U.S. patent application number 11/715215 was filed with the patent office on 2007-07-05 for hygiene monitoring.
This patent application is currently assigned to CHARM SCIENCES, INC.. Invention is credited to Stanley E. Charm, Cheryl B. Francisco, Robert J. Markovsky, Steven J. Saul.
Application Number | 20070154973 11/715215 |
Document ID | / |
Family ID | 27397083 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070154973 |
Kind Code |
A1 |
Saul; Steven J. ; et
al. |
July 5, 2007 |
Hygiene Monitoring
Abstract
A test device for hygiene monitoring by detecting glucose as an
indication of the presence of biological material.
Inventors: |
Saul; Steven J.; (Arlington,
MA) ; Francisco; Cheryl B.; (Fairhaven, MA) ;
Markovsky; Robert J.; (Brentwood, NH) ; Charm;
Stanley E.; (Boston, MA) |
Correspondence
Address: |
CHARM SCIENCES, INC.
659 ANDOVER STREET
LAWRENCE
MA
01843
US
|
Assignee: |
CHARM SCIENCES, INC.
|
Family ID: |
27397083 |
Appl. No.: |
11/715215 |
Filed: |
March 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10343582 |
Aug 1, 2003 |
|
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PCT/US01/24054 |
Aug 1, 2001 |
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11715215 |
Mar 7, 2007 |
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60222365 |
Aug 1, 2000 |
|
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60228369 |
Aug 28, 2000 |
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60267173 |
Feb 8, 2001 |
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Current U.S.
Class: |
435/14 |
Current CPC
Class: |
C12Q 1/54 20130101; G01N
33/523 20130101; C12Q 1/34 20130101; C12Q 1/48 20130101; C12Q 1/22
20130101; C12Q 1/42 20130101 |
Class at
Publication: |
435/014 |
International
Class: |
C12Q 1/54 20060101
C12Q001/54 |
Claims
1. A device for detecting biological material in a sample, which
device comprises: a) a material for receiving said sample; b) a
reagent for converting said biological material to glucose; and c)
a reagent for the colorimetric detection of said glucose.
2. The device of claim 1 wherein said reagent for converting
biological material to glucose comprises an enzyme.
3. The device of claim 1, wherein the said reagent for converting
biological material to glucose and the reagent for the colorimetric
detection of glucose are provided to a user on one or more zones on
a lateral flow test strip.
4. The device of claim 1, further comprising a sealed compartment
containing a surface wetting agent.
5. The device of claim 1 wherein said material for receiving a
sample comprises a swab.
6. The device of claim 1 further comprising one or more separate,
swab-puncturable, membrane compartments enclosing the reagent for
converting the biological material to glucose and the reagent for
the calorimetric detection of glucose.
7. The device of claim 1 wherein said biological material comprises
a carbohydrate.
8. The device of claim 1 wherein said biological material comprises
lactose.
9. The device of claim 2 wherein said enzyme comprises
.beta.-galactosidase.
10. The device of claim 7 wherein said carbohydrate comprises one
or more sugars.
11. The device of claim 7, wherein said carbohydrate comprises one
or more complex carbohydrates.
12. The device of claim 7, wherein said carbohydrate comprises
lactose.
13. The device of claim 2, wherein said enzyme comprises a
hydrolytic enzyme.
14. The device of claim 2, wherein said enzyme comprises
invertase.
15. The device of claim 2, wherein said enzyme comprises
amylase.
16. The device of claim 2, wherein said enzyme comprises
cellulase.
17. The device of claim 2, wherein said enzyme comprises
sucrase.
18. The device of claim 1, wherein said reagent for the
calorimetric detection of glucose comprises glucose oxidase.
19. The device of claim 1, wherein said reagent for the
calorimetric detection of glucose comprises peroxidase.
20. The device of claim 1 wherein the sample is from a surface.
21. The device of claim 1 wherein the sample is from a surface of
an inanimate object.
Description
REFERENCE TO PRIOR APPLICATIONS
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 10/343,582, filed Aug. 1, 2003, which
claims the benefit of International Application PCT/US01/24054,
filed Aug. 1, 2001; U.S. Provisional Patent Application Ser. No.
60/222,365, filed Aug. 1, 2000; U.S. Provisional Patent Application
Ser. No. 60/228,369, filed Aug. 28, 2000; and U.S. Provisional
Patent Application Ser. No. 60/267,173, filed Feb. 8, 2001; the
contents of each of which is hereby incorporated by reference in
their entirety.
BACKGROUND OF THE INVENTION
[0002] Cleanliness in industrial and health care settings is
critical. The surfaces of equipment used for food handling,
storage, or processing are major sources of microbial
contamination. Such contamination can lead to decreased shelf life
of products and, if pathogens are present, transmission of disease.
Microbial colonies develop rapidly. Continuous monitoring of
surfaces, e.g., hygiene monitoring, can help protect against the
spread of disease.
[0003] Historically, microbial culturing was used to determine the
presence of microorganisms. However, culturing is time consuming
and, therefore, the necessary "real time" feedback to sanitation
and food preparation personnel is not available. As a result, food
exposed to surfaces which were later found to contain potentially
harmful microorganisms could enter the food supply.
[0004] Lateral-flow chromatographic test strips have been used for
a variety of diagnostic purposes. The test described herein
utilizes a lateral-flow test strip to provide a means for rapid,
sensitive, user-friendly, hygiene monitoring of surfaces. Material
swabbed from a surface can be detected by reactions involving the
pathways described herein.
[0005] Recent attention has focused on the problems of biofilms.
Biofilms are created when microorganisms land on a surface and
attach to its microscopic cracks and crevices. Almost immediately,
the organism begins to produce a polysaccharide-like material which
in hours acts as a glue to stick bacteria and viruses to the
surface. Biofilms are more resistant to routine sanitizing
techniques than are their free-living counterparts. It is,
therefore, critical to generate rapid results, preferably within a
few minutes.
[0006] Food residues on surfaces are nutrients for rapid growth of
microorganisms and the potentially resultant biofilm. Such food
residues are also a source of cross-contamination to other food
products later exposed to the same contact surface. Therefore,
proper hygiene monitoring of a surface should include detection of
a broad range of contaminants, including both biofilms and residual
food.
[0007] During the 1990s various rapid and efficient test methods
and apparatuses were developed for the detection of contamination
on surfaces. Such methods do not detect microbes directly but
instead detect markers, such as ATP, which are indicative of either
the presence of microbes or the existence of residual food
contamination of a surface.
[0008] One such apparatus is the POCKETSWAB-(POCKETSWAB- is a
registered trademark of Charm Sciences, Inc. of Lawrence, Mass.),
which rapidly and efficiently detects ATP on surfaces. The
POCKETSWAB- apparatus detects ATP through the reaction of luciferin
and luciferase, which, in the presence of ATP, emits light. Light
emission is measured using a luminometer. It is desired, and the
primary object of this invention, to provide a rapid, visual test
for hygiene monitoring, and thereby avoid the need for a
luminometer or other reader.
[0009] There are various tests available in the field which provide
a rapid and visual result, thereby reflecting the degree of surface
cleanliness. Such tests are of interest for use in, for example,
restaurants and supermarkets, where an instrument for reading
results would not be acceptable, either because of the large volume
needed or because they could not be secured, or lack ease of
use.
[0010] One such test is marketed by Celsis International, PLC of
Cambridge, United Kingdom, under the trademark Spotchecka. The
SPOTCHECK.TM. employs a cyclic "comproproportionation" reaction to
detect ATP (see U.S. Pat. No. 6,043,047, issued Mar. 28, 2000, and
PCT International Publication No. WO 00/36139, published Jun. 22,
2000).
[0011] One example of a method for detection of inorganic phosphate
is described by N. Conrath et al, "A novel enzyme sensor for the
determination of inorganic phosphate", Analytica Chimica Acta 309
(195) 47-52 (1995), which is incorporated herein by this reference.
That method, however, requires skilled laboratory personnel, is
time consuming and requires equipment.
[0012] It is an object of this invention is to provide a
broad-spectrum test to rapidly monitor the hygiene of a surface by
detecting a variety of organic and inorganic materials, food
residues and microorganisms.
SUMMARY OF THE INVENTION
[0013] A new and improved user friendly, broad spectrum, test
apparatus, system and method adapted to provide a visual
determination of surface contamination is the object of the current
invention. The purpose of the test is to rapidly--within one
minute--detect the presence of certain biological materials that
are indicators of improper or inadequate sanitation and
cleanliness. Test results are qualitative; a positive result
indicates presence of residue and need to clean.
[0014] The invention comprises a method for the rapid, calorimetric
determination of hygiene monitoring of surface contamination, which
method comprises adding to a sample containing phosphate a
phosphorylase enzyme and a carbohydrate substrate to provide a
reaction product of free glucose and phosphate bound to a
saccharide; and detecting said free glucose in a colorimetric
reaction as a measure of surface contamination. Phosphorylases are
enzymes that help catalyze cleavage of a bond by orthophosphate. In
the case of maltose, maltose phosphorylase cleaves the
carbon-oxygen linkage between glucose molecules to produce glucose
and glucose-1-phosphate. That is, a phosphorylase enzyme such as
maltose phosphorylase, and carbohydrate substrate, such as maltose,
react with phosphate from the sample to produce .alpha.-D-glucose
and .beta.-D-glucose-1-phosphate.
[0015] The invention also comprises a method for the rapid,
colorimetric determination of hygiene monitoring comprising adding
to a sample containing phosphate maltose and maltose phosphorylase
and detecting the amount of glucose or phosphate in a calorimetric
reaction as a measure of surface contamination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a reaction scheme that is utilized for general
hygiene monitoring;
[0017] FIG. 2 is a reaction scheme that may be utilized when
sensitivity to animal tissue is of particular importance;
[0018] FIG. 3A is a schematic view of the VERICLEEN.TM. swab
removed from the test device;
[0019] FIG. 3B is a side perspective, see-through view of the
VERICLEEN.TM. swab;
[0020] FIG. 4 is a side perspective view of the VERICLEEN.TM. test
strip within the optional blister package with the package covering
pulled partially back to expose the wetting solution and
capillary-flow test strip;
[0021] FIG. 5 is a side perspective view of the VERICLEEN.TM. test
strip including a space or gap to prevent diffusion of color
reagents;
[0022] FIG. 6 is a side perspective view of the wetting-agent
dispenser attached to a test strip container; and
[0023] FIG. 7 is perspective view of the VERICLEEN.TM. test
strip.
DESCRIPTION OF THE EMBODIMENTS
[0024] The purpose of the rapid, one-step, assay test for hygiene
monitoring is to detect rapidly (within one minute, preferably
within 2 minutes) the presence of biological material as an
indicator of sanitation and cleanliness. The two materials
detected, within the same single service test, are glucose and
phosphate. Glucose is detected directly. Phosphate is detected
indirectly by the reaction of maltose phosphorylase with maltose to
produce glucose.
[0025] Enzymes are added to the assay system to increase the assay
spectrum by converting biological material to glucose or phosphate.
Materials are stablilized, purified to remove phosphate, adenosine
triphosphate (ATP), adenosine diphosphate (ADP), glucose, and
glucosidase, and placed in a POCKETSWAB.RTM. or lateral-flow test
strip format, both of which are single assay formats that permit an
easy testing format and rapid analysis.
[0026] Inorganic phosphate is ubiquitous in nature. It is involved
in energy metabolism and activation reactions in plants, animals,
and microorganisms. Inorganic phosphate is an important component
in many compounds and, as a major biological mineral, it is present
alone in significant amounts. Phosphates are used extensively in
the food industry, including also as additives in dairy products,
cereals, meats, and soft drinks.
[0027] Similarly, carbohydrates, including sugars, are present in
and on biological materials in significant amounts. For example,
glucose is present in blood, fruits, and honey. Acid phosphatase is
endogenous to certain animal tissue, and alkaline phosphatase is
present in feces. The detection of phosphate, phosphatase, and
glucose are markers of contamination. Other carbohydrates,
including sugars, can be converted to glucose for detection. For
example, lactose, a milk sugar, can be converted to glucose by
.beta.-galactosidase. Sucrose, which is used in processed foods and
is found in cane sugar, fruits, and as invert sugar in processed
foods, can be converted to glucose by sucrase or invertase.
[0028] The test utilizes several reaction pathways for the
detection of biological material. The primary detection pathway
uses maltose and maltose phosphorylase for the detection of
phosphate. Phosphate present in the sample reacts with maltose and
maltose phosphorylase to convert maltose into glucose and
glucose-1-phosphate. The glucose formed is detected
calorimetrically by methods well known in the art. The test also
uses additional pathways to increase sensitivity. These additional
pathways result in increased formation of either or both phosphate
and glucose.
[0029] The test strip consists of nitrocellulose adhesively bound
to a polystyrene backing, with one end having a sample pad
absorbent area in fluid-flow contact with the nitrocellulose, and
the other end having optionally attached to it, or incorporated
into it, a container of a surface-wetting reagent. The container of
surface-wetting agent can also be incorporated into the test strip
packaging. The wetting reagent container can be in the form of, for
example, a burstable pouch or peel-top container.
[0030] Within the test strip are reagents providing biochemical
pathways for the conversion of common surface biological material
to glucose or phosphate. Also located within the test strip are
reagents for the calorimetric detection of glucose. All reagents
are stabilized, purified to remove phosphate, ATP, ADP, glucose,
and glucosidase, and either sprayed onto the lateral-flow test
strip, incorporated into the absorbent pad, or mixed with wetting
solution.
[0031] Also within the test strip, preferably at the end opposite
the sample-absorbing end, there can be included a positive control
area for use in comparison of the color change and to confirm that
adequate sample flow occurred.
[0032] Various embodiments comprise reagents for three (optionally,
four or five or more) separate pathways.
Pathway 1:
[0033] A. Using apyrase to convert ADP and ATP to phosphate;
and
[0034] B. Using maltose phosphorylase to convert phosphate and
maltose to glucose and glucose-1-phosphate.
[0035] Phosphate can exist in free form on a surface or as
phosphate in ADP or ATP. The phosphate component of ATP/ADP can be
liberated by apyrase. Apyrase contains ADPase and ATPase activity
and, therefore, converts ATP or ADP to AMP and phosphate. The
liberated phosphate and the endogenous phosphate are detected by
the reaction of maltose with phosphate and maltose phosphorylase to
produce glucose and glucose-1-phosphate. The glucose formed from
the reaction of maltose with phosphate in the presence of maltose
phosphorylase is detected as described in Pathway 3.
Pathway 2: Sugar Conversion to Glucose
[0036] Carbohydrates, including sugars, are present in and on
biological materials in significant amounts. Many sugars can be
converted to glucose for detection. For example, lactose, a milk
sugar, can be converted to glucose by .beta.-galactosidase.
Sucrose, which is used in processed foods and is found in cane
sugar, fruits, and as invert sugar in processed foods, can be
converted to glucose by sucrase or invertase. The glucose formed by
these reactions is detected as described in Pathway 3.
Pathway 3: Detection of Glucose
[0037] Reactions for the calorimetric detection of glucose are well
known in the art. For example, .beta.-D-glucose+O.sub.2+H.sub.2O
are converted by glucose oxidase to
D-glucono-.delta.-lactone+H.sub.2O.sub.2. The hydrogen peroxide
formed from the above reaction, combined with a peroxidase enzyme,
such as horseradish peroxidase, converts a colorless substrate to a
dye that is readily visible. A mutarotase may be included to
enhance sensitivity by converting .alpha.-D-glucose associated with
phosphorylation to .beta.-D-glucose to react with the glucose
oxidase.
Other Pathways:
[0038] A: Glucose-phosphate, such as glucose-1-phosphate formed by
the reaction of maltose with phosphate and maltose phosphorylase,
Pathway 1 B, or present otherwise in the sample, can be cleaved by
phosphatase, for example, acid phosphatase, to produce both glucose
and phosphate. Providing phosphatase as a test reagent, therefore,
increases test sensitivity.
[0039] B: In certain environments, detection of phosphatase is
important. Glucose-phosphate, for example, glucose-6-phosphate, can
be provide as a test reagent for detection of phosphatase. If
phosphatase is present in the sample, glucose-6-phosphate will be
cleaved to produce glucose and phosphate for detection, as
previously described.
Sensitivity
[0040] The following test results compare Charm Sciences, Inc.'s
POCKETSWAB.RTM., read on both Charm Sciences, Inc.'s LUMINATOR.RTM.
and Firefly instruments, with this new one-step assay test,
referred to as the VERICLEEN.TM. test (VERICLEEN.TM. is a trademark
of Charm Sciences, Inc. of Lawrence, Mass.). The VERICLEEN.TM.
swabs, which do not require a reader, compares favorably to the
POCKETSWAB.RTM., which requires a reader.
Test Ingredients
[0041] Test results described herein were generated using the
following test ingredients incorporated into a single-service
device similar to that described in U.S. Pat. No. 5,827,675, issued
Oct. 27, 1998; and U.S. Pat. No. 5,965,453, issued Oct. 12, 1999
(TEST APPARATUS, SYSTEM ANVD METHOD FOR THE DETECTION OF TEST
SAMPLES). It should be noted that prior to use, all reagents must
be verified as free from glucose and phosphate contamination;
otherwise, they must be purified using dialysis, de-salting, enzyme
treatment, or other appropriate method.
EXAMPLE 1
[0042] Substrate composition: [0043] Maltose (approximately 470
.mu.g) [0044] EHSPT (approximately 125 .mu.g) [0045]
4-aminoantipyrine (approximately 62 .mu.g)
[0046] All 3 reagents are first lyophilized separately in a
stabilizing buffer, then optionally combined in a cellulose tablet
formation.
EXAMPLE 2
[0047] Enzyme composition: [0048] Maltose phosphorylase
(approximately 1.5 units) [0049] Horseradish peroxidase
(approximately 5 units) [0050] Glucose oxidase (approximately 12.5
units) [0051] .beta.-galactosidase (approximately 1 unit) [0052]
Apyrase (approximately 1.7 units)
[0053] All enzymes are lyophilized separately, then optionally
combined in a cellulose tablet formulation.
EXAMPLE 3
[0054] Liquid niblet: [0055] Sterile deionized water with 1 mM
CaCl.sub.2 and preservative.
Comparison of Charm Sciences, Inc.'s VERICLEEN.TM. vs.
POCKETSWAB.RTM.
[0055] Sample Preparation:
[0056] Solids: includes fruits, vegetables, ice cream, meats,
bread, and flour [0057] 20% extract: 2 grams food+8 mL deionized
water; pound with tissue masher for 30 seconds; let solids settle
[0058] prepare all dilutions from the 20% extract in deionized
water
[0059] Liquids: includes orange juice, milk, eggs, and soda [0060]
product "as is" is 100% extract [0061] prepare all dilutions from
the 100% extract in deionized water
[0062] Assay: [0063] 1. Inject 50 .mu.L extract directly into swab;
[0064] 2. Activate swab device by twisting; [0065] 3. For PKS
assay, count immediately on the Firefly analyzer; and [0066] 4. For
VERICLEEN.TM., set time counting up; note time when purple color
appears.
[0067] Blank testing:
[0068] run swab devices without removing swab TABLE-US-00001
POCKETSWAB .RTM. Blanks Firefly RLU Reading VERICLEEN .TM. Blanks 0
color development after 6 minutes 0 color development after 5.5
minutes 0 color development after 6 minutes
[0069] TABLE-US-00002 TABLE 1 Extract POCKETSWAB .RTM. VERICLEEN
.TM. Fruits and Concen- Firefly RLU Result (DNR = did not Vegtables
tration Reading run) Celery 20% 265187 DNR Celery 5% 74256 DNR
Celery 1% 19918 DNR Celery 0.1% 2943 purple in 30 seconds Cucumber
20% 270914 DNR Cucumber 5% 57434 DNR Cucumber 1% 41790 purple in 20
seconds Cucumber 0.1% 2965 purple in 2.5 seconds Carrot 20% 311245
purple in 15 seconds Carrot 5% 67449 purple in 30 seconds Carrot 1%
10318 DNR Carrot 0.1% 205 purple in 60 seconds Lettuce 20% 130230
DNR Lettuce 5% 30816 DNR Lettuce 1% 4689 purple in 30 seconds
Lettuce 0.1% 0 purple in 2 minutes Apple 20% 735903 DNR Apple 5%
162292 DNR Apple 1% 60205 DNR Apple 0.1% 2747 purple in 15 seconds
Orange 5% 3108565 DNR (fresh 1% 872245 DNR squeezed 0.1% 120521 DNR
juice) 0.01% 23387 purple in 40 seconds Orange 5% 3264303 DNR Juice
1% 819838 DNR (pasteurized) 0.1% 145470 DNR Orange Juice 0.01% 4961
purple in (pasteurized) 60 seconds
[0070] TABLE-US-00003 TABLE 2 POCKETSWAB .RTM. VERICLEEN .TM. Dairy
Extract Firefly RLU Result (DNR = did Products Concentration
Reading not run) Raw Milk 5% 50081 DNR Raw Milk 1% 29572 DNR Raw
Milk 0.1% 13732 purple in 45 seconds Raw Milk 0.01% 1547 purple in
4 minutes Pasteurized 50% 213870 purple in Milk 45 seconds
Pasteurized 5% 35594 pale purple in Milk 4 minutes Pasteurized 1%
6074 DNR Milk Pasteurized 0.1% 0 DNR Milk Whole egg 5% 2714 DNR
beaten Whole egg 1% 0 DNR beaten Whole egg 0.1% 0 purple in beaten
30 seconds Whole egg 0.01% 0 purple in beaten 45 seconds Soft serve
20% 47594 purple in ice cream 30 seconds Soft serve 5% 10241 purple
in ice cream 45 seconds Soft serve 1% 0 purple in ice cream 2
minutes Soft serve 0.1% 0 purple in ice cream 3.5 minutes
[0071] TABLE-US-00004 TABLE 3 Extract POCKETSWAB .RTM. VERICLEEN
.TM. Concen- Firefly RLU Result (DNR = did Meats tration Reading
not run) Raw Ground Beef 20% 279489 DNR Raw Ground Beef 5% 31154
DNR Raw Ground Beef 1% 3543 DNR Raw Ground Beef 0.1% 0 purple in 20
seconds Cooked Ground 20% 831281 DNR Beef Cooked Ground 5% 3629780
DNR Beef Cooked Ground 1% 4773347 purple in 30 Beef seconds Cooked
Ground 0.1% 1305347 purple in Beef 4 minutes Raw chicken 20% 457994
DNR breast Raw chicken 5% 43972 DNR breast Raw chicken 1% 0 purple
in Breast 60 seconds Raw chicken 0.1% 0 purple in breast 1.5
seconds Cooked chicken 20% 694045 DNR breast Cooked chicken 5%
3825860 purple in breast 45 seconds Cooked chicken 1% 4457393
Negative at breast 4 minutes Cooked chicken 0.1% 1730583 DNR
breast
[0072] TABLE-US-00005 TABLE 4 POCKETSWAB .RTM. VERICLEEN .TM.
Extract Firefly RLU Result (DNR = did Grains Concentration Reading
not run) White flour 20% DNR White flour 5% purple in 20 seconds
White flour 1% purple in 30 seconds White flour 0.1% purple in 2
minutes White bread 20% DNR White bread 5% DNR White bread 1% DNR
White bread 0.1% purple in 50 seconds
[0073] TABLE-US-00006 TABLE 5 POCKETSWAB .RTM. VERICLEEN .TM.
Extract Firefly RLU Result (DNR = did Soft Drinks Concentration
Reading not run) Coke 5% 0 DNR Coke 1% purple in 30 seconds Coke
0.1% purple in 60 seconds Coke 0.01% pale purple at 4 minutes
Sprite 5% 0 purple in 20 seconds Sprite 1% DNR Sprite 0.1% purple
in 60 seconds Sprite 0.01% DNR
[0074] The invention also comprises a method for increasing the
above-described hygiene monitoring test sensitivity to phosphate,
through a cycling reaction. Phosphatase, for example acid
phosphatase, which is included as a test reagent and may be present
in the sample, cleaves bound phosphate, such as glucose-1-phosphate
and glucose-6-phosphate into its component, glucose and phosphate
which both will be detected within the assay system.
Glucose-1-phosphate and glucose-6-phosphate may be from the sample
and also, in the case of glucose-1-phosphate, generated as a
reaction product of the carbohydrate substrate with phosphate in
the presence of an appropriate enzyme as previously described. The
reaction product glucose-1-phosphate is broken down by phosphatase
to produce more glucose for the color change reaction and more
phosphate to combine with the carbohydrate substrate as previously
described.
[0075] The invention also comprises a way to release phosphate and
glucose from various commonly found surface contaminants such as
adenosine triphosphate (ATP), adenosine diphosphate (ADP),
biofilms, carbohydrates, microbes, and phosphatase. A method for
releasing phosphate and glucose from such contaminants includes
using enzymes such as phosphatase, for example apyrase and glucose
phosphatase, and enzymes, such as hydrolytic enzymes, to breakdown
carbohydrates comprised of more than one saccharide (complex
carbohydrates) to release the glucose component, for example the
breakdown of lactose by .beta.-galactosidase, the breakdown of
sucrose by invertase and the breakdown of starch by amylase. A
method for releasing various molecules such as ATP, ADP and glucose
from microbes includes the use of lysing reagents. A mutarotase may
be included to enhance sensitivity by converting .alpha.-D-glucose
associated with phosphorylation to .beta.-D-glucose to react with
the glucose oxidase.
[0076] The presence of acid phosphatase on a surface is indicative
of contamination with certain animal tissue. A separate embodiment
of the invention includes additional way to detect phosphatase.
Such methods may be included within the assay system in
circumstances where detection of phosphatase is relatively more
important than increasing test sensitivity to phosphate, for
example when detection of animal tissue, such as muscle or blood,
is relatively more important compared to the general increase
sensitivity to phosphate. However, the above-described methods for
increasing test sensitivity to phosphate, by providing phosphatase
as a reagent, cannot practically be combined with the methods for
detecting phosphatase in a sample. Methods for detecting
phosphatase include providing glucose-6-phosphate or
glucose-1-phosphate as a test reagent. Phosphatase present in the
sample will cleave, for example glucose-6-phosphate, into is
components glucose and phosphate.
[0077] The invention involves a method for the rapid, colorimetric
determination of hygiene monitoring of surface contamination, which
method comprises: adding a phosphorylase enzyme and carbohydrate
substrate to a sample containing phosphate to create a reaction
product of free glucose and phosphate bound to a saccharide;
cleaving free phosphate from said bound phosphate; cleaving free
glucose from complex carbohydrates; releasing or extracting
intracellular phosphate; and changing the interconversion of
.alpha.-D-glucose to .beta.-D-glucose, wherein free glucose is
detected in a calorimetric reaction as a measure of surface
contamination.
[0078] The invention also involves a method for the rapid,
colorimetric determination of hygiene monitoring of surface
contamination including contamination with phosphate and
phosphatase, which method comprises: adding a phosphorylase enzyme
and a carbohydrate substrate to a sample containing phosphate to
create a reaction product of free glucose and phosphate bound to a
saccharide; cleaving free phosphate from bound phosphate; cleaving
free glucose from complex carbohydrates; releasing intracellular
phosphate; and catalyzing the interconversion of .alpha.-D-glucose
to .beta.-D-glucose, wherein free glucose is detected in a
calorimetric reaction as a measure of surface contamination.
[0079] The invention also comprises a device for rapid,
colorimetric hygiene monitoring, which test device comprises: a
capillary-flow test strip having two ends, the test strip includes
at a first end, a liquid sample absorbing material; reagents for
conversion of biological material to glucose, such as by providing
multiple biochemical pathways; and reagents for the calorimetric
detection of glucose, wherein a liquid sample from a material is
absorbed onto the first end and flows by capillary action to
contact reagents providing biochemical pathways for conversion of
biological material to glucose and calorimetric detection of
glucose.
[0080] The invention also comprises a device for rapid,
calorimetric hygiene monitoring having one or more reaction zones,
included on or in a capillary membrane, comprising reagents for
conversion of biological materials to glucose and phosphate and
reagents which, in the presence of phosphate and an appropriate
enzyme, convert certain biological materials to glucose; and
reagents for the calorimetric detection of glucose, wherein a
liquid sample from a material is absorbed onto the first end and
flows by capillary action through the test strip containing
reagents, which produces a visually detectable readout in the
presence of glucose.
[0081] The invention also comprises a device for hygiene
monitoring, by detecting biological material in a test sample from
or on a material, which test device comprises: a swab to collect
the test sample and a niblet comprising separate, swab-puncturable,
membrane compartments for one or more reagents and a solution; an
enzyme reagent composition, preferably in tablet form, comprising
glucose oxidase, horseradish peroxidase, maltose phosphorylase,
apyrase, .beta.-galactosidase and phosphatase; a substrate reagent
composition, preferably in tablet form, comprising
4-aminoantipyrene, mutarotase, EHSPT (the Trinder's reagent TOOS),
and maltose; and a liquid reagent comprising a buffering solution
and lysing solution, wherein the sample is contacted sequentially
with the liquid reagent, enzyme reagent, and substrate reagent and
a color change or lack thereof is observed as a measure of
hygiene.
[0082] The test apparatus of certain embodiments of the invention
is in the format of the POCKETSWAB.RTM., which is described in U.S.
Pat. No. 5,965,453, issued Oct. 12, 1999 (TEST APPARATUS, SYSTEM
AND METHOD FOR THE DETECTION OF TEST SAMPLES); U.S. Pat. No.
5,985,675, issued Nov. 16, 1999 (TEST DEVICE FOR DETECTION OF AN
ANALYTE); and U.S. Pat. No. 6,180,395, issued Jan. 30, 2001
(REAGENT CHAMBER FOR TEST APPARATUS AND TEST APPARATUS), which are
incorporated herein in their entirety. In these embodiments, the
apparatus incorporates a foam-tipped, or other absorbent-type swab
or wand for sample uptake from the surface to be monitored. The
swab may be premoistened with a wetting solution.
[0083] After sample uptake onto the swab, the swab is used to
puncture a series of "niblets" releasing and activating the
necessary reagents. The term "niblet" refers to a reagent chamber
in the form of a cylinder containing reagents and sealed on both
ends with a probe puncturable membrane.
[0084] In certain embodiments, the first niblet to contact the swab
includes cofactors and buffering compounds to optimize subsequent
reactions and antimicrobial or antifungal substance to prevent
contamination. In other embodiments, the first niblet may
additionally contain bacterial lysing reagents.
[0085] After the swab is contacted with the first reagent niblet,
the swab is contacted with the material in the second niblet by,
preferably, puncturing the membranes separating the first and
second niblet, thereby causing reagents from the first niblet to
flow into and mix with reagents in the second niblet. The second
niblet contains one or more reagents, preferably tablets,
containing enzymes, substrates, and biochemicals. Certain
embodiments include two tablets in the second niblet, generally
referred to as the "substrate tablet" and the "enzyme tablet."
[0086] In some embodiments, the substrate reagent composition
includes maltose, glucose-phosphates, such as glucose-1-phosphate
or glucose-6-phosphate, TOOS and 4-aminoantipyrine, and the enzyme
reagent includes maltose phosphorylase, horseradish peroxidase,
glucose oxidase, mutarotase, .beta.-galactosidase and apyrase. In
other embodiments, enzymes for converting other carbohydrates to
glucose, for example, amylase, sucrase (invertase) may be
included.
[0087] In another embodiment, glucose-6-phosphate is removed from
the substrate reagent. Such an embodiment will be less sensitive to
sample phosphatase. Instead, phosphatase is added to the enzyme
reagent or substrate reagent. The added phosphatase will cleave the
glucose-1-phosphate reaction product of maltose and phosphate to
produce additional phosphate and glucose, thereby enhancing test
sensitivity to phosphate through the additional cycling of the
phosphate group, from the glucose-1-phosphate, to be combined with
maltose and maltose phosphorylase. It will be appreciated that
although the reagents of particular embodiments are described as
"tablets" or "liquid," the reagents can be used in, and applied to,
the test device in a variety of forms, which forms are prepared
separately or in combination, including solid, liquid, powder,
freeze-dried emulsion, suspension, tablet, or any other form known
to those skilled in the art.
[0088] The test apparatus of certain other embodiments of the
invention is in the format of a capillary-flow test strip. The
strip consists of a capillary-flow capillary membrane, for example,
nitrocellulose membrane, adhesively bound to a backing, for example
a polystyrene backing, with one end having a sample pad absorbent
area in fluid flow contact with the membrane. Optionally, at the
sample pad end, or the other end, the strip could have attached, or
incorporated into the packaging, a container of a surface wetting
reagent. The container of surface-wetting agent may include a
wetting solution. The wetting solution container can be in the form
of, for example, a burstable pouch or peel-top container within the
test strip packaging. Alternatively, the surface-wetting solution
can be included in a bulk container, for example, a squeezable,
plastic, dispenser bottle or spray bottle, which is attached at the
end opposite the dispensing end, to a container within which
capillary-flow strips are placed. The wetting solution should,
optimally, be pH neutral, food compatible and non-interfering. The
capillary-flow strip container is formed of a light-blocking
material to protect the test strips. Applied to the test strip
capillary membrane are reagents for the production of glucose and
phosphate from common surface biological material and reagents for
the calorimetric detection of glucose.
[0089] In one example of the capillary-flow strip, reagents are
provided in two zones. The two reagent zones are applied to the
membrane portion of the test strip in fluid flow contact with the
absorbent pad. Optionally, the reagents can be applied by spraying
onto the strip. In one embodiment, the first reagent zone contains
a carbohydrate substrate, for example, maltose and
glucose-6-phosphate. In an alternative embodiment, the first
reagent zone contains the carbohydrate substrate and a phosphatase,
for example, acid phosphatase. The second reagent zone contains
reagents for the calorimetric detection of glucose, for example,
TOOS, glucose oxidase, mutarotase, horseradish peroxidase and
4-aminoantipyrene. Also included within the second reagent zone are
enzymes, such as maltose phosphorylase and apyrase and one or more
hydrolytic enzymes, such as .beta.-galactosidase.
[0090] The reagents in this device are able to flow in the membrane
reacting with the sample and each other. Sufficient capillary-flow
space is included beyond the second reagent zone for color reagents
to change color in the presence of a target material and provide an
easily read, visible result. The space beyond the second reagent
zone is sufficiently small to create a defined area for color
change to occur. Color change begins at the end of the capillary
membrane and spreads back. Providing space between the second
reagent zone and the end of the capillary membrane permits a wider,
more visible line to develop. At the end of the capillary membrane
is an air gap to prevent reagent flow beyond the end of said
membrane, a wetting solution for solubilizing surface food residue
and penetrating biofilm.
[0091] All reagents are stabilized, purified to remove phosphates,
glucose and maltose degrading enzymes, for example, glucosidase,
and either sprayed onto the capillary-flow membrane or incorporated
into the absorbent pad.
[0092] The test apparatus system and method will be described for
the purposes of illustration only in connection with a series of
illustrative test apparatus and test method employing various test
apparatus. However, it is recognized that those persons skilled in
the art may make various modifications, changes, additions, and
improvements to the test apparatus, system and methods without
departing from the spirit and scope of the invention.
EXAMPLE 4
[0093] The lateral or capillary-flow strip consists of a
nitrocellulose membrane onto which two regions, zones, or lines of
reagents have been applied using appropriate manufacturing
equipment, such as that made by BioDot, Inc. Optionally, the
membrane is overlapped with an absorbent cellulose paper pad. One
region consists of all enzymes needed for the color reaction, the
enzymes for release of phosphate and glucose, plus the two
substrates necessary for color development. Prior to use, all
enzymes must be purified to remove any sugar or phosphate
contamination, for example, by desalting. The second line contains
a preparation of glucose-free maltose and purified acid
phosphatase. The nitrocellulose is mounted onto a polystyrene
backing material and, optionally, packaged into a plastic,
blister-type device. If packaged into a blister-type device the
device, optionally, contains a bubble to which surface wetting
agent is added; the bubble may be sealed with peelable foil.
[0094] The reagents applied to the capillary membrane are the same
as those used as reagents or tablets for the swab-type assay.
However, the amount of each component required per test is
significantly lower for all reagents. The approximate amount of
each reagent and its corresponding amount used in the swab test are
listed in Table 6, in an embodiment including glucose-6-phosphate
and Table 7 in an embodiment including acid phosphatase.
[0095] Results generated with the embodiment described in Table 6
are listed in Appendix 1. TABLE-US-00007 TABLE 6 Amount per Test in
Capillary Flow Amount per Test Reagent Membrane in Swab Test TOOS
930 ng 125 mg 4-aminoantipyrine 460 ng 63 mg Maltose 2 mg 470 mg
Maltose phosphorylase 0.06 units 1.5 units Glucose oxidase 0.5
units 12.5 units Horseradish peroxidase 0.2 units 5 units
.beta.-galactosidase 0.04 units 0.8 units Glucose-6-phosphate 10 mg
100 mg Apyrase 0.2 units 1.5 units
Appendix 1
Test Data
[0096] TABLE-US-00008 I. Assay in which solution of food product is
allowed to air dry onto countertop then rehydrated with wetting
solution and wiped with test strip Time to Color Food Concentration
Change (s) Beef 2% 16, 15 Chicken 5% 10, 7 Fish 5% 20, 23 Ketchup
1% 3, 2 Milk 10% 35, 37 Orange juice 2% 4, 3 Egg 2% 4, 5
[0097] TABLE-US-00009 II. Assay in which wet solution of food
product is wiped from plastic surface with test strip Time to Color
Food Concentration Change (s) Beef 2% 8, 10 Chicken 5% 8, 7 Fish 5%
9, 8 Ketchup 1% 2, 2 Milk 10% 30, 32 Orange juice 2% 2, 3 Egg 2% 5,
5
[0098] TABLE-US-00010 TABLE 7 Amount per Test in Capillary-Flow
Amount per Test Component Membrane in Swab Test Glucose oxidase 0.7
units 12.5 units Horseradish peroxidase 0.3 units 5 units
.beta.-galactosidase 0.06 units 1 units Maltose phosphorylase 0.09
units 3 units Apyrase 0.3 units 5 units Acid phosphatase 0.01 units
0.1 units Maltose 4 mg 1400 mg 4-aminoantipyrene 700 ng 63 mg
N-ethyl-N-(2-hydroxy- 1400 ng 125 mg 3-sulfopropyl)-m-
toluidine
[0099] Materials are stabilized, purified to remove phosphate, ATP,
ADP, glucose and glucosidase, and placed in a POCKETSWAB.RTM. or
capillary-flow membrane format, both of which are single assay
formats that permit an easy testing and rapid analysis.
[0100] FIG. 1 illustrates the general use reaction scheme of the
invention, which is particularly useful when testing for a wide
range of contaminants. Maltose reacts with phosphate from the
sample in the presence of maltose phosphorylase to form
.alpha.-D-glucose and .beta.-D-glucose-1-phosphate.
.beta.-D-glucose forms color when combined with certain well-known
reagents. .alpha.-D-glucose naturally converts to .beta.-D-glucose.
The rate of natural conversion can be enhanced by mutarotase. The
.beta.-D-glucose-1-phosphate in the presence of acid phosphatase
(as a test reagent) is broken down into phosphate and
.beta.-D-glucose (Phosphatase from the sample will also break down
.beta.-D-glucose-1-phosphate.). The glucose enters into the color
reaction amplifying the color from the first glucose, and the
phosphate reacts with more maltose (a test reagent) to generate
more glucose and, ultimately, more phosphate and more color until
the maltose reagent is depleted, or the reaction is otherwise
inhibited. The above-described cycling of phosphate produces a
sensitive test for phosphate in the sample. The reaction scheme
also includes examples of conversion of common biological material
to glucose and phosphate. Lysing reagents release ATP and ADP from
microbes and apyrase cleaves the phosphate. Carbohydrates are
hydrolized to release glucose for detection.
[0101] FIG. 2 illustrates the reaction scheme particularly useful
for detecting contamination from raw or partially-cooked meat
products. Glucose-6-phosphate (or alternatively
glucose-1-phosphate) is added as a reagent to include an additional
source of phosphate and glucose when acid phosphatase is present in
the sample. In the embodiment represented by said reaction scheme,
acid phosphatase is not a reagent and is, instead, supplied from
the sample. If acid phosphatase is not present, the
glucose-6-phosphate does not enter the reaction, and only phosphate
from the sample generates glucose. Color is generated by the
glucose.
[0102] FIGS. 3A & B illustrate the invention in the format of
the swab-type device. In use of the swab-type device of the
invention, the swab 1 is removed from the body 3, by gripping the
swab handle 2, and a 4''.times.4'' surface, for example, a food
contact surface, is swabbed using the pre-moistened swab 1. The
swab 1 is then reinserted into the body 3 and screwed
longitudinally through the covering 9 of the microtube test unit 4
and through the covering 10 of the liquid reagent niblet 5 and into
the tablet niblet 6 containing an enzyme tablet 7 and a substrate
tablet 8. The liquid released to the bottom of the microtube test
unit 4 turns purple within 60 seconds if the surface is "dirty,"
for example, it has food residue contamination.
[0103] FIG. 4 illustrates the invention in the capillary-flow
strip-type device packaged within a blister package. In use of the
capillary-flow strip test-type device of the invention, the blister
package cover 12 is peeled back, and the surface-wetting solution
is released from the container 11 onto the surface to be monitored.
The user continues to peel back the covering 12 to expose the test
strip 13.
[0104] FIG. 5 is a side perspective view of the VERICLEEN.TM. test
strip. While handling area 14 the absorbent pad 15 is used to swab
the surface to be monitored. The sample is absorbed onto sample pad
15 and flows by capillary action through the first reagent zone 19,
to the second reagent zone 20 (Reagent zones 19 and 20 are depicted
for illustration.). In the preferred embodiment, reagent zones 19
and 20 are slightly visible or invisible. Sample flow stops at the
end of the capillary membrane 18. Positive results are reflected in
color change zone 17. Color change begins to form at the end of the
capillary membrane 18 and spreads back. A gap 21 is included
sufficiently wide to prevent unwanted diffusion of color into the
cover 22 of the backing 23, which cover 22 and backing 23 combined
make up the handling area 14. Line development at the top of the
capillary membrane, within approximately one minute, indicates
presence of residue and need to clean. All valid tests will change
color after approximately five minutes. Reagent zones 19 and 20
contain reagents depicted in the reaction scheme of either FIG. 1
or FIG. 2, depending on the requirements of the user.
[0105] FIG. 6 is a side perspective view of the wetting-agent
dispenser 24 attached to a light-blocking test strip container
25.
[0106] FIG. 7 illustrates an embodiment of the present invention
containing a strip of nitrocellulose adhesively bound to a
polystyrene support. Various reagents are sprayed and dried onto
the nitrocellulose in a series of discreet zones. FIG. 7 contains
seven such spaced-apart zones as follows:
[0107] Zone 1, 48, is comprised of apyrase and
.beta.-galactosidase. Apyrase cleaves any ATP/ADP liberating
phosphate that will flow in the solution along the test strip to
Zone 2. .beta.-galactosidase converts lactose to glucose. Zone 2,
46, comprises maltose for reaction with phosphate, in the presence
of the maltose phosphorylase to produce glucose. Zone 3, 44, is
comprised of maltose phosphorylase. Zone 4, 42, is comprised of
glucose oxidase for oxidation of glucose in a reaction that yields
peroxide. Zone 5, 40, is comprised of EHSPT
[N-ethyl-N-(2-hydroxy-3-sulfo-propyl)-n-toluidine], commonly
referred to as TOOS; Zone 6, 30, is comprised of 4-aminoantipyrine;
and Zone 7, 36, is comprised of horseradish peroxidase. Zones 5, 6,
and 7, 40, 30, and 36, respectively comprise reagents which, in the
presence of peroxide, produce a color precipitate which can be
observed visually.
[0108] It is also possible to combine the seven zones described
above into fewer zones, for example, three zones, containing
multiple reagents per zone.
[0109] In operation, the wetting reagent is released onto the
surface to be tested or absorbed onto the absorbent pad 15. Holding
the strip at the finger-hold area 32, the sample pad 15 is used to
swab the surface to be tested. Wetting the surface will suspend in
the liquid some of the material to be detected. Material to be
detected will be absorbed by the strip upon swabbing the wet
surface. The liquid absorbed onto the strip will then flow
laterally on the strip. Upon contact with a fluid-analyte, the
reactants in the zones are resolubilized and react with the
analyte(s) in the sample as described above. Color formation in the
horseradish peroxidase 36 reaction zone indicates a positive
sample, while no color indicates a clean sample.
* * * * *