was taken after 2 min incubation time.
[0211] Elastase substrate: The elastase substrate consists of an
Fmoc protected AAPV enzyme recognition motif (amino-acid sequence
AAPV) esterified to an Indoxyl moiety. It is soluble in organic
solvents, but completely insoluble in aqueous solution. After
enzyme cleavage, Indoxyl is released and immediately oxidized to
immobile blue Indigo dye (FIG. 8), visible in the reporter
area.
[0212] Optimized conditions led to the result shown in FIG. 7. In a
first step, the test circles were impregnated with a impregnation
mixture (0.25% (w/w) Nonidet, 2% (w/w) decanol in 0.05 M borate
buffer pH 8). Therefor the two-phase solution was mixed until
formation of an opalescent dispersion. This dispersion was
transferred in a glass container. The test circles were washed in
the impregnation mixture for 1-2 min. Thereafter the filter papers
were placed on a glass plate and dried for 1-2 h at 54.degree.
C.
[0213] In the next step elastase-substrate (10 mg/mL in acetone)
was pipetted on the circles 2 times in 2.5 .mu.L steps until a
final amount of 50 .mu.g per test circle (20 mm2) was applied (FIG.
7). After drying at room temperature an elastase assay was
performed by addition of 10 .mu.L test liquid (artificial wound
fluid 2% albumin, pH 7, with/without elastase). Color development
was observed and documented after 15 min incubation at room
temperature.
[0214] The pH indicator is a preparation of bromothymol blue in
chitosan, containing glutaraldehyde. The mixture is pipetted in the
reporter area, after drying leading to a dark yellow and immobile
indicator system. The color changes from slightly green (pH 7) to a
dark green (pH 8) within 30 minutes of liquid flow (artificial
wound fluid 2% albumin). See FIG. 14 for an example of a pH
indicator.
[0215] Immobilized bromothymol blue derived pH indicator after
running with approximately 300 .mu.L artificial wound fluid 2%
albumin with different pH values. pH indicator was applied in
amounts of 1.5 .mu.L per 10 mm2 in three pipetting steps of 0.5
.mu.L.
[0216] Production and functionality of the reporter area in
prototypes. In the reporter areas of the arms of the diagnostic
material for Lysozyme detection and the liquid control, 1.5 .mu.L
of the trapping mixture were printed. In the reporter areas for
Elastase and MPO detection as well as for the pH indicator, the
substrates were applied (FIG. 7, 9).
[0217] FIG. 9 shows a prototype with the reporter areas for
lysozyme, elastase and MPO detection, a pH indicator and the liquid
control. On the left the diagnostic material is shown, on the right
a magnification of the reporter areas. FIG. 9 (A) shows an example
for a prototype with the reporter areas before liquid
application.
[0218] FIG. 9 displays the diagnostic area after liquid application
(negative control, artificial wound fluid 0.5% albumin without
enzymes). FIG. 9(C) shows the diagnostic area after liquid
application (artificial wound fluid 0.5% albumin, 1 U/mL elastase,
10 .mu.g/mL MPO, 30000 U/mL lysozyme). The experiment was run over
2 h with a flow rate of 100 .mu.L/min for the first 10 min,
followed by 10 .mu.L/min. The experiment was repeated in n=10.
[0219] FIG. 9 shows a prototype with the reporter areas for
lysozyme, elastase and MPO detection, a pH indicator and the liquid
control. On the left the diagnostic material is shown, on the right
a magnification of the reporter areas. Color signals for the liquid
flow control are visible, so it is believed that the method of
visualization by trapping and accumulation works. The order of
reaction is generally MPO, then elastase, then lysozyme. Color
change of the pH indicator as well as the color development of the
MPO and elastase substrates is visible in the reporter area. The
in-place color change was established for these reactions and
functionality was demonstrated.
[0220] The inserts can be made in many forms including radial
designs (FIG. 10-12), linear designs and single spot approaches.
These vary in which layers and patterns are formed. It is generally
the goal to make the insert as small and non-occlusive as
possible.
[0221] One means to reduce occlusiveness is to reduce the area of
film layers. In the embodiments shown in FIG. 10-12, the only
occlusive layers are the lanes themselves. In this version, the
round bottom layer is replaced by only the adhesive. The advantage
of the round bottom layer is that tended to support a broader area
of the dressing being sampled into the device. The reduced bottom
layer has the advantage of permitting more vapor transfer.
Example 4: Lysozyme Responsive Testing Strip
[0222] In one embodiment of a means to detect lysozyme activity, a
strip of a wicking substance like filter paper is printed with both
dyed peptidoglycan (FIG. 15(D), a) and a trap material (quaternary
amine fixed with cross-linked PEI) (FIG. 15(D), b). Wound fluid is
applied to the base and allowed to wick up the carrier to point C
where it evaporates. Lysozyme, if present, degrades the dyed
peptidoglycan and transports anionic fragments to the trap (FIG.
15(D), b) where they form a line.
[0223] In FIG. 15, one embodiment of a lysozyme test strip (50)
comprises a Whatman filter 1001/85 that is cut into 0.5 cm.times.4
cm pieces having fixation areas (51), evaporation area (52), 3%
crosslinked, amino trap (53), substrate area (54), and a stitching
area (21) for wicking fluid from a wound. Side view (B) shows a
wound dressing comprising a test strip (50), base layer (55), and
stitching (21). Top view (C) shows the test strip (50) adhered to
wound dressing (56).
[0224] Integration of dyed peptidoglycan into a lysozyme responsive
testing strip (FIG. 15). In some embodiments, a testing strip
comprises a Whatman filter 1001/85 that is cut into 0.5 cm.times.4
cm pieces. 2 .mu.l of the quaternary amine trapping solution is
pipetted onto the cellulose filter 1.5 cm beneath the upper end of
the stripe. 2 .mu.l of a substrate formulation containing 4 mg dyed
peptidoglycan in 240 .mu.l 0.5% PEG6000 solution in H.sub.2O are
pipetted 1 cm above the lower end of the stripe. The modified strip
is incubated at 90.degree. C. for 30 minutes. The test strip is
then ready to use. Alternatively other dyed lysozyme substrates
(e.g. dyed chitosan derivatives) can be incorporated into the
testing system. In some embodiments, the testing strip comprises a
substrate spot, a quaternary amino trap, and a cellulose
matrix.
[0225] In some embodiments, integration of the lysozyme responsive
testing strip into a dressing for the online detection of early
stage wound infections.
[0226] Liquid transport system from the bottom side of the dressing
to the test strip is performed via a polypropylene yarn stitched
through the layers of the dressing and the first water impermeable
adhesive layer. While the stitching helps the process, it is not
essential and the same results are obtained without stitching,
albeit more slowly. The testing strip is embedded in between of two
water impermeable adhesive layers. An evaporating area is included
in the upper region of the strip. The detection unit releases the
coupled dye in region `a` which is then trapped in area `b` of the
testing stripe and gives a clear visible signal upon lysozyme
activity.
[0227] Material selection for the test strip: Different cellulose
based materials can be used as solid matrix for the test stripe.
Non-wovens containing a defined amount of cellulose can
alternatively be used. Schematic representation of the Lysozyme
test strip. Attachment of the detection system to the dressing
(FIG. 15). Base layer contains liquid transfer system to the
detection unit. Upper view of the combined base layer and detection
unit.
Example 5: Indicator Reactions
[0228] FIG. 16 shows examples of indicator reactions include a
substrate with at least two domains A and B, or A and C, connected
by a cleavage site (X), which is recognized by enzymes in wound
fluid, such as elastase (E or E2). In some embodiments,
peptidoglycan anchor (S) is attached to an enzyme substrate,
requiring digestion or breakdown of the peptidoglycan anchor (S) by
lysozyme (E1) before the cleavage site (X) on the substrate can be
accessed by an enzyme in the wound fluid. Products (P) of the
reactions are colored, giving rise to a color change detectable by
a user. In example I, upon exposure to elastase (E) in the wound
fluid, the substrate is cleaved at cleavage site X, releasing MPO
substrate (B), which can react with MPO in the wound fluid and
oxidize the substrate (B) to form a colored product (P). In example
II, lysozyme (E1) breaks down peptidoglycan anchor (S) to expose
cleavage site (X). Upon exposure to elastase (E2) in the wound
fluid, elastase cleaves the substrate at cleavage site (X) and
releases indole (C), which may be converted to indigo in the
present of oxygen, giving rise to a color change. In example III,
MPO substrate (B) may be used instead of indole (C) to yield a
colored product (P).
Example 6: Indicator Disk
[0229] FIGS. 10-12 show schematics of indicator inserts or disks.
FIG. 10(A) shows the top view of a diagnostic insert, comprising a
reporter area (60), reaction area (61), and evaporation area (62).
FIG. 10(B) shows the bottom layer, comprising an impermeable layer
of plastic film, preferably either white or transparent, with a
diameter of about 40 mm. The hole in the middle allows for liquid
transport and has a diameter of about 4 mm. The bottom layer is
covered with adhesive in the same shape underneath for an exact
fixation on a dressing. FIG. 10 shows the reaction material
comprising an adhesive layer (C) and a reaction layer (D) wherein
each arm may be a different substrate and/or pH system and where
the arms in each layer overlap to allow exact fixation. Indicator
disks can have any number or indicator arms, such as 4 or 5 arms of
indicators arranged radially as in FIG. 10. In some embodiments,
the indicator disks comprise 1 to 10 arms, or preferably 4 or 5
arms. FIG. 10(E) shows the cover, which preferably comprises an
impermeable white plastic foil with a diameter of 20 mm. The outer
ring may have an insider diameter of 25 mm and an outer diameter of
31 mm. The top layer may be covered with adhesive underneath for an
exact fixation on the reaction material.
[0230] In the embodiment shown in FIG. 11, (A) shows the bottom
layer, comprising a double sided and hydrophobic film (65) with a
diameter of 40 mm. A hole cut in the middle has a diameter of about
5-6 mm. Reference (66) shows the hydrophobic lanes on non-woven or
paper, either full sheet or cut out, placed on the adhesive film.
Reference (67) shows traps printed on non-woven or paper which is
adhered to the bottom layer with a back-flow trap (68). In (B), the
reaction layer comprises arms, each may have a different indicator
and color system as shown in (70). An evaporation cover (71) may be
printed, sprayed, or overlaid film. Reference (72) shows the
indicator disk affixed to a dressing, wherein outer dressing has a
window (shown as dashed line) for viewing the indicator change.
[0231] In another embodiment of the indicator disk, as shown in
FIG. 12, bottom layer (A) preferably comprises a white or
transparent impermeable plastic film (73) of diameter 40 mm. A hole
in the middle of bottom layer, comprising a diameter of 4 mm allows
for wound fluid transport. The bottom layer may be covered with
adhesive in the same shape (73) as the reaction material (77)
underneath for an exact fixation on wound dressing, double-sided
adhesive and hydrophobic. The reaction layer (77) is placed on top
of adhesive layer (73), at the bottom. Each arm of the reaction
layer may be 13 mm or 15 mm in length from the center, and about 5
mm wide. Cut access in the center of the disk may also comprise a
back-flow trap (75) to ensure fluid flows from the center outward
to evaporation area in the periphery of the insert. Reference (74)
shows hydrophobic lanes on non-woven or paper, fill sheet or cut
out, placed on adhesive. Reference (76) shows traps printed on
non-woven or paper with back-flow trap (75) in the middle. In some
embodiments, reaction material (77) comprises brilliant black
print, pH indicator, MPO substrate, elastase-peptide-indoxyl, and
lysozyme-peptidoglycan indicator, and any combination thereof on
arms of the indicator disk. Such substrates may be printed on the
reaction material or solid support material. Evaporation cover may
be printed, sprayed, or overlaid as a film over (78), shown as gray
box in (78). The reaction material may be covered by a transparent
or translucent film, with a window (79, dash-line box) to allow
detection of the reaction.
[0232] In some embodiments, a cover as shown in FIG. 10(E),
comprises a middle cover of impermeable white plastic film with a
diameter of 20 mm, an outer ring with an inside diameter of 25 mm
and an outer diameter of 31 mm, and a top layer covered with
adhesive in the same shape underneath for an exact fixation on the
reaction material.
[0233] As shown in FIG. 10(A), one embodiment comprises an
impermeable white plastic foil with an outer diameter of 31 mm,
inner diagnostic circle (60, reporter area) with diameter of 25 mm,
and the substrate cover (61) with diameter of 20 mm in embodiments
using a substrate cover. Evaporation area (62) is located at the
periphery of the indictor insert. A small evaporation area, such as
2.times.5 mm may be too small for a 7-day run, but is sufficient
for a smaller run, such as a one-day run. Visible signal resulting
from reactions can be detected in diagnostic area (60) or window
reporter area (FIG. 11 or FIG. 12). Such reporter areas can be
surrounded by an off-white layer to achieve maximum contrast to
color signals.
[0234] In another embodiment, the diagnostic reaction can be
performed on a solid phase in which liquid sample diffuses in the
vicinity of dyes that are absorbed onto the solid phase. Enzymes
carried in the sample can transform the dyes through contact in the
pores of the solid phase material. The changes are visible as color
changes. Due to the low volumes in use and the high concentration
of dye, the color change can be a sensitive indicator.
[0235] In a preferred embodiment indicator disks are prepared by
impregnating a filter paper with the reagents and then punching
disks prior to adhering them to a carrier to form a "stick" with a
reactive dye coated on to it. This stick can be brought into
contact with the sample and a color change observed.
[0236] In a more preferred embodiment, more than one indicator disk
type is placed onto the stick carrier such that multiple enzymes or
parameters can be detected in one test. Parameters that may be
determined include pH, lysozyme, elastase, Cathepsin G, MPO,
catalase and lipases. Such a stick should also contain a positive
control to indicate adequate sample wetting, and or sample
application including, in addition to wetting, also the presence of
protein.
[0237] In one preferred embodiment the indicator disks are aligned
in a line on a thin "stick" and the sample is applied to them in
sequence using a swab, gauze, or by pressing the stick into or onto
a sample, for example a used dressing.
[0238] In another embodiment, the indicator disks are aligned next
to each other on a broad support and their edges on one side are
cut such that the stick can be pressed with the cut edge to the
sample source (i.e. a used dressing or diluted wound fluid, or the
edge of a cleaning swab or gauze) such that liquid is taken up into
each of the disks at the front of the broad stick ("Fork"
format).
[0239] In another preferred embodiment the indicator disks are
placed inside a carrier box such that the sample swab can be
inserted into the box and then sealed inside by closing the box.
After closure, the sample swab can be moved and in the process,
contacts each sample disk in turn to wet them appropriately such
that the resulting reaction can be observed through windows
appropriately placed above each indicator disk. Such an arrangement
can preserve the swab for later microbiological examination and
simplify the handling of materials at or during a dressing
change.
[0240] Indicator disks are preferably prepared with reagents that
are capable of color change. Such reagents may be selected from
compounds such as p-aminophenol, ABTS (2,2inophenol, ABTS (strate.
In some embodiments, acid) diammonium salt), 3,3'-diaminobenzidine,
3,4 diaminobenzoic acid, DCPIP, N,N-dimethyl-p-phenylenediamine,
o-dianisidine, p-phenylenediamine, 4-chloro-1-naphthol,
o-phenylenediamine N-(4-aminobutyl)-N-ethylisoluminol,
3-amino-9-ethylcarbazole, 4-aminophthalhydrazide, 5-aminosalicylic
acid, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),
indoxyl, indigo, Fast Blue RR, 4-chloro-7-nitrobenzofurazan. In
some embodiments, the reactive layer comprises an arylamine. In
some embodiments, the reactive layer comprises an amino phenol. In
some embodiments, the reactive layer comprises an amino phenol an
aminophenol ether. In some embodiments, the reactive layer
comprises an indoxyl. In some embodiments, the reactive layer
comprises an a neutral dye. In some embodiments, the reactive layer
comprises a charged dye, e.g., a dye selected from remazole
brilliant blue, toluidine blue, reactive black 5, remazol brilliant
blue, reactive violet 5, and reactive orange 16, or a hydrolytic or
ammonolytic derivatives thereof, toluidine blue, reactive black 5,
or ahydrolytic or ammonolytic derivatives thereof; reactive violet
5, or hydrolytic or ammonolytic derivatives thereof; reactive
orange 16, or hydrolytic or ammonolytic derivatives thereof; a
dichlorotriazine-based reactive dye such as reactive blue 4,
reactive red 120, reactive blue 2, reactive green 19 and reactive
brown 10. In some embodiments, the dichlorotriazine-based reactive
dye appears black. In particular embodiments, the reactive layer
comprises compounds such as a reactive dye containing a
sulfonylethyl-hydrogensulphate-reactive-group. In some embodiments,
the reactive dye is reactive black 5, remazol brilliant blue,
reactive violet 5 or reactive orange 16, particularly reactive
black 5. In some embodiments, the reactive dye is remazol brilliant
blue, reactive violet 5, reactive orange 16, reactive black 5, or
remazol brilliant blue. Especially, the reactive layer comprises a
dye containing a sulfonylethyl-hydrogensulphate-reactive-group,
e.g., reactive black 5, remazol brilliant blue, reactive violet 5
or reactive orange 16, or a combination thereof; or a dye
containing a dichlortriazine reactive-group, e.g., reactive blue 4,
reactive red 120, reactive blue 2, reactive green 19 and reactive
brown 10, or a combination thereof.
[0241] In other embodiments, indicator disks are preferably
prepared with reagents that are capable of physical change, e.g.,
nanoparticle, colloidal gold particle or a luminol derivative.
[0242] In a preferred embodiment, MPO is detected using an analog
of Fast Blue, or a di-amino phenol as a color generating agent;
Elastase is detected using a peptide derived indicator including a
napthol phenol, indoxyl or a nitro-phenol; Lysozyme is detected
using an oligo saccharide conjugated to a dye or color generator,
or an oligosaccharide particle containing a charged dye in
particular said oligosaccharide may be selected from peptidoglycan
or chitosan derivatives. Purely as a representative example,
lysozyme may be detected by visualizing reactive black 5, remazol
brilliant blue, reactive violet 5 or reactive orange 16, reactive
blue 4, reactive red 120, reactive blue 2, reactive green 19 and
reactive brown 10, or a combination thereof bonded to a substrate
such as chitosan, N-acetyl chitosan;
oligo-.beta.-D-1,4-glucosamine; acetyl-D-glucopyranoside;
N-acetylglucosamine (GlcNAc); glucosamine dimer (GlcNAc).sub.2;
acetyl-chitosan; chitobiose octaacetate; a chitooligomer comprising
the structure (GlcNAc).sub.n wherein n=4, 5, or 6; a
chitooligosaccharide; 2-acetamido-2-deoxy-D-glucopyranoside;
2-deoxy-3,4,6-tri-O-acetyl-D-glucopyranoside; or a combination
thereof. Protease such as human neutrophil elastase or HNE) may be
detected by using a peptide substrate comprising a core sequence
Alanine-Alanine-Proline-Valine (AAPV) which is conjugated to one or
more of the aforementioned dyes.
[0243] In another embodiment, the reagents to detect these analytes
are subject to cleavage to yield a compound that is trapped on an
immobile portion.
Example 7. Use of an Indicator Dressing in the Context of a Wound
Therapy
[0244] A dressing containing an indicator disk as described above
is prepared in which the printed disks are inserted between the
absorbative outer layer of the dressing and the outer membrane or
film such that the reacted areas are visible. The dressing is
applied to a wound, be it chronic or surgical, such that sites of
secretion in the wound (deeper sites, sutures) are located under or
as near as possible to centers of the disks. See FIG. 17. Following
dressing application, the dressing will begin to absorb secretions.
In one embodiment, the first observation of wound status can be
made after the "flow control" has turned blue. This is an indicator
of the fact that sufficient liquid has entered the dressing to
saturate the reagent pads. If, at this stage one or more of the
biomarker indicators has already reacted, this would be an
indicator of the fact that a degree of inflammation or potential
infection was present in the wound at dressing change. One
biomarker reacting, with or without an indication of pH above
neutral, is likely sufficient to justify detailed wound hygiene
steps at the next change. Two biomarkers responding with or without
a pH above neutral is likely an indication that in an ideal
situation, the wound would be immediately re-dressed and
anti-microbial approaches initiated. Three biomarkers responding,
with or without pH would likely be an indicator that in an ideal
situation the dressing should be immediately changed and
anti-microbial hygiene, wound dressings and laboratory microbiology
should be initiated.
[0245] In a broad sense the indicators can respond immediately
after dressing change, after 1-2 days and after 2-5 days. Due to
the dynamics of flow, the reagents are intended to respond within
2-6 h of exposure to a threshold of enzyme activity, for example
0.5 U/mL elastase, however, long exposure to low enzyme levels,
i.e. 5 days, may also ultimately engender a signal. Thus, the user
can distinguish a low level of activity from an acute sign in that
the reporter area very slowly accumulates signal, i.e. very faint
at 3 or 4 days and only slightly more developed after 4 or 5 days.
This would be indicative of a wound deserving of close observation
and hygiene but not necessarily one in acute infection. Experience
with the particular patient would also inform the therapist. If the
same pattern was apparent over multiple dressing changes it would
suggest a stable situation but that any change in the degree of
reaction should be taken as an indication of a potential change in
wound status.
[0246] In contrast, a situation in which a strong signal suddenly
appears is potentially indicative of the onset of an acute
infection. Given that the indicator can change within 1-2 hours
once a threshold is crossed suggests that any sudden developments
reflect the current situation of the wound.
[0247] Where multiple infection indicator disks are placed within
the wound dressing, the position of those that react is an
indicator of where in the wound potential problems arise. Thus, the
absence of clear signals after 5 days would be an indication that
no thresholds have been crossed in that period and that current
therapy may be adequate. Weak signals that develop slowly may
indicate that hygiene could be improved. Moderate signals that
appear gradually after 5 days may be the first signs that an
infection is developing and should result in more elaborate
therapy. Strong signals that develop over 5 days would be
correspondingly more emphatic indications that therapy need be
improved, for example, by instituting silver dressings. The rapid
onset of a clear signal is, in turn, the indicator of an acute
issue that merits immediate attention.
[0248] As shown in FIG. 4(C), multiple reaction cells can be
applied to a wound dressing in some embodiments for detection of
microbial infection over an area. Amine back flow trap or filter or
leach-back trap (41) may be used to separate testing regions.
Example 8. Dressing Inserts that may be Applied to Any Dressing
[0249] In some embodiments, indicator insert may be freely placed
at a site of likely secretion or placed anywhere in a wound
dressing or a surgical dressing.
[0250] Diagnostic disks, as described above, can be incorporated
into a dressing during its manufacture. These inserts may be placed
between the outer absorbent and the outer film and equally spaced,
and glued in place during manufacture. However, the fixed spacing
may not be appropriate to a particular wound. In this example, the
reporter disks are prepared as independent materials that can be
put on any absorbent dressing below the outer film. For example,
the inserts are prepared as stand-alone disks, cut and sealed in
sterile outer envelope. Therapists using dressings, see reference
(92) in FIG. 17, without reporters may still insert these reporters
(90) into such dressings in so far as these are modular and require
the therapist to assemble the dressing from: wound contact
material, absorbent, and outer film or cover. The reporter disk can
fulfill its function in many ways, including so long as it is in
fluid contact with the wound fluids (91) and otherwise under an
appropriate outer dressing. An adhesive transparent outer disk is
one means of fixing and holding the reporter disk. Similarly, the
disk itself may have an adhesive bottom coat.
[0251] In another embodiment of diagnostic inserts, shown in FIG.
18, non-woven layer in a dressing carries or contains diagnostic
disks (705), wherein the dressing further comprises a film cover
layer (701), non-woven carrier of indicators (702), polyurethane
foam (703), and cellulose contact layer (704). As demonstrated by
the arrow in FIG. 18(A), wound fluid flows upward to diagnostic
disks (705) embedded in such dressing. FIG. 18(B) shows a side view
of the wound dressing with embedded diagnostic disks, wherein
quaternary amine coating (shown as dashed line) on foam surface
acts as trap for preventing return of diagnostic substances and
that wound fluid flows upward to diagnostic disks.
[0252] In a further embodiment of diagnostic disks in wound
dressing, as shown in FIG. 19(A), the side view representation (A)
shows an example disk for detecting MPO, wherein (720) is a paper
disk impregnated with the MPO substrate through dipping or spray
coating. Reference (721) is the paper or non-woven material that
acts as a carrier. Reference (722) shows an adhesive layer.
Reference (723) represents a disk containing glucose oxidase and/or
starch and an amylase, such as gamma amylase. FIG. 19(D) shows the
wound fluid mobilizes starch into glucose, which in turn is
oxidized by glucose oxidase to yield H.sub.2O.sub.2. This is used
by MPO in the wound fluid to convert the substrate to the
detectable blue form. FIG. 19(B) shows the side view of a disk for
detecting lysozyme, wherein particles of chitosan or peptidoglycan
are embedded in the paper disk on its lower side using a water
permeable adhesive layer that also serves to adhere the disk to the
foam layer below. Enzyme activity dissolves the particles and
releases dye that is trapped and is detectable in the top layer. In
FIG. 19(B), the paper disk (730) is a trap impregnated top layer.
In the presence of wound fluid, as shown by the upward arrow in
FIG. 19(C), the paper/non-woven disk acts as a carrier (721) so
that the wound fluid moves to the top layer, via stained
peptidoglycan particles (731) in the process. Reference (722) shows
an adhesive layer. Reference (732) shows an adhesive ring or
thermal weld that secures the disk to the non-woven carrier layer
(721). The dashed line in FIG. 19(C) represents quaternary amine
coating on foam surface under the diagnostic strips that acts as a
trap for preventing return of diagnostic substances. FIG. 19(E)
shows stained peptidoglycan particles slowly being dissolved by
wound fluid and the dye that is released is then captured in the
trap material while excess wound fluid flows to the sides, as
indicated by the arrows. In FIG. 19(E), the paper disk is
impregnated with trap material in the top layer.
[0253] In FIG. 20, the scaling up of the production of the disk
constructs is described. In the continuous process, the disks are
punched from a sheet comprised of sealing film, the adhesive, the
paper or non-woven carrier, which is protected by the top cover
sheet.
[0254] FIG. 21 shows different embodiments of paper disks. FIG.
21(A) shows the different layers involved in such embodiments,
namely, film cover on top, a non-woven carrier, a polyurethane
foam, and a cellulose contact layer. FIGS. 21(B) to 21(E) show
different variants of such analytic system with indicator disks.
FIG. 21(B) shows non-woven carrier of indicators with diagnostic
disks attached, including pH indicator on paper, paper disk printed
with starch, amylase, and glucose oxidase, and trap impregnated
paper disks. FIG. 21(C) shows partly printed non-woven and applied
paper disks, including trap printed and UV border or trap border
(910). FIG. 21(D) shows partly printed non-woven and gradient (911)
application of indicator disks. The gradient is formed by printing
concentric rings of substrate at different concentration, or with a
different pH mediator. Fully transformed, different substrate
concentrations lead to different color intensity. Alternatively,
using polymeric buffers in each ring can modulate the degree of
reaction requiring more activity to yield the same color. Suitable
buffers include polycarbonates and polysulfonates. The number of
concentric rings of color provides an indication of overall
activity and thus with reference to a color chart can assist in
assessing the degree of severity. FIG. 21(E) shows one embodiment
of the diagnostic disks with printed indicators (912) and reagents
applied on adhered paper disks. In these embodiments, the non-woven
functions as a carrier of indicators.
[0255] FIG. 22 shows different ways diagnostic disks (800) may be
attached to a dressing. For example, FIG. 22(A) shows continuous
adhesive that allows wound fluid to penetrate through the adhesive.
FIG. 22(B) shows ring or annular adhesive that allows wound fluid
to penetrate via the hole in the middle of the adhesive layer. FIG.
22(C) shows welding with UV printed border. FIG. 22(D) shows
welding with polyethylene component of non-woven.
Example 9: Dipstick--Traffic Light Format
[0256] Certain reagents have adequate affinity for paper or similar
solid phases and remain substrates for the biomarker enzymes of
interest. Where these substrates exhibit color change, the activity
of the enzymes can be observed by simply contacting the fluid
containing the markers with the impregnated paper. Capillarity
ensures the distribution of the fluid to the substrate. Each
impregnated disk can be separately added to a combined "dipstick"
which allows all disks to be used in a test (FIG. 23). One format
is the linear array of disks, although the layout may be easily
varied.
[0257] FIG. 23 shows indicator inserts or disks (820) specific for
various enzymes or microbial biomarkers and controls may be placed
in various combinations or arrangements to form various dipstick
devices. Each impregnated disk (820) can be separately added to a
combined dipstick that allows all indicator disks to be used in a
test. One format is the linear array of disks, although the layout
may be easily varied. Indicator disks may be separated by lanes or
borders (821).
[0258] In this example, the following disks are prepared:
[0259] 1. Fluid control: a 5 mm disk of double sided adhesive is
punched, and 50 .mu.g of a micronized Fast green powder is placed
on the adhesive in the center. A paper disk is placed over the
adhesive disk concentrically, such that the powdered dye is covered
by the paper. The resulting disk is then placed in the first
position on the carrier stick via the other side of the
adhesive.
[0260] 2. pH control. Filter paper is soaked in a mixture
containing bromothymol blue, chitosan and glutaraldehyde in ethanol
as reported above. The filter paper is dipped in the mixture,
allowed to drip dry, and is then dried on glass at 54.degree. C. 5
mm disks are then punched and the disks are attached to the carrier
with adhesive.
[0261] 3. MPO indicator. 5 mm paper disks are impregnated
sequentially with 1.5 .mu.L of the MPO fast blue substrate as
described above for the Dressing indicator. Once dried, one half of
the disk is impregnated with 10 .mu.g of glucose and the other half
of the disk is impregnated with 1 .mu.g of glucose oxidase in
buffer (PBS).
[0262] 4. Elastase indicator. Filter paper was impregnated with a
mixture (0.25% (w/w) Nonidet, 2% (w/w) decanol in 0.05 M borate
buffer pH 8) and dried for 1-2 h at 54.degree. C. 5 mm paper disks
are punched from the buffer treated paper and impregnated
sequentially with 2 times 2.5 .mu.L of the AAPV indoxyl substrate
(10 .mu.g/.mu.L in acetone) as described above for the Dressing
indicator.
[0263] 5. Lysozyme indicator. Filter paper is lightly sprayed (1.5
.mu.L per cm2) with a trap solution containing 3% W/V quaternary
amine trap and allowed to dry with the top surface identified. A 5
mm disk of double sided adhesive is punched, and 40 .mu.g of a
Brilliant Black stained Peptidoglycan is placed on the adhesive in
the center and allowed to dry. A paper disk is placed over the
adhesive disk concentrically, such that the PG-dye deposit is
covered by the paper. The resulting disk is then placed in the
fifth position on the carrier stick via the other side of the
adhesive. The resulting dipstick can have the sample applied to it
by means of swab, or gauze.
Example 10: Dipstick--"Fork" Format
[0264] In one embodiment a dipstick is prepared essentially as for
the above example with the exception that the reagent disks are
oriented to the base of a thicker carrying card or stick. The ends
of the reagent disks are trimmed at the last stage of production
such that they are flush with the bottom edge of the device. This
allows them to be pressed onto a surface to be sampled. The sample
then diffuses into the cut end of the disks to react. This format
is potentially more convenient for sampling surfaces like used
dressings.
Example 11: Dipstick--Box Format
[0265] In certain instances, suspected infection, or the risk of
contamination between patients through consumables and their
disposal demands a more secure system. In one embodiment, where
sampling is done via a swab, retention of the swab for subsequent
bacteriological evaluation may be desirable. Similarly, it may be
desirable to retain the result and display it to a colleague after
a dressing change. In this context, a means to retain the result
without risk of contamination is desirable. To this end, in one
embodiment, a sealable container or enclosure may be used for
accommodating a plurality of disks, such as 6 disks, in which a wet
swab can be placed and then closed such that it can apply the
sample to the paper disks but not contaminate any further objects.
One such design is illustrated along with its working principle
(FIG. 23). The key elements of the design are: the well for wetting
the swab; its closed sealable form; the sealing rings around the
stem of the swab; the pressure fins that push the swab to the disks
while also making it a one-way movement; the window to the disks;
the space for reference colors on the case, the possibility to
re-open in a microbiology lab.
Example 12: Surgical Site Detection
[0266] In another embodiment, the dressing is intended for the
treatment of surgical wounds and contains distinct linear regions
intended to be placed over the line of sutures. These linear
regions contain particularly high concentrations of reporter dye
such that even in the earliest phases of infection, the signal will
be apparent. In another embodiment, the dressing contains a
removable components such as a thread, or similar absorbent that
can be withdrawn and tested without removing the dressing (FIG.
24). Said removable component is placed in such a way as to be
located at or near the edges of the surgical wound. In another
embodiment, the surgical site dressing is essentially transparent
in the linear region both to allow observation of the sutures, and
the reporter dye. In a preferred embodiment, the transparent area
is covered by an opaque film that may be easily peeled back to
examine the wound. In another embodiment the covering and absorbent
material contains a trapping material such as a polymeric cation or
anion that is capable of binding and concentrating the dyes that
are released.
[0267] For example, in FIG. 24, sampling threads (100) are built in
or added to dressing for a wound or at a surgical site (92).
AQUACEL (4) is used in some embodiments of the dressing (92).
Sampling threads absorb wound fluid or fluid at surgical site (D).
A thread may be pulled out or extracted (E) from dressing without
having to remove or disturb dressing using an instrument or device
(101) such as a tweezer, hook, or thread hook device. The thread
can then be dissolved in a buffer for use in a diagnostic device
(102) using one or more indicator regents or indicator disks
described herein.
[0268] In some embodiments, a wound dressing comprises built in
sampling threads. In some embodiments, the sampling threads absorb
wound fluids and may be removed without disturbing the wound
dressing for detection of analytes in the wound fluid.
[0269] In some embodiments, the sample threads may be diluted in
buffer to dissolve markers for diagnosing the status of the
surgical site or wound.
[0270] In some embodiments, a thread hook device may be used to
remove a thread from a wound dressing.
Example 13: Manufacture of Dressing Inserts
[0271] The reporter inserts are manufactured by the sequential
placement of various materials on a solid carrier. This carrier can
be a cellulose, viscose, polyethylene, polyamide or other suitable
polymer or mixture of these components.
[0272] FIG. 25 shows indicator inserts may be manufactured or
printed in sheets or reels. FIG. 25 also shows the order of
printing, printing of lanes, order on which reagents are laid down,
and placement of reagents for printing disks in sheets or reels,
comprising adhesive or backing film as in FIG. 25(A), applying a
non-woven material as in FIG. 25(B), and printing reagents and
lanes on non-woven material as in FIG. 25(C). Completed or
assembled inserts, as show in FIG. 25(D) can be separated or cut
before sticking to a dressing or similar support materials.
[0273] In one embodiment the material is prepared in a reel to reel
format. The solid carrier is first printed with guide lanes that
penetrate the film to full thickness. Next, a bottom film that sits
under the polymer and does not penetrate it is printed, this
includes a hole in the center through which sample fluid enters.
Next trap material is printed, at half density around the entrance
site (back-flow trap) and at full density in the trapping sites for
the flow control and the lysozyme substrate. Next the flow control
ink is applied to the first position of the radial arms of the
disk, 10 to 50 .mu.g of Brilliant black in 1% methylcellulose is
typical. Next the pH reporter, as described above is printed in
position 2. Next, the MPO area is printed sequentially with
substrate, glucose and glucose oxidase as noted above. Next the
elastase substrate is applied in sequential prints to reach the
appropriate load. Next the lysozyme substrate is printed to
position 5 in the reagent level (as distinct from the trap level).
Finally a film is printed on the top of the construct but without
penetration of the solid carrier. This film occludes only the
radial arms from center to the end of the reporter window.
[0274] The resulting reel contains a continuous pattern of evenly
spaced reporter fields. These continuous printed fields can be
directly rolled into a dressing sandwich between absorbent and
outer film, or, they may be punch cut and packaged for separate
use.
[0275] FIG. 20 shows another embodiment of manufacturing paper
disks. FIG. 20(A) shows a side view of a continuous sheet,
comprising a cover film on top, paper in the middle, and backing
film at the bottom. Adhesive/particle matrix (901) may be applied
between the cover film/backing film and the paper layer (900). FIG.
20(B) shows a top view of cut sheets prepared for application to
non-woven carrier by removal of inter disk material prior to
placement on non-woven carrier.
Example 14: Manufacture of Reagents for Liquid Based Devices
[0276] In certain embodiments, it is desirable to place reagents in
devices in such a way as that they are stable, but readily soluble
for access to injected enzymes. One approach is to dry reagents on
disks of paper and include the disks in the devices.
[0277] Disks are prepared using either a continuous paper or
similar material or textile which is dipped, sprayed or printed, or
using pre-cut disks that are dipped or mixed in a reagent and
subsequently dried. See FIGS. 20, 25.
[0278] The densities of the reagents per 20 mm2 are:
[0279] MPO substrate (alkyl-fast blue) 0.6 .mu.g
[0280] Glucose 10 .mu.g, glucose oxidase 1 .mu.g
[0281] For elastase, paper is first impregnated with impregnation
mixture (0.25% (w/w) Nonidet, 2% (w/w) decanol in 0.05 M borate
buffer pH 8).
[0282] Thereafter the paper is sprayed with a solution of elastase
substrate corresponding to 2.5 .mu.g per mm2.
[0283] The paper so printed can be punched to yield disks
containing the reagents.
[0284] These disks can then be incorporated into the devices.
Example 15: Manufacture of Reagents for Liquid Based Devices
[0285] Alternatively, the reagents may be pressed into water
soluble "pellets" which are then included in the wells of the
devices. The pellets can contain a range of materials in addition
to those used on paper.
[0286] A liquid based diagnostic device uses pre-formulated
reagents to generate a colour in response to enzyme activity in a
sample. The sample may contain all or only some of the liquid
required. Where the sample is to be diluted, the device preferably
contains water or buffer suitable to dilute or render the sample
homogeneous. The resulting mixture is distributed to wells which
each contain a different reagent set. The reagents are a mixture of
buffer salts, energy source, substrate and associated chromophores
if not contained in the substrate. These reagents are ideally
delivered in a discreet entity like a tablet or similar that can be
placed in the wells. Here we describe the preparation of tablets
for enzymatic assays for elastase, lysozyme, MPO and protein
standard as internal standard. The tablets dissolve after addition
of wound fluid and release assay components to start the enzyme
reactions that lead to colour changes where positive.
[0287] A Perkin Elmer electro-hydraulic tablet press is used to
form the tablets as follows:
[0288] The pressing time per tablet is approximately 10 sec.
[0289] The diameter of the filled part of the pressing tool is 5
mm
[0290] Tablets are: 20 mg, 5 mm diameter, 1 mm deep
[0291] A vacuum is first applied for about 15 sec.
[0292] The applied vacuum is maintained until the removal of the
pressing tools.
[0293] The pressing pressure is adjusted to 2 t.
TABLE-US-00001 TABLE 1 List of tablet reagents for use in
liquid-based diagnostic devices. Amount in 20 mg Component (mg) MPO
Tablet Na.sub.2CO.sub.3 0.38 NaHCO.sub.3 0.54 Guajacol
(CH.sub.3O)C.sub.6H.sub.4OH = Substrate 1.53 Alternatively
diaminophenol Sodium percarbonat .times. 1.5 H.sub.2O.sub.2
(Na.sub.2CO.sub.3.cndot.1.5 H.sub.2O.sub.2) 0.02 Maltose
Monohydrate (C.sub.12H.sub.22O.sub.11.cndot.H.sub.2O) 17.53
Elastase Tablet Sodium Acetate (C.sub.2H.sub.3NaO.sub.2) 1.64
Sodium chloride (NaCl) 5.84
N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide 0.24
(C.sub.27H.sub.38N.sub.6O.sub.9) = Substrate Maltose Monohydrate
(C.sub.12H.sub.22O.sub.11.cndot.H.sub.2O) 12.28 Lysozyme Tablet
Potassium hydrogenphosphate (K.sub.2HPO.sub.4) 7.32 Potassium
dihydrogenphosphate (KH.sub.2PO.sub.4) 1.09 Peptidoglycan (von
Micrococcus lysodeicticus) as a 0.20 film or dyed with reactive
black as gross particles = Substrate Maltose Monohydrate
(C.sub.12H.sub.22O.sub.11.cndot.H.sub.2O) (Filler) 11.39 Internal
Standard Tablet Citric acid (HOC(COOH)(CH.sub.2COOH).sub.2 8.56
Sodium hydrogenphosphat (Na.sub.2HPO.sub.4) 1.54 Bromophenol blue
(C.sub.19H.sub.10Br.sub.4O.sub.5S) 0.06 Maltose Monohydrate
(C.sub.12H.sub.22O.sub.11.cndot.H.sub.2O) 9.84
Example 16: Standalone Device and Kit for Liquid Based Assay
[0294] Stand-alone devices and kit for detecting and measuring
wound infection using the compositions and device are described
herein. These devices and kits preferably comprise a sampling
component for collecting a sample and a test device. In some
embodiments, the test device comprises a housing surrounding a tube
to define an opening in the housing to receive the sampling
component, the housing having within it a sealed diluent chamber
which is connected to an opposite end of the tube and holding a
liquid diluent for removing the sample from the sampling tip to
form a test liquid. The tube is in liquid communication with a
reaction well which holds a reagent capable of indicating the
presence of the analyte. A driving mechanism drives the diluent
from the chamber past the sampling tip, into the tube and finally
to the reaction well.
[0295] In some embodiments, the kit for detecting an analyte in a
sample comprises: (i) a sampling component comprising a sampling
tip for collecting the sample and (ii) a test device, further
comprising: a housing surrounding a tube to define an opening in
the housing to receive the sampling component, the housing also
having disposed within it: a sealed diluent chamber connected to
the tube and holding a liquid diluent for removing the sample from
the sampling tip to form a test liquid; a reaction well in liquid
communication with the tube, the reaction well holding a reagent
capable of indicating the presence of the analyte within the test
liquid; and a driving mechanism capable of driving the diluent
through the device from the chamber, over the sample tip and into
the reaction well.
[0296] The kit operates by driving the diluent over the sample and
into a reaction well, a test solution is made by the flow of the
diluent over the sample. It is not necessary to first mix the
sample with the diluent to make a test solution and then move that
solution via a lateral flow strip to the reaction well. The driving
of the diluent past the sample and to the reaction well means that
the kit can be used with a minimum number of steps, for instance
taking the sample, inserting the sampling component into the
housing and activating the driving mechanism. This simple procedure
minimizes user error and thus minimizes false negative results and
misdiagnoses.
[0297] The sealed diluent chamber may contain a specified volume of
diluent so that an expected volume of test solution reaches the
reaction well or wells. In addition the pathway between the diluent
chamber and the reaction well is vented so that trapped air does
not affect the flow of test solution through the device or prevent
the test solution from reaching the reaction well.
[0298] The housing preferably has two parts which are capable of
moving with respect to each other while remaining connected to one
another. The action of moving the parts may provide the driving
mechanism by which diluent is moved through the device. The diluent
can be driven through the device by compression of the diluent
chamber which forces the diluent past the sample tip and to the
reaction well or wells. The compression of the diluent chamber can
occur when the parts of the housing are moved with respect to one
another such as by sliding one part past another.
[0299] In some embodiments, the housing comprises a locking
mechanism which locks the housing in position once the driving
mechanism has been activated and prevents reuse of the device. In
this way it is immediately apparent that the device has been used
and cannot be used again. This minimizes false results from, for
instance, a device that has been mistakenly activated in transit or
from reuse of a device whose reagents have been spent.
[0300] In some embodiments, the sampling component preferably
comprises a handle and a sampling tip, the handle preferably
comprising a seal which engages with the opening in the housing to
seal the tube when the sampling component is fully inserted in the
tube. The seal generally prevents escape of the sample and diluent
from the device reducing the chance of cross contamination from the
wound fluid. Preferably the seal and tube engage to lock the
sampling component in the device and prevent removal of the
sampling component once it has been used. This further reduces the
chance of cross contamination from the sampling component.
[0301] Preferably insertion of the sampling component in the device
releases the seal on the diluent chamber. Preferably the seal is a
ball valve or can be a film or membrane seal or a duck bill valve
or other non-return valve known in the art which is activated when
the sampling component is inserted in the device. The sampling
component preferably bursts, punctures or displaces the seal on the
diluent chamber when it is inserted in the device.
[0302] Preferably the tube is the same or similar size to the
sampling tip of the sampling component so that the act of inserting
the sampling tip into the tube causes it to be scraped along the
walls of the tube aiding the dispersion of the sample in the
diluent once it is released from the diluent chamber and is flushed
through the device. The sizing of the sampling tip to match the
tube also forces the diluent to be flushed through the tip when the
diluent is driven from the diluent chamber. Preferably the diluent
chamber is shaped like a bellows to assist in the compression of
the chamber. Alternatively the chamber can be a combination of a
plunger and tube similar to that found in a syringe or can be a
filled flexible sachet which is compressed by hand by the user or a
balloon which contracts when the seal is released.
[0303] In some embodiments, the kit comprises a sampling component
for collecting a sample and a test device. The test device
comprises a housing surrounding a tube to define an opening the
housing to receive the sampling component, the housing having
within it a sealed diluent chamber which is connected to an
opposite end of the tube and holding a liquid diluent for removing
the sample from the sampling tip to form a test liquid. The tube is
in liquid communication with a reaction well which holds a reagent
capable of indicating the presence of an analyte.
[0304] A driving mechanism drives the diluent from the chamber past
the sampling tip, into the tube and finally to the reaction
well.
[0305] FIG. 26 shows a cross section of a standalone device kit for
detecting an analyte in a sample. The sampling component (2)
comprises a handle (4) and a sampling tip (6) in the process of
being inserted into the housing through one end of a tube (10). The
sampling component (2) has a sealing means (12) which forms a seal
with the open end of the tube (10) while the sampling tip (6)
depresses the ball valve (14) to open the diluent chamber (16).
FIG. 27 shows a sampling tip fully inserted in the housing to seal
the component to the device. FIG. 28 shows a plan view of the
standalone device kit with the sampling component in place and
shows three viewing windows (20) to the left of the housing which
coincide with three reaction chambers (18) which contain a reagent
capable of indicating the presence of an analyte. The reaction
chambers may contain reagents capable of detecting different
analytes from for instance a wound fluid. The window on the right
of the housing when viewed from above is a control window which
indicates that the test has taken place. Housing (8) is in two main
parts which are slidably connected to each other. In FIG. 29, a
user of the device can slide a lower part of the housing (24) away
from the upper part of the housing (26) and in so doing cause a
lever (28) to compress the diluent chamber (16) and drive the
diluent out of the chamber, through the sampling tip (6) and up
tube (10) to manifold (30). A plan view (FIG. 30) of the standalone
device kit with housing slid apart, which results in windows (20)
and control window (22) indicating that a test has taken place. The
arrows (A) in FIG. 29 indicate the movement of the diluent through
the device to form a test solution. Diluent chamber, tube and
reaction chamber in the standalone device kit are shown in FIG. 31,
with the housing removed for clarity. FIG. 32 shows distribution of
test solution to each reaction chamber in a standalone device kit.
Test solution flows to each reaction chamber (18) from a central
node (32). The node (32) may also contain a non-return valve to
prevent test solution from flowing back into the device and causing
cross contamination.
[0306] The sampling component comprises a handle and a sampling tip
in the process of being inserted into the housing through one end
of a tube. The sampling component has a sealing means which forms a
seal with the open end of the tube while the sampling tip depresses
the ball valve to open the diluent chamber. The sampling tip, when
fully inserted in the housing to seal the component to the device,
allows the housing to be opened, releasing the diluent and allowing
the forcing means to operate.
[0307] The device also comprises three viewing windows in the
housing that correspond to three reaction chambers which contain a
reagent capable of indicating the presence of an analyte. The
reaction chambers may contain reagents capable of detecting
different analytes from for instance a wound fluid. Some
embodiments include a control window which indicates that the test
has taken place and that the sample was sufficient to make the test
viable.
[0308] The user of the device can slide a lower part of the housing
away from the upper part of the housing and, in so doing, cause a
lever to compress the diluent chamber and drive the diluent out of
the chamber, through the sampling tip and up tube to manifold. If
the device is not activated, that is if the seal on the diluent
chamber has not been broken, it is not possible for the housing to
open. The opening of the housing causes the viewing windows to be
positioned over the reaction wells and enable the result to be
viewed by the user. This provides a safety measure as it ensures
that proper operation of the device in order to obtain a reliable
result.
[0309] Once activated, the test solution flows to each reaction
chamber from a central node. In some embodiments, the node
comprises a non-return valve and filter to prevent test solution
from flowing back into the device and between reaction chambers,
which can cause cross contamination. The pathway for the flow of
diluent through the device is preferably provided with vents at the
reaction chamber end.
Example 17. Devices with Separate Sample Preparation Chamber
[0310] FIG. 33 shows a diagnostic swab device with housing. In one
embodiment the swab device comprises a resealable housing (80),
further comprising locator and locking pins (82), a viewing window
(81) for observing visible signals from reagent disks placed in
disk holders (83), and a groove (85) for placing the swab. Side
view of FIG. 33(C) shows the housing (80). To use, a user touches a
sample with the swab, places the swab in the housing (80) in groove
(85), pull on the stem of the swab as shown by the arrow in (D) so
that the sample on the swab slides on the strip (86) and transfers
the sample to reagent or indicator disks (83). The results may be
viewed through viewing window (81). The swab may also be kept in
the housing (80) for analysis later.
[0311] FIG. 34 shows one embodiment of a thread hook sample
preparation device (200), comprising a needle-like tip and a handle
or plunger (201), wherein the tip further comprises a hook for
extracting a thread from a dressing without disturbing the dressing
as shown in FIG. 34(A). Upon extracting a thread from the dressing,
thread hook device (200) may be inserted into a sample preparation
chamber or diluent chamber (202) containing a diluent for
dissolving or diluting microbial biomarkers or wound fluid from the
thread FIGS. 34(B) and 34(C). The plunger (201) of the thread hook
device may be depressed downward in the sample preparation chamber
(202) so that the tip of the needle breaks a seal as shown in FIG.
34(D) at the bottom of the sample preparation chamber (203) in
order to release the sample solution into a device for analysis of
wound fluid or surgical site.
[0312] FIG. 35 shows one embodiment of a swab sample preparation
device (300), comprising a swab (302) with a handle or plunger
(301) may be used to touch a sample for testing. The swab device
(300), after sampling a bodily fluid or wound fluid, is placed
inside a sample preparation chamber (202) containing a buffer for
dissolving or diluting the wound fluid or bodily fluid as seen in
FIG. 35(A). The swab device is agitated or mixed inside the sample
preparation chamber to further release the fluid sample into the
sample preparation chamber as shown in FIG. 35(B). The plunger
(301) of the needle is depressed downward as shown in FIG. 35(C) to
break the seal (203) at the bottom of the sample preparation
chamber, allowing the sample fluid to flow into a reaction chamber
containing reagents or indicator inserts or disks for detecting
microbial infection in the sample taken by the swab. In some
embodiments as shown in FIG. 35(D), gas is removed using Goretex
membranes (204) which are gas and vapor permeable, but not
permeable to liquid water. Said membranes can be used to degas both
the sample as it is injected and to vent the fluid chambers where
the assay takes place.
[0313] FIG. 36 shows a sample preparation chamber adapted to
indicator testing. Sample preparation chamber (202) is adapted for
dissolving or diluting a sample for testing further and comprises a
resealable top (401) and a breakable seal (402) at the bottom of
the chamber (203), where the sample preparation chamber connects to
a reaction chamber or diagnostic device. When a swab device or a
thread hook device is plunged downward or depressed downward in the
chamber, it causes the seal (402) at the bottom to break, releasing
sample fluid into a diagnostic device connected to the chamber.
[0314] In a further embodiment, FIG. 37, a diagnostic device (500)
or analysis system is adapted to connecting to the sample
preparation chamber (202) at one end, allowing sample fluid to flow
from the sample tip (300) upon breaking the seal (203) at the
chamber connector, which allows the sample fluid to flow from the
sample preparation chamber (202) into reaction chambers (502) for
analysis. Absorbent material (501) positioned after the reaction
chambers (502) helps to draw the sample fluid from the sample
preparation chamber (202) into the reaction chambers (502).
Reaction chambers may contain reagents, reagent tablets, reagent
disks, or indicator inserts, as described herein.
[0315] In so far as liquid phase tests are desired, they may be
conducted using a variety of means but ultimately rely on the
formation of a visible signal in a low volume of liquid (e.g. 100
.mu.L). The methods differ in terms of how one acquires, dilutes
and introduces the sample. In this example, we introduce the sample
using an adapted syringe-like configuration. The sample may be a
swab, piece of gauze or contaminated thread from a dressing. The
swab (FIG. 35) is placed in a plunger configuration and then the
plunger forms a handle with which the swab can be mixed with an
extraction buffer or a diluent in a sample preparation chamber. The
plunger then allows the removal of fluid by sealing against the
stem of the swab and the sides of the chamber simultaneously; a
goretex insert in the plunger allows gas removal as the plunger
descends. Where the sample is a thread or piece of gauze, the swab
is replaced by a hook, however, the principle is the same as the
stem of the hook is placed within the plunger.
[0316] The sample preparation chamber contains buffer which is
mixed with the sample on the swab/hook. The chamber is sealed at
the Luer-Lock style connector and this seal is broken either when
the Luer is placed in a receptacle, or when the swab or hook is
pushed through the bottom of the chamber (FIG. 35). The assay
device entrance includes a standard female Luer with a Luer lock
like surround to ensure good sealing. The modified chamber engages
irreversibly with the female Luer lock and on depression of the
plunger, the fluid is transferred gas free into the device via a
fluid distribution network. Each chamber in the device contains a
reagent tablet (see previous example for the reagents). Each
chamber is vented via a goretex patch sonic welded over the
chamber. As soon as the chamber is filled (from bottom to top),
fluid flow preferably stops. The vented gas passes by a filter
before reaching the atmosphere. The arrival of fluid dissolves the
reagent pills and allows the reaction to start. The degree of
reaction over a given time is determined by comparison to a chart
of colors. The result is largely binary, clear color or not. The
more markers associated with color, the more likely the potential
infection. Thus, wound fluids from uninfected wounds do not cause
color change. Those from infected wounds cause at least one marker
to change color and more often all three markers within 5
minutes.
[0317] FIG. 38 shows one embodiment of a diagnostic device or a
transfer system, comprising a chamber or vessel (601) containing a
buffer, such as saline, a resealable top (600), a plunger or
similar device (602) with a gas outlet and a hook or sample tip at
one end for transferring sample into the a sample preparation
chamber or a diluent chamber (601), and at least one reaction
chamber (606) capable of analyzing a sample fluid from the chamber
(601). To conduct such analysis, the plunger or piston (602)
containing a sample at the end is inserted into the sample
preparation chamber (601), or a sample is placed in the diluent
chamber (601), so that the sample may be diluted or dissolved in
the buffer in the chamber (601). The assembly of a plunger (602)
inserted in a diluent chamber (601) is shown in (607).
[0318] The chamber (601) may further comprise a Luer-Lock or slip
tip (605) for connecting to a reaction chamber (604) or an analysis
system. After connecting plunger unit (601, 602) to reaction
chamber (604), one may depress the plunger (602) downward to break
a seal at the end of the chamber (601), releasing sample fluid from
the sample preparation chamber into reaction chambers (604),
wherein individual reaction chamber (606) may have a different
reporter or color system for detecting an analyte. The plunger
(602) can further comprise membrane that pushes water and lets out
gas, thus degassing the sample fluid as one depresses the plunger
into the chamber. The reaction chambers may be filled in parallel,
and the last chamber contains an aerosol filter and a pressure exit
to atmosphere. Pressure, equalization, reaction chamber filling and
aerosol filtering can be achieved through membrane exits. In some
embodiments, reaction chambers contain reagent tablets or reagent
disks. Top membranes can be welded in place using ultrasound.
Lenses that enlarge the view of the reaction chambers are used in
some embodiments. The connection to the reaction chamber or
transfer system (604) includes a rough filter and a penetrator for
breaking the buffer seal on connection at 605. Reaction chambers
can be closed at the top and bottom by clipping on.
[0319] The conformation of the reaction vessels can be flexibly
organized. One example is shown in FIG. 39, which shows another
embodiment of an analysis system (604). Reaction chambers (606) can
be arranged in a radial manner instead of in a linear arrangement.
A fan- or radial-shaped analysis system (604) is adapted to use
with a sample preparation chamber (601) with a plunger (602) system
for driving a sample solution into reaction wells or chambers.
Different views of such analysis system (604) are shown in (B).
(608) shows a top view of a series of reaction chambers arranged in
a radial arrangement. In some embodiments, the reaction chamber
unit (610) may be removable from housing (609). This removable
feature facilitates a user in refilling, inserting, or exchanging
reagents in individual reaction chambers within the reaction unit
(610).
[0320] In this example, the reagents used are water soluble and are
formulated as tablets using excipients such as PEG, maltose and
sorbitol as carriers. The tablets are formulated with the
appropriate amounts of buffer salts in the bulk mixture to result
in optimal pH upon dissolution. For supply of hydrogen peroxide,
sodium percarbonate is used. As an MPO substrate, a soluble Fast
Blue derivative, i.e the product of reaction with succinic
anhydrice, is used, alternatively, guacol, diamino phenol or
similar may be used. For Elastase, AAPV nitrophenol amide is
employed, alternatively, AAPV-indoxyl with a diazonium salt
enhancer. For Lysozyme, the substrate is a labelled peptidoglycan
particle, however, the well contains a positively charged membrane
at the viewing interface. This membrane is derived on one half with
the trap, and the contrast between the two sides in the main
indicator of reaction indicates the degree of reaction.
[0321] In some embodiments, such as FIG. 24, sampling threads (100)
are built in or added to dressing for a wound or at a surgical site
(92). AQUACEL (4) is used in some embodiments of the dressing (92).
Sampling threads absorb wound fluid or fluid at surgical site (D).
A thread may be pulled out or extracted as shown in FIG. 24(E) from
dressing without having to remove or disturb dressing using an
instrument (101) such as a tweezer, hook, or thread hook device.
The thread can then be dissolved in a buffer for use in a
diagnostic device (102) using one or more indicator regents or
indicator disks described herein.
[0322] In some embodiments, a wound dressing comprises built in
sampling threads. In some embodiments, the sampling threads absorb
wound fluids and may be removed without disturbing the wound
dressing for detection of analytes in the wound fluid.
[0323] In some embodiments, the sample threads may be diluted in
buffer to dissolve markers for diagnosing the status of the
surgical site or wound.
[0324] In some embodiments, a thread hook device may be used to
remove a thread from a wound dressing.
[0325] While preferred embodiments of the disclosed technology have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
disclosed technology. It should be understood that various
alternatives to the embodiments of the disclosed technology
described herein may be employed in practicing the disclosed
technology. It is intended that the following claims define the
scope of the disclosed technology and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *