U.S. patent application number 12/618343 was filed with the patent office on 2010-05-20 for apparatus and method for detecting glycol.
Invention is credited to Melanie Margarete Hoehl, Peter James Lu, Alexander H. Slocum.
Application Number | 20100124758 12/618343 |
Document ID | / |
Family ID | 42170737 |
Filed Date | 2010-05-20 |
United States Patent
Application |
20100124758 |
Kind Code |
A1 |
Hoehl; Melanie Margarete ;
et al. |
May 20, 2010 |
APPARATUS AND METHOD FOR DETECTING GLYCOL
Abstract
A method and apparatus are provided for detecting contaminants,
such as ethylene glycol and diethylene glycol, in various
materials, including household products, and medicines. The
contaminants can be detected using enzyme assays that produce
measurable changes in light absorption and/or light
fluorescence.
Inventors: |
Hoehl; Melanie Margarete;
(Cambridge, MA) ; Lu; Peter James; (Cambridge,
MA) ; Slocum; Alexander H.; (Bow, NH) |
Correspondence
Address: |
Grossman, Tucker, Perreault & Pfleger, PLLC
55 South Commercial Street
Manchester
NH
03101
US
|
Family ID: |
42170737 |
Appl. No.: |
12/618343 |
Filed: |
November 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61199289 |
Nov 14, 2008 |
|
|
|
Current U.S.
Class: |
435/26 ;
250/459.1; 356/432; 356/51; 435/28; 435/288.7 |
Current CPC
Class: |
G01N 21/643 20130101;
G01N 21/78 20130101; G01N 21/33 20130101; G01N 2333/904 20130101;
C12Q 1/32 20130101 |
Class at
Publication: |
435/26 ; 435/28;
435/288.7; 250/459.1; 356/51; 356/432 |
International
Class: |
C12Q 1/32 20060101
C12Q001/32; C12Q 1/28 20060101 C12Q001/28; C12M 1/34 20060101
C12M001/34; G01N 21/64 20060101 G01N021/64 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in part with government support
under an award from CIMIT, whose parent award is from the
Department of Defense U.S. Army Medical Research and Material
Command. The cooperative agreement number is W81XWH-07-2-0011. The
Government may have certain rights in the invention.
Claims
1. A method of detecting at least one of ethylene glycol and
diethylene glycol in a sample suspected of containing glycol, the
method comprising: reacting a glycol with NAD+ in the presence of
an alcohol dehydrogenase to produce NADH; oxidizing NADH with an
oxidase to produce hydrogen peroxide; oxidizing a fluorogenic
substrate in the presence of the hydrogen peroxide and a peroxidase
to convert the fluorogenic substrate to a fluorescent form;
irradiating the sample at a first wavelength; detecting light
emission at a second wavelength; and providing a signal
corresponding to the amount of light detected.
2. The method of claim 1 wherein reacting a glycol includes
generating an aldehyde.
3. The method of claim 1 wherein the alcohol dehydrogenase is a
yeast alcohol dehydrogenase.
4. The method of claim 1 wherein oxidizing a fluorogenic substrate
includes converting N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red)
or Amplex Ultrared into its fluorescent form.
5. The method of claim 1 wherein the glycol is reacted with the
NAD+ in an alkaline environment.
6. The method of claim 1 wherein the glycol is reacted with the
NAD+ at a pH of greater than or equal to 7.5.
7. The method of claim 1 wherein the glycol is reacted with the
NAD+ at a pH between 7.3 and 9.
8. The method of claim 1 wherein the glycol is reacted with the
NAD+ in a Tris-HCl buffer having a pH of about 7.8.
9. The method of claim 1 wherein the glycol is reacted with the
NAD+ in a buffer selected from the group consisting of Tris,
bicine, Tris Base HCl, bicine NaOH, alkaline pH "Good's," and
phosphate buffers.
10. The method of claim 1 further comprising the step of fitting
the signal to V(t)=.beta. exp(t/.tau.)+V0.
11. The method of claim 10 further comprising the step of
normalizing a time constant for the sample by a time constant for
pure glycol.
12. A method of detecting ethylene glycol and diethylene glycol in
a sample suspected of containing glycol, the method comprising:
reacting the sample with a coenzyme in the presence of an alcohol
dehydrogenase to form a measurement solution; electroluminescently
generating a single wavelength ultraviolet light; illuminating the
measurement solution with the single wavelength ultraviolet light;
detecting ultraviolet light transmitted through the measurement
solution; and producing a signal corresponding to the amount of
light detected.
13. The method of claim 12 wherein the signal comprises a voltage
signal.
14. The method of claim 13 comprising normalizing the voltage
signal by dividing the voltage signal by a second voltage signal
recorded at time equals zero.
15. The method of claim 14 comprising fitting the normalized
voltage signal to V(t)=1-a*exp(b*t).
16. The method of claim 15 comprising determining an initial slope
of the normalized voltage signal, -dV/dt, at time equals zero.
17. A device comprising: first and second cuvette spaces, each
cuvette space comprising; a single wavelength light source
constructed and arranged to illuminate at least a portion of the
cuvette space; a second light source at a wavelength different from
the first, the second light source constructed and arranged to
illuminate at least a portion of the cuvette space; a light
detector positioned to detect light transmitted from the second
light source through the cuvette space; and a fluorescence detector
positioned to receive light emitted from the cuvette space at a
wavelength different than that emitted from either light
source.
18. The device of claim 17 wherein the device is powered by a
portable battery.
19. The device of claim 17 wherein the device is capable of
measuring fluorescence simultaneously in two separate samples.
20. A method of detecting a contaminant in a sample comprising:
intermittently generating an electroluminescent ultraviolet light;
intermittently generating an electroluminescent visible light;
detecting a quantity of ultraviolet light transmitted through the
sample; detecting a quantity of light fluoresced from the sample at
a wavelength different than that of the ultraviolet light and the
visible light; and determining the concentration of the contaminant
using both the amount of light transmitted and the amount of light
fluoresced.
21. The method of claim 20 comprising repeating steps one and two
at least twice and extinguishing the ultraviolet light prior to
generating the visible light and extinguishing the visible light
prior to generating the ultraviolet light.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/199,289 filed on Nov. 14, 2008, the
contents of which are hereby incorporated by reference herein.
BACKGROUND
[0003] 1. Field of Invention
[0004] This invention relates to a method and apparatus for
detecting contaminants and specifically to a method and apparatus
for providing an assay for detecting glycols in consumer
products.
[0005] 2. Discussion of Related Art
[0006] Contamination of various products and materials can cause
serious injuries to people and property. For example, contamination
of common household products and medicines by poisons such as
ethylene glycol (EG) and diethylene glycol (DEG) has killed
thousands worldwide in recent years. In addition, contamination of
process materials, such as the boiler and feed water used in
nuclear reactors can cause corrosion and premature failure of
expensive machinery. It is therefore desirable to be able to detect
contaminants, such as EG and DEG, in various materials before they
can cause harm.
SUMMARY
[0007] The invention provides a method and apparatus for
determining the presence of and/or amount of one or more
contaminants in a test sample.
[0008] In one embodiment, a method of detecting at least one of
ethylene glycol and diethylene glycol in a sample is provided that
includes reacting a glycol with NAD+ in the presence of an alcohol
dehydrogenase to produce NADH, oxidizing NADH with an oxidase to
produce hydrogen peroxide, oxidizing a fluorogenic substrate in the
presence of the hydrogen peroxide and a peroxidase to convert the
dye to a fluorescent form, irradiating the sample at first
wavelength, detecting light emission at a second wavelength, and
providing a signal corresponding to the amount of light
detected.
[0009] In another embodiment, a method of detecting ethylene glycol
and diethylene glycol is provided that includes reacting the sample
with a coenzyme in the presence of an alcohol dehydrogenase to form
a measurement solution, electroluminescently generating a single
wavelength ultraviolet light, illuminating the measurement solution
with the single wavelength ultraviolet light, detecting ultraviolet
light transmitted through the measurement solution, and producing a
signal corresponding to the amount of light detected.
[0010] In another embodiment, a device is provided comprising first
and second cuvette spaces, each cuvette space comprising a single
wavelength light source constructed and arranged to illuminate at
least a portion of the cuvette space, a second light source at a
wavelength different from the first, the second light source
constructed and arranged to illuminate at least a portion of the
cuvette space, a light detector positioned to detect light
transmitted from the second light source through the cuvette space,
and a fluorescence detector positioned to receive light emitted
from the cuvette space at a wavelength different than that emitted
from either light source.
[0011] In another embodiment, a method of detecting a contaminant
in a sample is provided that includes intermittently generating an
electroluminescent ultraviolet light, intermittently generating an
electroluminescent visible light, detecting a quantity of
ultraviolet light transmitted through the sample, detecting a
quantity of light fluoresced from the sample at a wavelength
different than that of the ultraviolet light and the visible light,
and determining the concentration of the contaminant using both the
amount of light transmitted and the amount of light fluoresced.
[0012] The methods and apparatuses disclosed herein may include one
or more of a number of different features. For example, any of the
following elements could also be implemented into embodiments of
the invention: generating an aldehyde; the alcohol dehydrogenase is
a yeast alcohol dehydrogenase; oxidizing a fluorogenic substrate
includes converting N-acetyl-3,7-dihydroxyphenoxazine (AMPLEX
RED.RTM.) or AMPLEX ULTRARED.RTM. into its fluorescent form; glycol
is reacted with the NAD+ in an alkaline environment; glycol is
reacted with the NAD+ at a pH of greater than or equal to 7.5;
glycol is reacted with the NAD+ at a pH between 7.3 and 9; glycol
is reacted with the NAD+ in a Tris-HCl buffer having a pH of about
7.8; glycol is reacted with the NAD+ in a buffer selected from the
group consisting of Tris, bicine, Tris Base HCl, bicine NaOH,
alkaline pH "Good's," and phosphate buffers; the step of fitting
the signal to V(t)=.beta. exp(t/.tau.)+V0; the step of normalizing
a time constant for the sample by a time constant for pure glycol;
the signal comprises a voltage signal; normalizing the voltage
signal by dividing the voltage signal by a second voltage signal
recorded at time equals zero; fitting the normalized voltage signal
to V(t)=1-a*exp(b*t); determining an initial slope of the
normalized voltage signal, -dV/dt, at time equals zero; the device
is powered by a portable battery; the device is capable of
measuring fluorescence simultaneously in two separate samples;
repeating steps one and two at least twice and extinguishing the
ultraviolet light prior to generating the visible light and
extinguishing the visible light prior to generating the ultraviolet
light.
[0013] Other advantages, novel features, and objects of the
invention will become apparent from the following detailed
description of the invention when considered in conjunction with
the accompanying drawings. For purposes of clarity, not every
component is labeled in the drawings, nor is every component of
each embodiment of the invention shown where illustration is not
necessary to allow those of ordinary skill in the art to understand
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 illustrates an enzyme reaction pathway used in one
embodiment of the invention.
[0015] FIG. 2 shows an enzyme reaction pathway used in one
embodiment of the invention.
[0016] FIG. 3 shows an absorbance method according to one
embodiment of the invention.
[0017] FIG. 4 shows the reactions and enzyme pathways for one
embodiment of a fluorescent method.
[0018] FIG. 5 shows a fluorescence method according to one
embodiment of the invention.
[0019] FIG. 6a is a plot of voltage versus time according to one
embodiment of the invention.
[0020] FIG. 6b is a plot of normalized enzyme activity versus
ethylene glycol concentration according to one embodiment of the
invention.
[0021] FIG. 7a is a plot of voltage versus time according to one
embodiment of the invention.
[0022] FIG. 7b is a plot of normalized enzyme activity versus
diethylene glycol concentration according to one embodiment of the
invention.
[0023] FIG. 8 is a schematic of a detector according to one
embodiment of the invention.
[0024] FIG. 9 is a circuit diagram of a detector according to one
embodiment of the invention.
[0025] FIG. 10 shows an arrangement of light sources and light
detectors according to one embodiment of the invention.
[0026] FIG. 11 is an isometric view of a measurement device
according to one embodiment of the invention.
[0027] FIG. 12a is a side view of a measurement device according to
one embodiment of the invention.
[0028] FIG. 12b is a section view of a measurement device according
to one embodiment of the invention.
[0029] FIG. 13 is a bottom isometric view of a measurement device
according to one embodiment of the invention.
[0030] FIG. 14 is a bottom isometric view of a measurement device
according to one embodiment of the invention.
DETAILED DESCRIPTION
[0031] Contamination of household and consumer products with
poisons such as ethylene glycol (EG) and/or diethylene glycol (DEG)
is a lethal public health hazard that episodically kills hundreds
to thousands at a time. If not detected and treated promptly,
ingestion of even a small amount can result in central nervous
system depression, cardiopulmonary compromise, and kidney failure.
This contamination has led to several mass-poisonings around the
world in the past few decades.
[0032] At present there is no simple, specific method to detect the
relevant levels of contaminants such as EG and DEG on an industrial
scale, particularly in third-world countries that may be most
vulnerable to contaminated goods and have the health systems least
capable of responding. Standard general test methods, such as gas
chromatography or chromatography/mass spectrometry, are expensive,
can be slow, have specific power requirements and require
specially-trained staff, so they are rarely deployed even in
developed countries for identifying contamination in commercial
products. Described herein are several devices and procedures that
can provide reliable, robust and/or inexpensive tests to detect
contaminants, such as EG and DEG, in a wide range of household and
other materials.
[0033] "Detect" or "detecting" means to determine the presence or
amount of a target compound or class of compounds. For example,
"detecting" EG in a sample can mean identifying the presence of EG
and/or a threshold level of EG in the sample and/or determining the
quantitative amount or concentration of EG present in the
sample.
[0034] "Single wavelength" means 80% of output falls within a 20 nm
range. For example, a "single wavelength" light can have 80% of its
output fall between 350 and 370 nm.
[0035] "Electroluminescently generating" means generating light
with an electroluminescent device such as a light-emitting diode
(LED) or a laser.
[0036] "Sample" includes any substance that may contain a
contaminant and/or a species of interest.
[0037] This invention provides methods and devices for detecting
one or more contaminants in a substrate. The methods and devices
may be based on enzyme assays that produce measurable changes in
light absorption and/or light fluorescence according to the
concentration of contaminants present. For example, in one aspect,
an absorbance method is provided that uses a kinetic assay to
produce light absorbance changes that vary with contaminant
concentration. The light absorbance changes may be measured using a
low-cost, single-wavelength device. In another aspect, a
fluorescence method is provided that uses a kinetic assay to
produce light fluorescence changes. The light fluorescence changes
may be determined using a low-cost, single-wavelength device. Any
of these methods and devices may be provided in kit form that
includes, for example, a package, any combination of the reagents
disclosed herein, sample preparation materials, reaction vessels
and/or cuvettes, a detection device and instructions for use.
Reagents may be in a stabilized form and may be sealed in capsules
or ampoules.
[0038] In one set of embodiments, the substrate may be any type of
material, including: liquids, gels, sols, suspensions, foams,
emulsions, and dispersions. In a further set of embodiments, the
substrate may be any type of household product or industrial
material. Among household products, the substrate may be, for
example, any one or combination of: medicine, food, an alcoholic
beverage, a non-alcoholic beverage, lotion, cleaning agent, air
freshener, or any other household product that may come in contact
with humans or other living things.
[0039] In another embodiment, the substrate tested may have any
rheology, density, and thermal properties. For example, the
substrate may have Newtonian or non-Newtonian rheology. The
substrate may also have any specific gravity that is typical of
industrial materials and household products. In a typical
embodiment, the specific gravity will be about 1.0. Regarding
thermal properties, the substrate may be thermally conductive or
thermally insulating.
[0040] In one embodiment, the contaminants detected may be any
compound that includes one or more hydroxyl groups. In another
embodiment, the contaminants may be any type or combination of
alcohol, glycol, and/or glycerol. In another embodiment, the
contaminants may be ethylene glycol and/or diethylene glycol. In
another embodiment the contaminants may be ethylene glycol and/or
diethylene glycol that may be tested for in the presence of other
hydroxylated compounds such as alcohols, glycerol and propylene
glycol.
[0041] In a further embodiment, the contaminants may be detected in
the presence of one or more other hydroxylated compounds that are
not contaminants. For example, the substrate may be a beverage
containing ethyl alcohol, such as wine, and the contaminant
detected may be a glycol, such as ethylene glycol and/or diethylene
glycol. In another embodiment the contaminants may be ethylene
glycol and/or diethylene glycol that may be tested for in the
presence of other glycols such as propylene glycol and
glycerol.
[0042] The concentration of contaminants detected may range from
below FDA limits up to 100 percent contaminant. In one embodiment,
ethylene glycol is detected in concentrations ranging from below
about 1 weight percent up to about 100 weight percent. In a further
embodiment, diethylene glycol is detected in concentrations ranging
from below about 3 weight percent up to about 100 weight
percent.
[0043] In a further aspect, this disclosure provides an enzyme
assay for detecting one or more contaminants in a substrate. One
class of useful enzymes for the assay is the dehydrogenases. In
some embodiments, the enzymes may include alcohol dehydrogenase
and/or aldehyde dehydrogenase. For example, the enzymes may include
yeast alcohol dehydrogenases such as yeast alcohol dehydrogenase
USB 10895.
[0044] In an additional embodiment, the enzyme assay may include a
coenzyme. The coenzyme may be, but is not limited to, NAD+. For
example, the coenzyme may be NADP+.
[0045] In one embodiment, the enzyme assay includes one or more
buffers. For example, the buffers used may be Tris or bicine
buffers. Additionally, the buffers may be Tris Base HCl, bicine
NaOH, bicine HCl, one or more of alkaline pH range "Good's
buffers," and/or phosphate buffer. The pH of the assay materials
may range from about 4.0 to about 10.0. In one set of embodiments,
the pH may between about 6.0 and 10.0, between 7.0 and 9.0, between
7.5 and 8.6, or about 7.8.
[0046] The temperature of the assay materials during testing may
range from between about zero to about 100 degrees C. In one
embodiment, the temperature of the assay materials ranges from
between about 10 to about 40 degrees C. Additionally, the
temperature of the assay materials may be near room temperature
(about 20 degrees C.) and maintained within about .+-.0.5 degrees
C.
[0047] In one set of embodiments, contaminants such as EG and DEG
can be detected with an absorbance method 100 (FIG. 1) using a
kinetic assay. Absorbance method 100 allows the measurement of
concentrations of EG and DEG from below the limits established as
safe by the U.S. Food and Drug Administration (FDA) to levels
beyond those detected in various historical contamination
incidents. In one set of embodiments, absorbance method 100 can
convert analytes such as EG and DEG into their respective aldehydes
in the presence of yeast alcohol dehydrogenase, as shown in FIG. 1
for EG and FIG. 2 for DEG. In these reactions, the coenzyme NAD+
can be converted to NADH. EG and/or DEG concentrations are
determined by monitoring the increase in concentration of NADH,
which may be obtained by measuring absorption at about 340 nm using
a spectrophotometer.
[0048] In one embodiment, shown in FIG. 3, absorbance method 100
begins at step 110 with the collection of a test sample containing
one or more glycols. At step 120, the test sample is combined with
an enzyme, such as yeast alcohol dehydrogenase, and a coenzyme,
such as NAD+. The test sample, enzyme, and coenzyme are placed in a
cuvette at step 130. At step 140, the one or more glycols in the
sample are converted to one or more aldehydes, and NAD+ is
converted to NADH to form a measurement solution. At step 150, a
single wavelength light can be electroluminescently generated using
a device such as a light emitting diode (LED). At step 160, the
cuvette and measurement solution are illuminated with the single
wavelength light. At step 170, the amount of light transmitted
through the cuvette and measurement solution is detected. At step
180, a signal corresponding to the amount of light detected is
provided and at step 190, the concentration of glycol in the sample
is determined from the amount of light detected. The absorbance
change in this kinetic assay can be proportional to the amount of
contamination present.
[0049] One or more of steps 110-190 described above and in FIG. 3
may be optional and the order of steps 110-190 may not be
important. Additionally, while method 100, as described above, may
be used to detect glycol, it may also be used to detect any other
contaminants that may include one or more hydroxyl groups.
[0050] In a further embodiment, the signal from step 180 may be
normalized by the value of the signal at time equals zero.
Specifically, the signal may be, for example, a voltage, and the
voltage at all times may be divided by the voltage at time equals
zero. In this way, the background is normalized so that, for
example, if the intensity of light generated at step 150 varies
from month-to-month, this variation need not affect analytical
results. The voltage at time equals zero may be determined by
extrapolation if voltages are not available and/or recorded until
after that time.
[0051] Data from step 180 may be further processed by fitting it
with a functional form. In one embodiment, the functional form may
be an exponential function, such as V(t)=1-a*exp(b*t), where V may
be the normalized voltage (relative to the voltage at t=0), t is
time, and a and b are constants to be determined, for example,
through a least square curve-fit. Once a functional form as been
fit through the time history data, the data may be further analyzed
to, for example, extrapolate voltage values to times when
measurements were not taken. Additionally, the functional form can
be used to, for example, obtain the differences and/or ratios
between voltages at different times. The voltage at t=0 can be the
voltage measured at t=0 or can be, for example, the voltage
extrapolated to time t=0 rather than the actual voltage measured at
t=0.
[0052] The test sample may be heated to drive off certain
non-target contaminants or centrifuged prior to being placed in the
cuvette. Centrifugation may be used when the sample contains
particles such as silicate particles (in toothpaste for example)
that can scatter light and interfere with optical measurements.
[0053] Embodiments utilizing yeast alcohol dehydrogenase can
require relatively large substrate concentrations (e.g., 1.5-1000
mM) to achieve reasonable reaction rates. With yeast alcohol
dehydrogenase, the Michaelis-Menten constant, KM, defined as the
substrate concentration for half-maximum enzyme activity, is large
for both EG and DEG. In order to detect small amounts of substrate
quickly, a kinetic assay that measures the initial rate of
absorption can be used instead of waiting until the reaction
reaches completion and quantifying the overall absorption change.
This can provide accurate results in less time than waiting for an
endpoint-assay.
[0054] Absorbance method 100 may be applied to aqueous solutions
having various pH values and ionic strengths. In some embodiments,
the pH is maintained between 7 and 9. Repeatable, accurate results
have been obtained with pH near 8.0. In one embodiment, absorbance
method 100 utilizes buffers such as Tris-HCl. In other embodiments,
biocompatible buffers such as phosphate or bicine may be used.
[0055] Absorbance method 100 may be used to measure contaminant
concentrations in solutions having any temperature between about
zero and 100 degrees C. Preferably, the temperature is between
about 10 and 40 degrees C. Excellent results have been obtained
with the temperature held constant, for instance, within about 1
degree C., such as 26.+-.0.5 degrees C.
[0056] For one embodiment of absorbance method 100, an
Alcohol-Dehydrogenase-NAD reagent may be prepared from a
commercially available kit for ethanol determination (no. 331-CMA;
Sigma Chemical Co., St. Louis, Mo. 63178). To an NAD-ADH Multitest
Vial (Sigma no. 331-10), 5.3 mL of Tris-HCl buffer, pH 8.8 (Biorad,
0.1 M diluted with ddH2 from 1.5M) are added. The resulting
composition of the reagent (per liter) is roughly: 1.5.times.10 5 U
of alcohol dehydrogenase (EC 1.1.1.1), 1.89 mmol of NAD, and 100
mmol of Tris-HCl (pH 8.8). The reagent may be stable for at least 8
hours at room temperature. In a typical measurement, ethylene
glycol substrate may be added to the NAD-ADH solution at a ratio 1
to 2 (for example 300 and 600 microliter; or 120 and 240
microliter). The absorbance change may be monitored, for example,
for 10 min at 340 nm.
[0057] In another set of embodiments, contaminants such as EG and
DEG can be detected using a fluorescence method 200 that employs a
coupled-enzyme assay to produce changes in fluorescent emission.
For example, contaminants such as EG and/or DEG may be used to
indirectly convert a fluorogenic substrate (dye) into its
fluorescent form, and the concentration of EG and/or DEG can be
determined from the amount of light fluorescing from the
sample.
[0058] FIG. 4 illustrates the reactions and enzyme pathways for one
embodiment of fluorescence method 200. In reactions a and b, EG and
DEG, respectively, are reacted with alcohol dehydrogenase to
produce an aldehyde and NADH. By using yeast alcohol dehydrogenase
as the particular type of alcohol dehydrogenase, it has been found
that interference from the presence of glycerol, propylene glycol,
and/or polyethylene glycol may be minimized or eliminated. In
reaction c, the NADH may then be converted back to NAD+ with NADH
oxidase, which generates one equivalent of hydrogen peroxide for
each equivalent of NADH. The hydrogen peroxide may then cleave to
create two free radicals. In reaction d, the free radicals, in the
presence of horseradish peroxidase (HRP), convert a dye, such as a
fluorogenic substrate, from its non-fluorescing form into its
fluorescing form. Fluorogenic substrates include materials that can
be converted from non-fluorescing to fluorescing forms in the
presence of free radicals and a peroxidase. Fluorogenic substrates
may be from the resazurin/resorufin family and examples include
N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) available from
Invitrogen A36006) and its derivatives and related compounds such
as Amplex UltraRed. Peroxidases can include HRP, such as type 1 and
type 2.
[0059] FIG. 5 illustrates another embodiment of fluorescence method
200 for detecting contaminants such as glycols. At step 210, a
sample containing a glycol is collected. At step 212, the glycol is
converted to an aldehyde and NAD+ is converted to NADH. At step
214, NADH is oxidized to produce a stoichiometric amount of
hydrogen peroxide. At step 216, in the presence of hydrogen
peroxide, Amplex UltraRed dye is oxidized with horseradish
peroxidase to produce a measurement solution containing resorufin.
At step 218, a single wavelength light having a frequency of about
530 nm is electroluminescently generated with, for example, an LED.
At step 220, the measurement solution is illuminated with the
single wavelength light. At step 222, the amount of light
fluoresced from the measurement solution at a different wavelength,
about 590 nm, is detected. At step 224, a signal is provided
corresponding to the amount of light detected. At step 226, the
presence or concentration of glycol is determined. At step 228, a
decision can be made about whether or not to continue with the
measurements.
[0060] One or more of steps 210 through 228 described above and in
FIG. 5 may be optional and the order in which the steps occur can
be varied. Additionally, while method 200, as described above, may
be used to detect glycol(s), it may also be used to detect any
other contaminants having a hydroxyl group. In another set of
embodiments, a background subtraction procedure can be performed on
the signal from step 224. Specifically, a change in signal between
two different time points can be determined by, for example,
subtracting the signal at an earlier time from the signal at a
later time. For example, if the signal is a voltage, the absolute
voltage change between three minutes and eight minutes is the
voltage at eight minutes minus the voltage at three minutes. Using
this approach, a constant background of fluorescence may be
removed, allowing for greater test sensitivity.
[0061] Many types of household materials and medicines, such as
toothpaste and cough syrup may contain either glycerol or propylene
glycol. The tests described herein are able to analyze these
samples for EG and DEG without significant interference from
glycerol and/or propylene glycol. In other cases, sample
preparation may be helpful. While absorbance method 100 and
fluorescence method 200 may be used to analyze any type of
substrate material, the test sample preparation procedures may
depend on the specific type of substrate to be analyzed. For
example, when preparing toothpaste samples for fluorescence method
200, the toothpaste samples may be dissolved in buffer and
centrifuged. For instance, 3 g of toothpaste sample may be
dissolved in 20 ml of Tris-HCl 0.1 M pH 7.8 buffer by vortexing the
mixture. The sample may then be centrifuged for 10 minutes at 3000
rpm in order to settle out silicate particles that can scatter
light and interfere with measurements. The sample may then be
further diluted with buffer. Table 1 shows the total amount of
buffer that may be added for a given amount of toothpaste and DEG
concentration.
TABLE-US-00001 TABLE 1 Toothpaste sample preparation. wt % DEG mM
DEG DEG (.mu.l) toothpaste (.mu.l) total buffer (ml) 100 569 135 0
2.365 80 455 108 180 2.212 60 341 81 360 2.059 40 228 54 540 1.825
30 171 40.5 630 1.830 20 114 27 720 1.753 10 57 13.5 810 1.680 5 28
6.7 849 1.650 3 17 4.05 873 1.623 1 6 1.35 891 1.608 0 0 0 900
1.600
[0062] When preparing other substrates besides toothpaste, such as
cough syrup and allergy syrup, for fluorescence method 200, the
sample may be mixed with 0.1 M Tris-HCl pH 7.8 buffer. The sample
mixture may then be shaken vigorously before usage. Measurement
samples may be prepared freshly each day. For viscous samples that
are difficult to pipet accurately, such as paracetamol syrup, it
may be more practical to weigh out or alternatively predilute the
sample with buffer before pipetting it into the mixture. Table 2
shows the total amount of buffer that may be added for a given
amount of sample and DEG concentration.
TABLE-US-00002 TABLE 2 Sample preparation. Wt % DEG mM DEG DEG
(.mu.l) sample (.mu.l) total buffer (ml) 100 569 135 0 2.365 80 455
108 27 2.365 60 341 81 54 2.365 40 228 54 81 2.365 30 171 40.5 94.5
2.365 20 114 27 108 2.365 10 57 13.5 121.5 2.365 5 28 6.7 127.3
2.365 3 17 4.05 130.95 2.365 1 6 1.35 133.65 2.365 0 0 0 135
2.365
[0063] In some cases, the analysis may be directed to EG rather
than DEG. EG sample preparation may be qualitatively the same as
described for DEG in Tables 1 and 2, above. However, since EG may
be more reactive than DEG in the presence of yeast alcohol
dehydrogenase, smaller concentrations of EG can be employed.
Examples of sample concentrations include 0-112 mM EG and
corresponding amounts of household product (e.g. toothpaste, cough
syrup, paracetamol syrup) to make 0-100 wt % ethylene glycol
samples.
[0064] In one embodiment of a fluorescence method 200, an
Alcohol-Dehydrogenase-NAD-NADH Oxidase reagent may be prepared as
follows. First, 15 mL of Tris-HCl buffer pH 7.8 (BM-318 Boston
Bioproducts, 0.1 M, diluted from 1.0M with ddH20) may be added to
an NAD Vial (Sigma Aldrich, 50 mg no. 331-10). Second, a Yeast
Alcohol Dehydrogenase (USB/Affymetrix 10895) stock solution of 1.2
KU/ml may be prepared by diluting. This may be done right before a
measurement. Stock solutions may be prepared freshly and need not
be frozen down between measurements. Third, a Horseradish
Peroxidase Type 1 (Sigma Aldrich P8125-5KU) stock solution of 1000
U/ml may be prepared using Tris-HCl buffer, pH 7.8 (BM-318 Boston
Bioproducts, 0.1 M, diluted from 1.0M with ddH20). This stock
solution may be split up and stored in small vials at -20 degrees
Celsius for up to about two months. Before each measurement, one
vial of stock solution may be defrosted and diluted to 10 Units/ml
using 100 mM Tris HCl buffer (from Boston Bioproducts). Fourth,
NADH Oxidase (EMD Chemicals, 481925-5U) stock solution may be
prepared by diluting 5 Units of enzyme in 800 microliter of
Phosphate Buffer pH 7.4 (Sigma Aldrich P3619-1GA). Aliquots of 100
microliters may be stored in centrifuge tubes at -20 Celsius. Stock
solutions may be defrosted between experiments and may be discarded
after they have been defrosted. Fifth, Amplex Ultra Red (Invitrogen
A36006) stock solutions may be prepared freshly before each
measurement by diluting a vial of 1 mg Amplex Ultra Red in 400
microliter DMSO (EMD OmniSolv MX1456-6) and vortexing it for
several seconds to dissolve the dye.
[0065] In a further embodiment, to start the reaction with
fluorescence method 200, the measurement sample, a hydrogen
peroxidase solution, and the Alcohol-Dehydrogenase-NAD-NADH Oxidase
reagent may be combined in a cuvette. 120 microliter of a sample
containing, for example, DEG may be added to a plastic cuvette (VWR
97000-586) using a 1 ml pipettor. A new pipette tip may be used for
each sample to avoid cross-contamination. Using a 10 microliter VWR
pipettor, 3.5 microliter of a 10 U/ml Horseradish Peroxidase Type 1
solution may be injected into each cuvette. A new pipette tip may
be used to inject the Horseradish Peroxidase Type 1 solution. To
start the reaction, 240 microliter of an
Alcohol-Dehydrogenase-NAD-NADH Oxidase reaction mixture may be
added to each cuvette. The same tip of the 1 ml pipettor may be
used to add the reaction mixture. The
Alcohol-Dehydrogenase-NAD-NADH Oxidase reaction mixture, enough for
eight cuvettes, may be made of: 1.95 ml NAD, 13 microliter Amplex
Ultrared, 6.5 mictoliter NADH Oxidase, and 40 microliter Alcohol
Dehydrogenase. In another embodiment, multiple samples may be run
in parallel in separate cuvettes. In addition, 1.95 ml of the
Alcohol-Dehydrogenase-NAD-NADH Oxidase reaction mixture may be made
freshly before each run and may be mixed thoroughly before starting
the reactions.
[0066] Coupled enzyme assays often involve different enzymes that
function on different substrates and have different pH and
temperature optima. With a single enzyme, optimizing activity
typically involves selecting only the proper pH, temperature and
concentrations of enzyme and substrate, a relatively
straightforward task. However, it has been found for the
coupled-enzyme reactions described herein the parameter space
becomes unpredictable. The concentration of the first enzyme and
substrate may be selected so that the resulting product will be in
a concentration range that the second enzyme can act upon
significantly and without saturation. The product of the second
enzyme, a function of the concentrations of both the second enzyme
and the product of the first enzyme, should similarly fall in a
useful concentration range for the third enzyme, and so forth.
Identifying effective concentrations for all of the enzymes and
substrates involved is not straightforward as the most effective
concentration or conditions for one enzyme may have a deleterious
effect on the second enzyme. For example, if the concentration of
the product of one enzyme is too low for the next one, no activity
will register; conversely, if the concentration of the product of
one enzyme is too high, then the activity rate of the next maximum
enzyme will limit production of its product. Unless the enzyme
activities are comparable, it may not be possible to use a coupled
enzyme assay to determine the concentration of a contaminant, such
as DEG or EG.
[0067] Any test for EG and/or DEG may be subject to a possibility
of interference from other glycols, particularly with glycerol and
propylene glycol. With several alcohol dehydrogenases from
different species, the interference from glycerol and propylene
glycol may lead to such high detected activity that the
concentrations of DEG and/or EG may not be determined accurately.
However, it has been found that one particular alcohol
dehydrogenase, yeast alcohol dehydrogenase (USB 10895), may yield a
higher activity for higher concentrations of DEG, even in samples
with a large fraction of glycerol or propylene glycol.
[0068] The pH of the assay mixture may be any value between 4 and
10. In one embodiment, the pH is between 7 and 9. In a further
embodiment, the pH is about 7.8.
[0069] The concentration of alcohol dehydrogenase may be any
appropriate value such as above 5.5 U/mL and in one set of
embodiments is near or at 16.5 U/mL. The concentration of NAD may
be any value above 0.7 mg/mL and in one set of embodiments is near
or at 2.22 mg/mL. The concentration of NADH oxidase may be any
value above 4.5 mU/mL and may be, for example, at or near 14.1
mU/mL. The concentration of HRP may be any value above 35 mU/mL but
preferably is at or near 97.2 mg/mL. The concentration of Amplex
Ultrared may be any value above 4 mg/L but in a preferred
embodiment is at or near 11.3 mg/L.
[0070] Alcohol dehydrogenase (ADH), and indeed many or all
dehydrogenases, are capable of converting OH groups to aldehydes.
It has been found that alcohol dehydrogenase acts very efficiently
on methanol (with one carbon) and ethanol (with two carbons), for
which it originally evolved in biological organisms. It acts on
other alcohols, as well, and on glycols, compounds with multiple OH
groups. Its activity on ethylene glycol (two carbons) may be
expected, and the lower activity rate acting on DEG may also be
expected, which with four carbons has a higher molecular weight. We
may therefore expect that alcohol dehydrogenase would have a
comparable, if not faster, rate of activity on three-carbon
propylene glycol and glycerol. Using an alcohol dehydrogenase to
detect DEG in the presence of glycerol and propylene glycol may
therefore not be expected to work due to interference from these
compounds; by contrast, given the high reactivity rate of EG with
ADH, we may expect higher rates of activity for propylene glycol
and glycerol, relative to DEG. However, as the methods described
herein reveal, with a certain type of ADH, the concentration of DEG
can be detected and quantified, even in the presence of propylene
glycol and glycerol.
[0071] For a given enzyme, there exists an optimal set of
conditions for activity, in particular temperature and pH. The
assays described above may be run at room temperature
(approximately 25 degrees C.) even though this may not be the
optimum temperature for these reactions. Slight variations in room
temperature (i.e. in the range of 20-25 deg C.) may not affect the
results significantly. When running a single-enzyme reaction, such
as the single alcohol dehydrogenase reaction, a pH is selected that
maximizes activity. The optimal pH range for each enzyme used in
the above assays may be found in the literature. However, when
these optimal ranges are not the same for each enzyme used in a
coupled assay, the coupled reaction may not succeed because the
enzymes may be incompatible. For example, enzymes in the wrong pH
conditions may be unstable, and can disintegrate. For instance,
placing an acid-optimized enzyme in a highly alkaline environment,
in many cases, will denature that enzyme, causing it to lose its
proper structure and render it incapable of functioning at all. In
particular, alcohol dehydrogenase has maximum activity at pH 9.0,
well in the alkaline regime; at pH 8.0, its activity is 10% of the
maximum. By contrast, NADH oxidase has maximum activity at an
acidic pH of 6.5. Coupling the product of an alcohol dehydrogenase
reaction, ideally performed in an alkaline environment, to a
reaction involving NADH oxidase, which should be run in an acidic
environment, may not be expected to work. Horseradish peroxidase is
even more acidophilic, having an optimal pH range of 5 to 7. There
are different ways to solve this problem. The product from the
first alcohol dehydrogenase reaction could be isolated, purified,
and then reintroduced into a reaction under conditions optimal for
NADH oxidase; however, this isolation and purification is
impractical for a portable device, such as device 400, both in
terms of cost and complication. Alternatively, for a "one-pot"
reaction, a single set of conditions under which the enzymes
operate well enough may not be expected, let alone guaranteed, to
exist. For the methods disclosed herein the reactions are run at a
pH that is outside the optimum range for all of the enzymes
involved.
[0072] FIG. 6a provides a photocopy of a plot of voltage output
from a light detector versus time, for various concentrations of
EG, as obtained with absorbance method 100. As shown, with
absorbance method 100, output voltage may decrease in an
exponential decay manner, with more rapid decay occurring with
higher glycol concentrations. The voltage at all times may be
divided by the voltage at time equals zero. In this way, the
background is normalized so that, for example, if the intensity of
light generated at step 150 varies from month-to-month, this
variation may not affect analytical results. The voltage at time
equals zero may be determined by extrapolation if voltages are not
available and/or recorded until after that time.
[0073] The rate of voltage change may provide an indication of
glycol concentration. For example, FIG. 6b provides a photocopy of
a plot of the rate of voltage change, -dV/dt, at time equals zero
for various substrates and EG concentrations, where V is the raw
voltage normalized by its value at time equals zero. As shown, the
initial rate of voltage change may increase with EG concentration.
Also, measurement results may be insensitive to the type of
substrate being tested, since the results in FIG. 6b for water,
toothpaste, cough syrup, paracetamol syrup, and antifreeze are
nearly identical. For reference, FIG. 6b also includes vertical
lines indicating the EG concentrations associated with mass
poisonings in Panama and Nigeria and the EG concentration of
antifreeze (about 45 percent). It is also notable that most of
these samples contained propylene glycol at levels up to 30% and
the testing procedure did not provide false positive results under
these circumstances.
[0074] FIG. 7a provides a photocopy of a plot of voltage output
from a light detector versus time, for various concentrations of
DEG, as obtained with fluorescence method 200. As shown, with
fluorescence method 200, output voltage may increase over time in
an exponential growth manner. In addition, exponential growth may
occur even when the DEG concentration is zero percent, possibly due
to the presence of a small amount of NADH in thermal equilibrium,
which in turn may drive the production of the fluorogenic substrate
into its fluorescent form.
[0075] In one embodiment, the output voltage from a light detector
is measured as a function of time. For example, in a typical
measurement, output voltage may be recorded about once per second.
In addition, voltage may be recorded beginning about three minutes
after the reaction is started and ending about 10 minutes after the
reaction is started. The voltage measured by the detector for the
first ten minutes after mixing may conform to a functional form
where V.sub.0 is the baseline, constant voltage for the detector,
which can be measured, for example, with a water-filled cuvette.
Different concentrations of EG and/or DEG yield different values of
the enzyme activity, manifest as the characteristic time constant
.tau..sub.H. However, due to variations in the activity of the
different enzymes, and the chemical amplification of the signal,
variations of up to .+-.20% of the absolute enzyme activity may
occur for different measurements of the same sample under
ostensibly identical conditions.
[0076] To minimize such measurement errors, for both absorbance
method 100 and fluorescence method 200, measurements may be
performed simultaneously on multiple devices and results may be
normalized. For example, a test sample may be measured in one
device and a sample of 100 percent DEG (standard) may be
simultaneously measured in another device. Enzyme activity values
may be determined from these measurements, and the time constant
resulting from the enzyme activity value for the test sample,
.tau..sub.H, can be divided by the time constant resulting from the
enzyme activity for the 100 percent DEG sample,
.tau..sub.H.sup.100%, to obtain a normalized enzyme activity
.tau.'.ident..tau..sub.H/.tau..sub.H.sup.100%.
[0077] With this multi-detector strategy and normalization, the
normalized enzyme activity is monotonically correlated with the
concentration of DEG, even in the presence of glycerol and
propylene glycol, for a range of household products and medicines,
as shown in FIG. 7b. FIG. 7b is a plot of normalized enzyme
activity versus DEG concentration, as obtained with fluorescence
method 200, for various household products and medicines.
[0078] For both absorbance method 100 and fluorescence method 200,
described above, it may be desirable to heat certain test samples
prior to measuring them. For example, when alcohol dehydrogenase is
used as an enzyme, ethanol can be removed from the sample liquid
prior to mixing with the buffer solution. In one embodiment,
ethanol may be removed by heating the sample above the boiling
point of ethanol (78.4.degree. C.). For example, the sample may be
heated in a microfluidic channel using a vapor permeable polymer
membrane such as polydimethylsiloxane or Nafion. In addition, the
gums and particles present in materials such as toothpaste can
interfere with measurements. In one embodiment, these gums and
particles are degraded by placing the test sample in boiling water
for about 10 minutes.
[0079] In one aspect, a device is provided that can quantify light
absorbance and/or fluorescence using single-wavelength illumination
and photodiode detection. The device may include a plastic housing
that precisely holds the illumination and detection components in
place, ensuring that the relative positions of these components and
the sample cuvette are the same over time, in order to achieve
reproducible measurements. The device may be manufactured and
assembled at far less cost than traditional laboratory fluorometers
and spectrophotometers. It may be battery powered and therefore
completely portable. It may also include two complimentary halves
that may be molded identically with each half constructed and
arranged to hold a standard 1 cm cuvette, and where the two halves
may be snapped together to form a single double-sided instrument
that can simultaneously measure two samples.
[0080] FIG. 8 schematically illustrates one embodiment of a
low-cost, single wavelength meter 240 that may be used with
absorbance method 100 and/or fluorescence method 200 described
above. Meter 240 consists of a light source 250, a cuvette 252
containing a measurement sample, and a light detector 254. Light
source 250 illuminates cuvette 252, and light detector 254 detects
the amount of light exiting from a portion of cuvette 252.
[0081] The light generated with light source 250 may be a single
wavelength light. Such light may be generated using an
electroluminescent device such as an LED or a laser. Light source
250 may be chosen so that the generated range of wavelengths is
located near the peak absorption of a product of enzyme activity,
such as NADH. NADH has peak absorption at about 362 nm. In that
case, the light may be UV with an emission peak at about 365 nm.
Light source 250 may also be chosen so that the generated range of
wavelengths is located near the peak excitation frequency of a
fluorescent material. For example, when Amplex Ultrared fluorescent
dye is used, the light may be green and have an emission peak at
about 527 nm.
[0082] Light detector 254 generates a time-dependent voltage 256
proportional to the amount of light received. Voltage 256 may be
recorded with a voltmeter or similar device and converted to a
digital signal using an analog-to-digital converter 258.
Analog-to-digital converter 258 may be connected using a digital
data link 260 to a microprocessor 262. Microprocessor 262 may be
programmed to convert the digital signal to the concentration of
contaminants, such as EG and/or DEG. Finally, a digital readout 264
may be used to display the results, such as measured concentration,
from microprocessor 262.
[0083] When used with absorbance method 100, described above, the
voltage from light detector 254 may decay exponentially over time.
In a kinetic assay, the initial rate of this decay is directly
proportional to the amount of contaminant, such as EG and/or DEG,
present in the sample. As a result, meter 240 may be capable of
detecting contaminants in a short amount of time (e.g., less than
30 minutes, less than 20 minutes or less than 10 minutes).
[0084] A circuit diagram for an embodiment of meter 240 is shown in
FIG. 9. Meter 240 may be driven by a 5V power supply 266, which may
be provided either with AC power stepped down through a power
regulator (not shown), or DC power with, for example, several
batteries. Light source 250 may be connected to a variable resistor
268 so that the amount of light generated can be adjusted, if
desired. The light passes through cuvette 252 and strikes light
detector 254 to produce voltage 256.
[0085] Meter 240 may be less expensive than traditional analytical
optical instruments, such as a spectrophotometer. One reason for
the high cost of traditional instruments is that they have the
ability to generate precise wavelengths of light over a very large
range. While this flexibility is needed to analyze the wide range
of samples and reactions that might be encountered, it is not
needed to analyze the reactions that occur with absorbance method
100 and fluorescence method 200, described above, where the optical
properties of the materials to be detected are already known. In
the latter situation, single wavelength light sources may be used,
thereby avoiding the far more expensive and complicated lamp and
diffraction-grating pairing found in spectrophotometers.
[0086] The NADH absorption method, described above, is particularly
amenable to a single wavelength approach. NADH has a broad
absorption peak, centered at about 340 nm, so that light
transmission measured at 340 nm should provide the most sensitive
results. It has been found however that wavelengths in the 350-360
nm range also can yield useful results. Light over this range of
wavelengths can be produced by frequency-tripled Nd:YAG and Nd:YVO4
lasers (which cut the original 1064 nm wavelength into 3, yielding
a 355 nm beam) are relatively low-cost and can generate large
amounts of power. In addition, LEDs that generate around a
milliwatt of power in the 360 nm range are available at relatively
low cost.
[0087] In addition to an inexpensive light source, meter 240 may
include other inexpensive components. For example, light detector
254 may be a TSL257-LF photodiode from TAOS. In addition,
analog-to-digital converter 258, microprocessor 262, and digital
readout 264 can be combined into a single low-cost, low power
microprocessor, such as the MSP430 series from Texas Instruments.
By using such items, meter 240 can be produced at far less cost
than a traditional spectrophotometer. Additionally, meter 240 may
be built in a handhold form and readily implemented in tough field
environments.
[0088] FIG. 10 schematically illustrates an embodiment of a device
300 capable of measuring light absorbance and light fluorescence at
the same time. Device 300 may be used to perform measurements for
absorbance method 100 and/or fluorescence method 200, described
above. Device 300 combines a fluorescence detector 301, a UV light
detector 302, a green light detector 303, a green LED 304, a UV LED
305, and a cuvette 311. Cuvette 311 may contain a measurement
sample. Green LED 304 and UV LED 305 shine light into cuvette 311.
Fluorescence detector 301, UV light detector 302, and green light
detector detect light received from cuvette 311.
[0089] Green LED 304 may be used for both absorbance and
fluorescence measurements. Green LED 304 shines a single-wavelength
green light into cuvette 311. For absorption measurements, green
light detector 303, located on the opposite side of cuvette 311
from green LED 304, detects the amount of green light from green
LED 304 that passes through measurement sample and cuvette 311. For
fluorescence measurements, a fluorescence detector 301 detects the
amount of red light fluoresced from the measurement sample within
cuvette 311.
[0090] Green LED 304 may be chosen so that the emission peak of the
green light is within the excitation peak of the fluorescent dye
used for fluorescent method 200. For example, in one embodiment,
green LED 304 may have an emission peak at about 527 nm, which
falls within the excitation peak of Amplex Ultrared. In addition,
the emission peak of green LED 304 may be near an absorption peak
of the fluorescent dye used for fluorescent method 200. For
example, Amplex Ultrared dye has strong absorption in the green.
Therefore, when green LED 304 has an emission peak at about 527 nm,
green LED 304, used in conjunction with Amplex Ultrared dye, may be
suitable for both fluorescence and absorption measurements.
[0091] Because fluorescence may be isotropic, the intensity of
fluoresced light from cuvette 311 could be nearly independent of
the angle at which fluorescence detector 301 is placed relative to
the light beam from green LED 304 used for excitation. Fluorescence
detector 301 may therefore be placed at any angle with respect to
the light beam. For example, as shown in FIG. 10, fluorescence
detector 301 may be placed at nearly ninety degrees from the
incoming beam path generated by green LED 304, thereby increasing
the signal-to-noise ratio of the detected fluorescence
emission.
[0092] For the purposes of absorption measurements, device 300 may
also include a UV LED 305. UV LED 305 can generate and send a
single-wavelength UV light into cuvette 311. A UV light detector
302, located on the opposite side of cuvette 311 from UV LED 305,
detects the amount of UV light from UV LED 305 that passes through
measurement sample and cuvette 311.
[0093] UV LED 305 may be chosen so that it has an emission peak
near the absorption peak of a reactant and/or a product of a
chemical reaction. For example, UV LED 305 may have an emission
peak near about 365 nm because NADH, a product of the enzyme
reactions in FIG. 4, absorbs strongly near that wavelength.
[0094] In another embodiment, to improve the accuracy of absorption
and fluorescence measurements, device 300 may include longpass
filter 306, shortpass filter 307, and bandpass filter 308. Filters
306, 307, 308 may improve the signal-to-noise ratio by preventing
unwanted light from reaching fluorescence detector 301, UV light
detector 302, and green light detector 303. For example, bandpass
filter 308 may be placed between green light detector 303 and
cuvette 311 so that only green light can impact green light
detector 303. In addition, shortpass filter 307 may be placed
between UV light detector and cuvette 311 so that only UV light can
impact UV light detector 302. Finally, longpass filter 306 may be
placed between fluorescence detector 301 and cuvette 311 so that
only fluoresced light can reach fluorescence detector 301.
[0095] To further improve the signal-to-noise ratio, device 300 may
also include a shortpass filter 309 located between UV LED 305 and
cuvette 311, and a bandpass filter 310 located between green LED
304 and cuvette 311. Bandpass filter 310 may allow only green light
to reach cuvette from the vicinity of green LED 304. Shortpass
filter 309 may allow only UV light to reach cuvette 311 from the
vicinity of UV LED 305.
[0096] To ensure accurate and reproducible measurements, the
relative positions of the illumination sources, sample cuvette and
detectors should be carefully maintained over time. In a laboratory
fluorometer, these components are mounted in a
precisely-manufactured housing that would be too delicate and
expensive for field use. Therefore, to reduce costs and improve
durability, the holder for samples and optics may be fabricated
from a precision injection molded plastic to provide for precise
and repeatable alignment of cuvettes in a portable device.
[0097] FIG. 11 shows one embodiment of a device 400 that includes
identical molded halves 1a and 1b. Molded halves 1a and 1b each
include sensor holding structures 7a, 7b, buttons 2a, 2b, and
indicators 3a, 3b, 4a, 4b. Sensor holding structures 7a, 7b include
sample holder receivers 6a, 6b. Molded halves 1a and 1b may be made
of any solid material or combination of solid materials. For
example, molded halves 1a and 1b may be made of one or more types
of plastic such as polystyrene, polypropylene, ABS, polyurethane,
polyethylene, polyamide (Nylon), or polyacetal (DELRIN.RTM.).
[0098] Device 400 may be used to simultaneously measure two
separate samples. For example, molded half 1a may be used to
measure a reference sample, and molded half 1b may be used to
measure a test sample. These two samples may be measured
concurrently or in sequence. A cuvette having a cap 5a and
containing the reference sample may be inserted into sample holder
receiver 6a, and a cuvette having a cap 5b and containing the test
sample may be inserted into sample holder receiver 6b. Measurements
may be initiated by pressing buttons 2a and/or 2b.
[0099] Indicators 3a, 3b, 4a, and 4b may indicate the presence or
absence of contaminants in the cuvettes. For example, indicators 3a
and 3b may be red and may illuminate if a contaminant is detected
in one or both of the cuvettes. Similarly, indicators 4a and 4b may
be green and may illuminate if no contaminants are detected in one
or both of the cuvettes. Indicators 3a, 3b, 4a, 4b may be
illuminated using, for example, LEDs. Buttons 2a and 2b and
indicators 3a, 3b, 4a, 4b may be mounted to a circuit board 20 in a
base 8 of device 400.
[0100] FIG. 12a is a side view of device 400 and FIG. 12b is a
section view in which circuit board 20 can be seen. Each molded
half 1a, 1b of device 400 includes a green LED 29b, a UV LED 29A, a
green light detector 31b, a UV light detector 31c, and a
fluorescence detector 31a. These LEDs 29b, 29a and detectors 31b,
31c, 31a may be connected to circuit board 20 with leads 21, 22.
Circuit board 20 may also contain (not shown) the basic circuits
needed to control LEDs 29b, 29a and process signals from detectors
31b, 31c, 31a. A reference sample 15c in a cuvette 15b may be
prepared and inserted into sample holder receiver 6a. Similarly, a
test sample may be inserted into sample holder receiver 6b.
[0101] FIGS. 13 and 14 show a bottom isometric view of molded half
1a. Molded half 1a includes LED pockets 28a, 28b and detector
pockets 33a, 33b, 33c. LED pockets 28a, 28b contain green LED 29b
and UV LED 29a. Green LED 29b, may be, for example, of the type
having CREE's model number LC503FPG1-15P-A3. UV LED 29a may be, for
example, of the type having LED Supply's model number
L5-0-U5TH15-1. Detector pockets 33a, 33b, 33c contain green light
detector 31b, UV light detector 31c, and fluorescence detector 31a.
One or more of green light detector 31b, UV light detector 31c, and
fluorescence detector 31a may be TAOS's TSL257 high-sensitivity
light-to-voltage converter. These LEDs and detectors may be
identical to those shown in devices 240 and 300.
[0102] In another embodiment, device 400 also includes filters 32a,
32c, 32b, 30a, 30b and focusing slits 34a, 34c, 34b, 27a, 27b. To
reach green light detector 31b, light from green LED 29b passes
through filter 30b, focusing slit 27b, cuvette 15b, focusing slit
34b, and filter 32b. To reach UV light detector 31c, light from UV
LED 29a passes through filter 30a, focusing slit 27a, cuvette 15b,
focusing slit 34c, and filter 32c. To reach fluorescence detector
31a, light fluoresced from the measurement sample passes out of
cuvette 15b, through focusing slit 34a, and filter 32a. Focusing
slits 34a, 34c, 34b, 27a, 27b may help to spatially constrain the
light, and may be shaped, for example, as slits and/or
pinholes.
[0103] Signals from detectors 31b, 31c, 31a may be processed on
circuit board 20, as described previously, and if one or more
contaminants are present indicators 3a, 3b may light up. If no
contaminants are present, indicators 4a, 4b may light up.
[0104] Filters 32a, 32c, 32b, 30a, 30b may be made of clear plastic
filter material. The material may be, for example, of the
inexpensive type often used for theatre lighting. The filter
material can be die cut to the desired size to fit into pockets
28a, 28b. For a sense of scale, green LED 29b and UV LED 29a may
each be about 5 mm diameter, and filters 32a, 32c, 32b, 30a, 30b
may be about 0.5 mm thick, 5 mm wide, and 20 mm long.
[0105] To improve measurement accuracy, LEDs 29b, 29a and detectors
31b, 31c, 31a may be secured in place with adhesive and/or a piece
of compressible material such as foam rubber stuffed in LED pockets
28a, 28b and/or detector pockets 33a, 33b, 33c. The foam rubber may
also help isolate the LEDs 29b, 29a and detectors 31b, 31c, 31a
from stray light. Circuit board 20 may be pre-drilled with either
press-fit connectors for leads 21, 22 so the board can be removed,
or leads 21, 22 can poke through plated vias to be soldered into
place. If circuit board 20 has to be removed, leads 21, 22 may be
desoldered.
[0106] The modular design of device 400 allows two identical molded
halves 1a and 1b to be combined to make the instrument body. Molded
halves 1a and 1b have skirts 39a and 39b that form a pocket for the
circuit board when the two halves are assembled. To provide
alignment, as shown in FIG. 13, tabs 40 and 41 can mate with
corresponding tab 42 and surface 44 by placing molded halves 1a, 1b
face-to-face but rotated with respect to each other, and then
twisting molded halves 1a, 1b until tabs 40, 41, 42 seat. Round 43
helps to allow the twist-and-seat motion to occur. An adhesive can
be applied before assembly, or the presence of a screwed-down
circuit board 20 and a snapped-in place bottom cap 36 can hold the
instrument together.
[0107] Standalone fluorometers typically involve a single sample
cuvette. It is not possible to run multiple samples at the same
time (as is possible on a plate reader, a large, sophisticated and
expensive piece of equipment that has never been made portable).
The high cost of these instruments is due at least partially to the
selectability of excitation and emission wavelengths with high
spectral resolution, using diffraction gratings. Because the
substrates, enzymes and dyes described above are well characterized
spectrally, single-wavelength excitation sources (e.g., LEDs) may
be selected along with specific filters to isolate emission and
excitation wavelengths. This allows the construction of multiple
low-cost detectors, so that having several samples run at the same
time now becomes practical and efficient. This means that a
standard reference sample of 100% DEG can be run at the same time
as a test sample, using the same enzymes and under the same
conditions of temperature, time, pressure and other environmental
parameters. By running the two samples in parallel, the results for
the test sample may be normalized, as described above, thereby
removing fluctuations due to accumulated differences in enzyme
activity. This measure may be reproducible through different
batches of all enzyme components, and it allows contaminant
concentrations to be measured more accurately than may be possible
with a single detector and sample.
[0108] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0109] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0110] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0111] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified, unless clearly
indicated to the contrary.
[0112] All references, patents and patent applications and
publications that are cited or referred to in this application are
incorporated in their entirety herein by reference.
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