U.S. patent application number 14/244437 was filed with the patent office on 2014-08-07 for reagentless ceria-based colorimetric sensor.
This patent application is currently assigned to CLARKSON UNIVERSITY. The applicant listed for this patent is Daniel Andreescu, Emanuela Silvana Andreescu, Cristina R. Ispas, Maryna Ornatska. Invention is credited to Daniel Andreescu, Emanuela Silvana Andreescu, Cristina R. Ispas, Maryna Ornatska.
Application Number | 20140220608 14/244437 |
Document ID | / |
Family ID | 47293507 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140220608 |
Kind Code |
A1 |
Andreescu; Emanuela Silvana ;
et al. |
August 7, 2014 |
Reagentless Ceria-Based Colorimetric Sensor
Abstract
A colorimetric reagent in the form of nanoparticles, composite
nanoparticles, and nanoparticle coatings, including methods of use,
methods of preparation, deposition, and assembly of related devices
and specific applications. The colorimetric reagent comprises
cerium oxide nanoparticles which are used in solution or
immobilized on a solid support, either alone or in conjunction with
oxidase enzymes, to form an active colorimetric component that
reacts with an analyte to form a colored complex. The rate of color
change and the intensity of the color are proportional to the
amount of analyte present in the sample. Also described is the use
of ceria and doped ceria nanoparticles as an oxygen
storage/delivery vehicle for oxidase enzymes and applications in
biocatalytic processes in anaerobic conditions of interest in
biomedicine and bioanalysis. Further described are a variety of
related applications of the disclosed technology including clinical
diagnosis, in vivo implantable devices, food safety, and
fermentation control.
Inventors: |
Andreescu; Emanuela Silvana;
(Potsdam, NY) ; Ornatska; Maryna; (Potsdam,
NY) ; Ispas; Cristina R.; (Brea, CA) ;
Andreescu; Daniel; (Potsdam, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Andreescu; Emanuela Silvana
Ornatska; Maryna
Ispas; Cristina R.
Andreescu; Daniel |
Potsdam
Potsdam
Brea
Potsdam |
NY
NY
CA
NY |
US
US
US
US |
|
|
Assignee: |
CLARKSON UNIVERSITY
Potsdam
NY
|
Family ID: |
47293507 |
Appl. No.: |
14/244437 |
Filed: |
April 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13156755 |
Jun 9, 2011 |
8691520 |
|
|
14244437 |
|
|
|
|
Current U.S.
Class: |
435/11 ; 435/14;
435/25; 435/288.7 |
Current CPC
Class: |
Y10T 436/206664
20150115; C12Q 1/26 20130101; G01N 21/8483 20130101; C12Q 1/54
20130101; G01N 21/78 20130101; C12Q 1/60 20130101; G01N 31/228
20130101; G01N 33/587 20130101 |
Class at
Publication: |
435/11 ; 435/25;
435/14; 435/288.7 |
International
Class: |
C12Q 1/60 20060101
C12Q001/60; C12Q 1/54 20060101 C12Q001/54; C12Q 1/26 20060101
C12Q001/26 |
Claims
1. A system for the colorimetric determination of an analyte in a
sample, the system comprising: a sample; and a colorimetric reagent
comprising a plurality of ceria nanoparticles and a plurality of
analyte-specific oxidase enzyme molecules; wherein a measureable
change in an optical property of the colorimetric reagent is
associated with the presence of the analyte in said sample.
2. The system of claim 1, wherein at least a portion of the
colorimetric reagent is immobilized on a support.
3. The system of claim 1, wherein the colorimetric reagent further
comprises a stabilizing agent.
4. The system of claim 1, wherein the colorimetric reagent further
comprises a linking agent.
5. The system of claim 1, further comprising: an optical sensor
adapted to measure said optical property.
6. The system of claim 1, wherein the plurality of ceria
nanoparticles comprise cerium oxides in at least two different
valence states.
7. The system of claim 1, wherein the plurality of ceria
nanoparticles are doped with a metal.
8. The system of claim 7, wherein the metal is gold.
9. The system of claim 1, wherein the colorimetric reagent does not
comprise a dye.
10. The system of claim 1, wherein the analyte-specific oxidase
enzyme molecule is glucose oxidase, and the analyte is glucose.
11. The system of claim 1, wherein the analyte-specific oxidase
enzyme molecule is alcohol oxidase, and the analyte is ethanol.
12. The system of claim 1, wherein the analyte-specific oxidase
enzyme molecule is cholesterol oxidase, and the analyte is
cholesterol.
13. The system of claim 1, wherein the analyte-specific oxidase
enzyme molecule is lactate oxidase, and the analyte is lactate.
14. The system of claim 1, wherein the plurality of ceria
nanoparticles have a diameter ranging from about 2 nm to about 20
nm.
15. The system of claim 1, wherein the colorimetric reagent is
contacted by the sample under anaerobic conditions.
16. A system for the colorimetric determination of an analyte in a
sample, the system comprising: a sample; a colorimetric reagent
comprising a plurality of ceria nanoparticles doped with a metal,
and a plurality of analyte-specific oxidase enzyme molecules; and
an optical sensor adapted to measure an optical property of the
colorimetric reagent, wherein a change in said optical property is
associated with the presence of the analyte in said sample.
17. The system of claim 16, wherein at least a portion of the
colorimetric reagent is immobilized on a support.
18. A kit for the colorimetric determination of an analyte in a
sample, the system comprising: a colorimetric reagent comprising a
plurality of ceria nanoparticles and a plurality of
analyte-specific oxidase enzyme molecules; and an optical sensor
adapted to measure an optical property of the colorimetric reagent,
wherein a change in said optical property is associated with the
presence of the analyte in said sample.
19. The kit of claim 18, wherein at least a portion of the
colorimetric reagent is immobilized on a support.
20. The kit of claim 18, wherein the plurality of ceria
nanoparticles comprise cerium oxides in at least two different
valence states.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/156,755, filed on Jun. 9, 2011, and entitled
"Reagentless Ceria-Based Colorimetric Sensor," the entire
disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to components, methods, and
analytical devices for fast point of care or field colorimetric
analysis. More specifically, the invention relates to the use of
nanoparticles as active color agents to fabricate colorimetric
assays for the detection of substances and to their practical
applications in a variety of fields including clinical diagnosis,
environmental and food.
[0004] 2. Description of the Related Art
[0005] The invention consists of a colorimetric assay (including a
method, test device, test strip, detection kit, biosensor) for the
visual analysis of chemical substances in various samples. The
method incorporates a new concept based on a novel colorimetric
component. The colorimetric component refers to nanoparticles of
cerium oxide, or ceria, which change the color in response to the
presence of a particular analyte, and of cerium oxide nanoparticles
in close contact with specific oxidase enzymes, in response to a
substrate or product of the enzymatic reaction. The nanoparticles
can be used in solution, or attached to a solid support to
construct a device. The device is fabricated by immobilizing cerium
oxide nanoparticles with and without enzymes onto a solid support.
Examples of suitable solid supports include but are not limited to
paper, ceramics, membrane, packaging materials, polymeric support,
cotton swab, patch, test tube, wipe, gas or fluid collection
device, sponge, and lens. The device can be used to determine
quantitatively the presence and the relative concentration of
chemicals including, but not limited to, hydrogen peroxide,
reactive oxygen species and free radicals, ethanol, glucose,
cholesterol, billirubin, glutamate, lactate, and various
antioxidants.
[0006] Hydrogen peroxide is a key component in many chemical,
biological, pharmaceutical, clinical, environmental and food
processes. The availability of a reliable, efficient and economic
method for its detection is of great practical importance. For
example, the use of hydrogen peroxide has become a popular method
of water treatment because peroxide is effective in elimination of
bacteria and moulds. Cheap and effective monitoring methods of
hydrogen peroxide in pools, hot tubs and drinking water are needed.
Monitoring peroxide concentrations is also important in hospitals
where hydrogen peroxide is used as a sterilizing and cleaning
solution. Because hydrogen peroxide is prone to decomposition,
losing its antibacterial potency overtime, periodic confirmation of
the concentration of these solutions is necessary. At the same
time, hydrogen peroxide is an indicator of inflammation and it is
known that wound-induced extracellular H2O2 may reach
concentrations of 0.5-50 mM near the wound area. Fast detection
kits of hydrogen peroxide could also find potential uses for
screening of hydrogen peroxide-generating bacteria, (e.g. lactic
acid bacteria), present in healthy human micro flora and in food
industry (e.g. milk products, sausage production). In addition,
hydrogen peroxide is the by-product of many enzymatic reactions of
oxidase enzymes (e.g. glucose oxidase, lactate oxidase, glutamate
oxidase, cholesterol oxidase, alcohol oxidase, etc) and its
detection represents the basis of numerous enzyme biosensors e.g.
for quantitative determination of glucose, lactate, glutamate,
cholesterol, ethanol, etc). Quantitative analysis of hydrogen
peroxide as well as oxidase enzyme substrates listed above has been
accomplished using electrochemical methods based on direct
reduction or oxidation of H2O2 at the surface of a chemically or
enzymatically modified working electrode. Such systems are used in
conjunction with reference/counter electrodes and a
potentiostat.
[0007] Several colorimetric test strip devices for these substances
have been reported. These devices use colorimetric reagents (e.g.
soluble dyes), fluorescent compounds, redox reagents and enzymes,
to quantitatively determine specific substances in a sample. In
previous reports, an oxidase enzyme (e.g. glucose oxidase,
cholesterol oxidase, alcohol oxidase, etc), in solution or
immobilized onto a solid surface constituting the sensing area, is
used to catalytically transform the enzyme substrate (glucose,
cholesterol, ethanol, etc) to hydrogen peroxide. The enzymatically
generated hydrogen peroxide is subsequently measured using
chromogenic substances and spectrophotometric analysis. The
response of the sensor is based on a color change of a dye, added
in solution, in response to a chemical and/or enzymatic reaction.
The intensity of the color is typically compared to that of several
standard color charts obtained with known concentrations of
analyte. In many cases these test strips involve the use of
multiple compartments and separate reagents (chromogens for the
color change, enzymes, co-substrates, etc) that need to be added in
order to initiate the desired colored reaction. In most cases, ABTS
(2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonate) is used as a
chromogenic compound for the detection of hydrogen peroxide. In
previously developed paper based enzymatic assays, the soluble dye
(e.g. KI) migrates to the sensing spot by capillary action. In the
new colorimetric assay described here, redox active ceria
nanoparticles are used as a chromogenic indicator for H2O2,
eliminating the need for both the organic dye and the peroxidase
enzyme.
[0008] Traditionally, cerium oxide or ceria, or CeO.sub.2, has been
used in catalytic applications like automotive combustion engines,
industrial chemical synthesis and solid oxide fuel cells. Recently,
ceria has found new applications in biomedicine due to its
interesting catalytic and radical scavenging properties, and its
low toxicity. The hydroxylated cerium (IV) ions form a reddish
orange complex with hydrogen peroxide. This interesting property
first discovered by de Boisbaudran was used as the most sensitive
test for cerium ions but it has not been utilized as a test for the
analysis of other compounds. Later studies have shown that this
reaction has two stages: (1) oxidation of Ce(III) to Ce (IV), and
(2) complexation of Ce(IV) ion with two molecules of hydrogen
peroxide. Ceria nanoparticles are comprised of cerium oxides in
mixed valence states both as Ce(III) and Ce(IV) with lower size
particles having higher percentage of Ce(III) valence states. This
invention is the first to: (1) use ceria nanoparticles as
chromogenic indicators in an enzyme assay; (2) immobilize ceria
onto a solid support such as paper; and (3) integrate this concept
to construct a paper bioassay for the detection of glucose for
point-of-care ("POC") diagnostics.
[0009] An electrochemical hydrogen peroxide sensing system is
disclosed in Patent No. US 2009/0071848 entitled: "Cerium oxide
nanoparticle regenerative radical sensor" by Seal et al. (Seal, Cho
et al. 2006). Electrochemical detection of hydrogen peroxide is
also described in two research papers (Ispas, Njagi et al. 2008)
(Mehta, Patil et al. 2007). In these systems, ceria was used as
electrode coating and the signal was obtained by electrochemical
means. Such system can be used only in combination with an
electrochemical transducer and involves the use of
reference/counter electrodes and specialized electrochemical
instrumentation (e.g. potentiostat). The sensor is limited to the
detection of hydrogen peroxide and superoxide radicals. It is also
used in conjunction with platinum, which is an expensive catalyst
for hydrogen peroxide by itself. In addition, that sensor might be
prone to interferences from other electrochemically active species
which can be oxidized or reduced at the applied potential such as
hydrogen peroxide.
[0010] The relevant art is described in further detail in the
following references, all of which are hereby incorporated by
reference: Babko, A. K. and A. I. Volkova (1954), "The colored
peroxide complex of cerium," Ukrains'kii Khemichnii Zhurna 20:
211-215; Beach, E. F. and J. J. Turner (1958), "An enzymatic method
for glucose determination in body fluid," Clinical Chemistry 4(6);
462-475; Dungchai, W., O. Chailapakul, et al. (2010), "Use of
multiple colorimetric indicators for paper-based microfluidic
devices. " Analytica Chimica Acta 674(2): 227-233; Ispas, C., J.
Njagi, et al. (2008), "Electrochemical studies of ceria as
electrode material for sensing and biosensing applications,"
Journal of the Electrochemical Society 155(8): F169-F176; Martinez,
A., S. Phillips, et al. (2007), "Patterned Paper as a Platform for
Inexpensive, Low-Volume, Portable Bioassays," Angewandte Chemie
International Edition 46(8): 1318-1320; Mehta, A., S. Patil, et al.
(2007), "A novel multivalent nanomaterial based hydrogen peroxide
sensor," Sensors and Actuators a-Physical 134(1): 146-151; Seal,
S., H. Cho, et al. (2006), "Cerium oxide nanoparticle regenerative
free radical sensor," USA, University of Central Florida, USA. WO
2006130473, US 20090071848: 19 pp; Trinder, P. (1969),
"Determination of blood glucose using 4-amino phenazone as oxygen
acceptor," Journal of Clinical Pathology 22(2): 246; Wolfgang, G.,
U. B. Hans, et al. (1973), "Reagent composition and process for the
determination of glucose," Germany, Boehringer, Mannheim GmbH, U.S.
Pat. No. 3,721,607; and Yu, P., S. A. Hayes, et al. (2006), "The
phase stability of cerium species in aqueous systems--II. The
Ce(III/IV)-H2O-H2O2/O-2 systems. Equilibrium considerations and
pourbaix diagram calculations." Journal of the Electrochemical
Society 153(1): C74-C79.
BRIEF SUMMARY OF THE INVENTION
[0011] It is therefore a principal object and advantage of the
present invention to use ceria as a chromogenic indicator in an
enzyme assay.
[0012] It is another object and advantage of the present invention
to provide a system that immobilizes ceria onto a solid
support.
[0013] It is yet another object and advantage of the present
invention to construct a ceria-based bioassay for the detection of
glucose for point-of-care ("POC") diagnostics.
[0014] Other objects and advantages of the present invention will
in part be obvious, and in part appear hereinafter.
[0015] In accordance with the foregoing objects and advantages, the
present invention takes advantage of the color change of ceria
nanoparticles in the presence of hydrogen peroxide and antioxidants
such as ascorbic acid, thus making possible their detection; the
disclosed system is based on colorimetric detection. The
concentration can be distinguished with the naked eye without
special transduction systems.
[0016] The invention also demonstrates the use of ceria
nanoparticles in conjunction with oxidative enzymes, thus providing
selectivity through biocatalysis. The invention discloses a new
capacity of ceria nanoparticles when used in conjunction with
oxidase enzymes: the oxygen storage capacity for biocatalysis. This
property provides possibilities for operation of oxidase enzymes in
anaerobic conditions, thus expanding the range of analytes that can
be detected and the application field of this new device. The
disclosed device can thus be adapted for detecting a variety of
analytes and be used in numerous applications; the system can
detect virtually any oxidase enzyme substrate in both aerobic and
anaerobic conditions.
[0017] Taking advantage of the property of cerium oxide
nanoparticles as described above, disclosed is a new colorimetric
test strip design in which ceria nanoparticles are used as color
indicator reagent instead of a soluble dye. In another embodiment
of the invention, the new colorimetric component (based on ceria
nanoparticles) is used in solution. In yet another embodiment of
this invention, all the necessary components are conveniently
attached to a solid support to form the sensing surface and
construct a new type of ceria based device, test strip, biosensor.
The new device described in the present invention is inexpensive,
easy to fabricate and reagentless: the only needed step for
analysis is sample addition to bring the analyte in contact with
the sensing surface. In a preferred embodiment, the system
comprises a single compartment of active sensing material
consisting of a layer of immobilized cerium oxide nanoparticles, or
cerium oxide nanoparticles co-immobilized with oxidase enzymes. The
sensing materials can be deposited onto a solid support, such as:
paper, ceramics, packaging materials, wipes, glass, plastic, cotton
swabs, patches, bag, lenses, gas or fluid collection device or
other porous substrates. The same principle can also be used by
placing all components of the assay in solution, in a test tube.
This new device would be used primarily for analysis of liquid
samples but the application is not restricted to liquids; analysis
of gaseous samples such as breath ethanol is also possible. The
device is regenerable and can be used for analysis of multiple
samples. In addition, the device can function in conditions of
oxygen depletion such as anaerobic fermentation environments, in
vivo implantable conditions, analysis in deep oceans, space,
etc.
[0018] There are many embodiments of the invention. In some
embodiments the invention include a colorimetric ceria component as
a color reagent that can be used in solution for direct
colorimetric detection of: (1) hydrogen peroxide, (2) antioxidants
(such as ascorbic acid, gallic acid, vanillic acid, caffeic acid,
trolox. resveratrol, quercetin, etc) and (3) for detection of
hydrogen peroxide formed by specific enzymes. It also includes the
use of colorimetric ceria in immobilized state, attached to solid
supports alone or in conjunction with enzymes. In another
embodiment, the invention includes the operation of the described
ceria based systems incorporating oxidase enzymes, both in solution
and immobilized, either in aerobic or anaerobic conditions. In
another embodiment, this invention includes descriptions of the
fabrication of ceria-based colorimetric test devices and ceria
based biosensors. Examples of specific applications of the new
disclosed devices for semi-quantitative detection of analytes in
various samples including clinical: whole blood, urine, saliva,
human breath, etc, environmental and food samples, including
applications in food packaging; both liquid and gaseous samples can
be determined. Applications to in vivo implantable devices are also
included.
[0019] Therefore, in accordance with the foregoing objects and
advantages, the present invention provides a method for the
colorimetric detection of hydrogen peroxide in a sample comprising
the steps of: (i) providing a colorimetric reagent comprising a
plurality of ceria nanoparticles; (ii) contacting the colorimetric
reagent with the sample to form a mixture; and (iii) detecting an
optical property of the mixture, where a change in the optical
property of the mixture is associated with the presence of hydrogen
peroxide in the mixture.
[0020] According to a second aspect of the present invention is
provided a method for the colorimetric detection of an analyte in a
sample comprising the steps of: (i) providing a colorimetric
reagent comprising a plurality of ceria nanoparticles and a
plurality of analyte-specific oxidase enzyme molecules; (ii)
contacting the colorimetric reagent with a sample to form a
mixture, where at least some of the plurality of oxidase enzyme
molecules react with the analyte to form hydrogen peroxide; and
(iii) detecting an optical property of the mixture, where a change
in the optical property of the mixture is associated with the
presence of hydrogen peroxide or antioxidants in the mixture. In a
preferred embodiment, the change in the optical property is
proportional to the concentration of the analyte in the sample. The
colorimetric reagent can be in solution or immobilized to a
support. In a preferred embodiment, the colorimetric reagent does
not comprise a dye. In yet another embodiment of the present
invention, the step of contacting the colorimetric reagent with a
sample to form a mixture comprises contacting the colorimetric
reagent with a sample under anaerobic conditions
[0021] According to a third aspect of the present invention is
provided a method for the colorimetric detection of an analyte in a
sample comprising the steps of: (i) providing a colorimetric
reagent comprising a plurality of ceria nanoparticles and a
plurality of analyte-specific oxidase enzyme molecules; (ii)
contacting the colorimetric reagent with a sample to form a
mixture, where at least some of the plurality of oxidase enzyme
molecules react with the analyte to form hydrogen peroxide; (iii)
detecting an optical property of the mixture, where a change in the
optical property of the mixture is associated with the presence of
hydrogen peroxide in the mixture; and (iv) comparing the optical
property of the mixture to a pre-determined value. In a preferred
embodiment, the plurality of ceria nanoparticles comprise cerium
oxides in at least two different valence states, and have a
diameter ranging from about 2 nm to about 20 nm. Further, in yet
another embodiment of the present invention the ceria nanoparticles
are doped with a metal selected from the group consisting of
platinum, gold, palladium, manganese, osmium, gadolinium, samarium,
niobium, dysprosium, erbium, germanium, holmium, indium, iridium,
molybdenum, neodymium, rhodium, tantalum, tungsten, yttrium,
zirconium, ytterbium, thulium, terbium, praseodymium, and
combinations thereof.
[0022] According to a fourth aspect of the present invention, the
analyte-specific oxidase enzyme molecule is selected from the group
including, but not limited to, alcohol oxidase, glucose oxidase,
cholesterol oxidase, glutamate oxidase, galactose oxidase, lactate
oxidase, and combinations thereof. In a preferred embodiment, the
analyte is selected from the group including, but not limited to,
ethanol, glucose, cholesterol, glutamate, galactose, billirubin,
lactate, and combinations thereof.
[0023] According to a fifth aspect of the present invention is
provided a method for the colorimetric detection of an analyte in a
sample comprising the steps of: (i) providing a colorimetric
reagent comprising a plurality of ceria nanoparticles and a
plurality of analyte-specific oxidase enzyme molecules; (ii)
contacting the colorimetric reagent with a sample to form a
mixture, where at least some of the plurality of oxidase enzyme
molecules react with the analyte to form hydrogen peroxide; (iii)
detecting an optical property of the mixture, where a change in the
optical property of the mixture is associated with the presence of
hydrogen peroxide in the mixture; and (iv) reusing the colorimetric
reagent. In a preferred embodiment, the colorimetric reagent can be
used after a certain time period, and/or after a period of
heating.
[0024] According to a sixth aspect of the present invention is
provided a system for the colorimetric determination of an analyte
in a sample, the system comprising: (i) a sample; and (ii) a
colorimetric reagent comprising a plurality of ceria nanoparticles
and a plurality of analyte-specific oxidase enzyme molecules. In a
preferred embodiment, the colorimetric reagent is immobilized on a
support such as cellulose paper, cotton, silk and/or synthetic
materials such as porous glass, cross-linked polymers and
co-polymers, contact lenses, or plastic/glass test tubes. In yet
another embodiment of the present invention, the colorimetric
reagent further comprises a stabilizing agent and/or a linking
agent.
[0025] According to a seventh aspect of the present invention is
provided a method for the colorimetric detection of an antioxidant
in a sample comprising a predetermined amount of analyte, the
method comprising the steps of: (i) providing a colorimetric
reagent comprising a plurality of ceria nanoparticles and a
plurality of oxidase enzyme molecules specific to the analyte; (ii)
contacting the colorimetric reagent with a sample to form a first
mixture, wherein at least some of the plurality of oxidase enzyme
molecules react with the analyte to form hydrogen peroxide; (iii)
detecting an optical property of the first mixture; and (iv)
comparing a change in the optical property of the first mixture to
a change in an optical property of a second mixture under the same
conditions but in the absence of said antioxidant, wherein a
decrease in the change in the optical property of the first mixture
compared to the change in the optical property of the second
mixture is associated with the presence of said antioxidant.
[0026] According to an eighth aspect of the present invention is
provided a method for the colorimetric detection of an antioxidant,
the method comprising the steps of (i) providing a colorimetric
reagent comprising a plurality of ceria nanoparticles; (ii)
contacting the colorimetric reagent with a sample to form a
mixture; and (iii) detecting an optical property of the first
mixture, wherein a change in the optical property of the mixture is
associated with the presence of the antioxidant.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0027] The present invention will be more fully understood and
appreciated by reading the following Detailed Description in
conjunction with the accompanying drawings, in which:
[0028] FIG. 1A depicts ceria-based colorimetric detection of
hydrogen peroxide showing the color change of ceria nanoparticles
in colloidal state in the presence of various concentrations of
hydrogen peroxide; as the figure shows, the color change is
concentration dependent.
[0029] FIG. 1B is a graph of the calibration curve.
[0030] FIG. 1C depicts the effect of particle size (20 nm versus
100 nm particles).
[0031] FIG. 2A is a schematic representation of ceria based
colorimetric device on a paper substrate for semi-quantitative
analysis of hydrogen peroxide.
[0032] FIG. 2B is a ceria-based colorimetric device on a paper
substrate for semi-quantitative analysis of hydrogen peroxide, with
the intensity of the color quantified using the ImageJ
software.
[0033] FIG. 3 is a schematic of the working principle of the ceria
nanoparticle bioassay for the detection of oxidase enzyme
substrates showing detection of glucose as a model example; the
particles and the enzyme are co-immobilized onto a paper platform
and the only step needed to perform the analysis is the addition of
the analyte. The visible color change from white-yellowish to dark
orange in the presence of glucose is due to the change of the
oxidation state and formation of surface complexes onto the ceria
nanoparticle surface, induced by the enzymatically produced
hydrogen peroxide.
[0034] FIG. 4A is a schematic representation of immobilization of
ceria nanoparticles onto paper using a silanization procedure,
using a multi-layered sequence consisting of ceria nanoparticles,
silica, chitosan and enzyme. Other supporting materials and
procedures can also be used. The invention is not restricted to
paper nor to this immobilization method; this are only provided
here as an example.
[0035] FIG. 4B is a ceria-glucose oxidase based colorimetric device
on a paper substrate for semi-quantitative determination of
glucose. To fabricate the assay, the enzyme is immobilized onto the
paper platform, in the close proximity to the ceria
nanoparticles.
[0036] FIG. 5 is a graph of the UV-VIS spectrum of a glucose
oxidase activity assay in the presence and absence of ceria in
aerobic and anaerobic conditions showing that in the presence of
ceria, glucose oxidase is able to catalyze the oxidation of glucose
to hydrogen peroxide even in the absence of oxygen.
[0037] FIG. 6 is a graph of the calibration curve for glucose in
human serum with the corresponding colorimetric images for each
concentration tested. The intensity of the color obtained with the
ceria papers in buffer in the absence of glucose was 41(+/-3.79)
for n=3. In the same conditions but after addition of serum, the
intensity was 74(+/-5.69), indicating that glucose is present in
the serum sample. The concentration of glucose in serum determined
from the calibration curve using the standard addition method was
3.71 mM.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The colorimetric test device described herein is the first
reporting the use of cerium oxide nano-particles as a color
indicator agent for quantitative analytical purposes. The present
invention takes advantage of the color changes of ceria
nanoparticles as a result of a redox process and complex formation.
In particular, cerium oxide particles with a diameter ranging from
2 nm to 20 nm in colloidal solution or immobilized onto a solid
support change the color almost instantaneously from white to
reddish-orange in the presence of hydrogen peroxide or
antioxidants. The color change is due to a change in the oxidation
state of cerium from Ce(III) to Ce(IV) and formation of a cerium
(IV) complex with hydrogen peroxide. This change is proportional
with the concentration of hydrogen peroxide (FIGS. 1A and 1B) or
antioxidants such as ascorbic acid, gallic acid, vanillic acid,
caffeic acid, trolox, resveratrol, and/or quercetin, for example,
in the reaction. This process is used as a basis for fabrication of
a test strip comprising a colorimetric detection tool for analytes
in various samples. The intensity of the color varies with the
particle size (FIG. 1C): that is, a more intense color--and thus
higher sensitivity and lower detection limit--is obtained with
smaller size cerium oxide nanoparticles, which have a higher
percentage of the Ce(III) oxidation state and a higher exposed
surface area.
[0039] The device includes a sensing surface comprising of an inert
adsorbent support material, containing cerium oxide nanoparticles
as the colorimetric reagent, or cerium oxide nanoparticles
co-immobilized with oxidase enzymes and stabilizing agents (an
example is shown in FIG. 4). The supporting material may be made of
one or more materials including natural materials such as cellulose
paper, cotton, silk and/or synthetic materials such as porous
glass, cross-linked polymers and co-polymers, contact lenses, or
plastic/glass test tubes. The sensing layer containing the
colorimetric nanoparticles may incorporate stabilizing agents such
as surfactants and/or hydrogels. The sensing layer reacts with the
analyte, resulting in a color change. The analyte may be hydrogen
peroxide, free radicals, and/or antioxidants. It may also be a
substrate of an oxidase enzyme such as ethanol, glucose,
cholesterol, glutamate, galactose, and/or lactate, as an example.
These substrates, in the presence of oxygen and its specific enzyme
(alcohol oxidase, glucose oxidase, cholesterol oxidase, glutamate
oxidase, galactose oxidase, lactate oxidase, etc), generates
hydrogen peroxide (an example using glucose is shown in FIG. 3).
The generated hydrogen peroxide will then react with the
immobilized ceria changing its color in a concentration dependent
manner.
[0040] In another aspect of this invention, the described test
strip can be used for indirect detection of substances that inhibit
the colorimetric reaction (i.e. the redox and complexation process
of ceria) or the enzymatic reaction, thus preventing the formation
of the color. Example include, but are not limited to, analysis of
antioxidants which inactivate free radicals, or analysis of ions
like Ag.sup.+, Hg.sup.2+, Cu.sup.2+ which inactivate enzymes like
glucose oxidase.
[0041] The device has a simple design, is inexpensive, can be
mass-produced and used on site by non-skilled personnel. To
manufacture the test device, the active components can be fixed
(adsorbed, entrapped in polymers or sol-gel glasses, cross-linked
or printed) directly onto a solid support such as paper, ceramic,
glass, plastic, or other solid substrates. It can also be
incorporated or attached to cotton swabs. An example of
immobilization procedure using a multilayer deposition sequence is
shown in FIG. 4A. A layer of the active components can be used to
coat the internal surface of a transparent glass, contact lenses or
plastic test tube that would, in this case form the device
itself.
[0042] The test device described in this method involves applying a
sample to the active surface area of the colorimetric test device
based on ceria and allowing the color to develop. The presence and
the amount of analyte are determined by measuring the intensity of
the color or the rate of the color change and comparing it to a
calibration test. This can be done either visually or by
instrumental means.
[0043] Semi-quantitative visual analysis can be performed in a time
period ranging from several seconds to several min, depending on
the analyte. For hydrogen peroxide and antioxidants, the color
develops immediately after bringing the sample in contact with the
sensor (dipping, placing a drop onto the active surface, blowing
gas containing the analyte, etc). For the enzymatic detection the
response time is slightly higher (typically, up to several
minutes). The time necessary for the development of the color can
vary with the different additives, stabilizing agents and solid
support used, and with, the nanoparticles size and available
surface. The color is stable for several hours or days (depending
on the analyte) in ambient conditions. It can be reversed when left
at room temperature for several hours or within several minutes by
slight heating, after the adsorbed hydrogen peroxide is decomposed.
The assay can be reused for multiple cycles (at least ten times
without losing analytical performance) for the detection of
analytes. The measurement of color change can be made visually and
comparing it with control test strips of known concentrations. More
precise quantification can be performed with specialized
instruments for spectrophotometric analysis. For example, the
ceria-based colorimetric component defined herein can be used for
photometric detection of glucose in solution mixed with glucose
oxidase and measured at 450 nm vs calibration plot. The intensity
of the color can also be evaluated with specialized imaging
software like the ImageJ software.
[0044] In addition to the size of the ceria nanoparticles, other
variabilities in the particles can result in variable outcomes. For
example, ceria nanoparticles from different manufacturers and/or
suppliers can have variable properties other than size, including
but not limited to properties resulting from surface
characteristics or the procedure and/or compounds and additives
used to create the nanoparticles. Variabilities in the
nanoparticles other than size may also affect the intensity, rate,
or other characteristic of the color change in the presence of
hydrogen peroxide. One of ordinary skill in the art would recognize
that variations in the characteristics of the ceria nanoparticles
will likely have some effect on the properties of the interactions
and chemical reactions described herein.
[0045] An example of test device in the present invention, in a
very simple form is shown in FIGS. 2 and 4 where Whatman filter
paper was used as supporting material for the colorimetric
ceria-based composition. The paper support is loaded with the color
changing ceria nanoparticles (in FIG. 1 for detection of hydrogen
peroxide) and ceria nanoparticles and enzyme respectively (glucose
oxidase (GOX) in FIGS. 3 and 4 for detection of glucose, as an
example). The enzymatic test sensor was used for detection of
glucose in a concentration range from 0.5-500 mM. The ceria and the
enzyme can be deposited in a composite form using a polymeric or a
silica-gel linker, and can contain stabilizing agents, additives;
it can also be covered with stabilizing layers of polymers,
hydrogels, porous silica-gels, etc.
[0046] This invention is not limited to any one application (i.e.
color forming in the presence of hydrogen peroxide, antioxidants,
or hydrogen peroxide formed in an enzymatic reaction, and color
inhibiting reagents). In general, any system that reacts with ceria
and determines its color change as a result of a redox process, or
any system that generates hydrogen peroxide and gives a color
change proportional to the quantity of the analyte can be
employed.
[0047] In another aspect, this invention includes operation in
anaerobic conditions of these systems (e.g. sensors based on
oxidase enzyme). In the present invention, ceria nanoparticles act
as an oxygen reservoir or oxygen storage/delivery vehicle. Oxidase
enzymes use molecular oxygen as a co-substrate. In the absence of
oxygen, these enzymes are not able to catalyze the oxidation of
their specific substrates. The use of cerium oxide nanoparticles,
in the intimate contact or proximity of oxidase enzymes provides
the oxygen needed to carry out the oxidation reaction, even in
anaerobic conditions as shown in FIG. 5. The oxygen present on the
ceria surface initiates the enzymatic reaction generating hydrogen
peroxide. The formed hydrogen peroxide reacts with ceria, changing
the oxidation state and releasing oxygen in the process, thus
providing the oxygen necessary for the enzymatic reaction:
2CeO.sub.2+H.sub.2O.sub.2.fwdarw.Ce.sub.2O.sub.3+O.sub.2+H.sub.2O
CeO.sub.2+H.sub.2O.sub.2.fwdarw.Ce(OH).sub.2+O.sub.2
CeO.sub.2+2H.sub.2O.sub.2.fwdarw.Ce(OH).sub.4+O.sub.2
[0048] The aspect described above is not limited to any one enzyme.
In general, any enzyme that requires oxygen as a co-substrate can
be employed. Further, the aspect described above refers to
nanoparticles of: ceria, ceria doped and binary and tertiary
mixture of ceria with other metal oxides. Doped forms and mixtures
of ceria based materials have higher oxygen storage capacity due to
defective structures. Ceria doped particles refers to ceria doped
with any, or a combination of the following elements: platinum,
gold, palladium, manganese, osmium, gadolinium, samarium, niobium,
dysprosium, erbium, germanium, holmium, indium, iridium,
molybdenum, neodymium, rhodium, tantalum, tungsten, yttrium,
zirconium, ytterbium, thulium, terbium, and praseodymium. Mixtures
of ceria based metal oxides and ceria based composites include
binary or tertiary mixtures of ceria with any of the following
components and alike: titania, yttria, zirconia, gadolinia,
samaria, niobia, etc.
[0049] The aspects described above apply to any system in which
oxygen is required for biocatalysis and bioanalysis in conditions
of oxygen depletion. Two such examples are listed here but the
invention is not limited to those: implantable devices with
immobilized biocatalysts designed for in vivo uses where there is a
limited oxygen concentration, anaerobic fermentors for
quantification of fermentation parameters. For such applications,
the materials described herein can be used in solution or
immobilized onto solid supports. Both optical and electrochemical
detection systems can be used. Examples of solid supports are:
paper, electrodes, glass, etc.
[0050] The devices described in the present invention can be
disposable (used for one analysis), or can be used multiple times
after regeneration (several hours after use the devices regenerates
by itself by decomposing the hydrogen peroxide; slight heating can
be used to increase the decomposition rate).
[0051] The methods and devices described herein can also comprise
the direct detection of antioxidant without the presence of
hydrogen peroxide. Antioxidants can include any antioxidant that
creates a color change in the sensor, including but not limited to
ascorbic acid, gallic acid, vanillic acid, caffeic acid, trolox,
resveratrol, and/or quercetin, for example. For example, a series
of experiments were conducted using ascorbic acid to study the
interaction of ceria with an antioxidant. It was observed that when
ascorbic acid was added to 4% ceria, APTS papers, a pink color
ensued. It was of interest to determine whether this color was
concentration dependant, so 15, 10, 5, 4, 3, 2, and 1 mM ascorbic
acid were added to a series of papers and three trials for each
concentration were done. The addition of antioxidant to the ceria
sensors resulted in a color change, with the strongest response
seen at 1 mM and greater.
[0052] In another series of experiments, when ascorbic acid was
added to 4% ceria sensors, which were yellow from hydrogen peroxide
addition, the color decreased until all ceria-peroxide complexes
were broken. It was observed that when a high concentration of
H.sub.2O.sub.2 was used, such as 100 mM, the color was merely
reduced, rather than changed to pink, within the same range of
concentrations used above. In fact, this method appeared to be much
more sensitive, since a significantly large color reduction was
seen even with addition of 10 nM ascorbic acid to 100 mM H2O2
containing 4% ceria papers. In contrast, 25 uM of ascorbic acid on
the 4% ceria papers alone showed little color production.
[0053] Applications
[0054] There are many applications of this invention. The disclosed
device is particularly suitable for on-site detection in any
applications involving uses of hydrogen peroxide, antioxidants as
well as for point-of-care diagnosis. Two main areas are identified
where detection of hydrogen peroxide is important: (1)
cleaning/water monitoring where concentrations of hydrogen peroxide
are significant and (2) physiological/biomedical applications,
where hydrogen peroxide is present in lower concentrations and
often as a secondary product of enzymatic reactions. Sensor patches
for monitoring the quality of food products are also possible.
Ceria based sensor strips for the detection of the antioxidant
content (for example food antioxidants) to determine the
antioxidant capacity are also covered by this invention. Examples
of applications are provided below to illustrate the invention, but
these are not construed as limiting the scope of the invention. The
particular materials, analytes, type of sample, amounts thereof,
products, physical testing equipment in these examples, as well as
other conditions and details, are to be interpreted to apply
broadly in the art and should not be construed to unduly restrict
or limit the invention in any way.
[0055] Materials Used
[0056] Potential materials to be used in one or more of the
following examples are: (1) ceria nanoparticles -colloidal solution
with particles having a diameter ranging from 2-20 nm
(non-agglomerated, smaller size particles are preferred as they
will provide higher surface area and higher intensity of the color
and therefore lower detection limit and higher sensitivity),
hydrogen peroxide, antioxidants such as ascorbic acid, gallic acid,
vanillic acid, caffeic acid, trolox, resveratrol, quercetin, etc
Watman filter paper, glass test tube, chitosan, alginic acid,
calcium chloride (CaCl.sub.2), carboxymethylcellulose (CMC),
aminopropyltriethoxysilane (APTS), tetraethylsilane (TES),
tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate (TMOS),
sodium tripolyphosphate (NaTPP), glucose, glucose oxidase, lactate,
lactate oxidase, glutamate, glutamate oxidase, xanthine, xanthine
oxidase, billirubin, billirubin oxidase, ethanol, alcohol oxidase,
lactose, galactose, galactosidase. The optimum amounts of active
components (e.g. nanoparticles, enzymes, additives) can be varied
to tailor the desired performance of the device in the useful
concentration range according to a particular sample or
application. A suitable, optimal configuration can be determined
experimentally.
[0057] Example of Preparation of a Ceria Based Substrate.
[0058] Used here to illustrate the concept is commercial Watman
filter paper. To create ceria-modified paper as a sensing surface:
defined sensing spots of Whatman filter paper are immersed for 10
minutes in 3% cerium oxide aqueous colloidal nanoparticle solution
and then dried at 100.degree. C. The so-prepared ceria-modified
sensing paper is immersed in 5% APTS in ethanol for 10 minutes and
then dried at 100.degree. C. for 10 additional minutes. The ceria
based paper is used for measuring hydrogen peroxide containing
samples. Alteration of the amount of ceria or size of nanoparticles
can be used to tailor the sensitivity of the sensing area to the
useful concentration range, according to specific sample and
application and can be determined through experimentation.
[0059] Enzyme-modified ceria paper: fresh ceria paper prepared as
described above is soaked in 1% chitosan, prepared in 0.5% succinic
acid solution for 10 minutes, and air dried for 5-10 minutes. The
following treatment is used to immobilize the enzyme: (1) the paper
is immersed in 5% glutaraldehyde for 1 minute, (2) air dried for
5-10 minutes, (3) enzyme (e.g. 9 mg/mL glucose oxidase) is added
directly to each test spot and allowed to react for 5 minutes, (4)
rinsed with water and (5) air dried.
[0060] A Ceria-Paper Based Colorimetric Test device for Determining
Hydrogen Peroxide.
[0061] A defined spot of ceria-modified support is exposed to or
dipped in a sample containing hydrogen peroxide (e.g. cleaning or
disinfecting compositions). Upon contact, ceria is immediately
changing the color to reddish-orange depending on the amount of
hydrogen peroxide present in that composition (FIG. 2). The color
change is compared visually with that of standard ceria-based paper
spots obtained with known amounts of hydrogen peroxide or
quantified using imaging software (ImageJ, Adobe, etc) as shown in
FIG. 2.
[0062] A Ceria-Paper Based Colorimetric Test Device for Determining
Antioxidant Content.
[0063] A defined spot of ceria-modified support is exposed to or
dipped in a sample containing antioxidants (e.g. tea, food
extracts). Upon contact, ceria is immediately changing the color to
dark brown or pink-red, depending on the type of sample and
concentration of antioxidants. The color change is compared
visually with that of standard ceria-based paper spots obtained
with known amounts of antioxidants and quantified using imaging
software (ImageJ, Adobe, etc).
[0064] A Test Device for Determining Ethanol Concentrations in
Fluids and Human Breath.
[0065] A device comprising of ceria nanoparticles and alcohol
oxidase in solution, co-immobilized or fixed onto a test tube (e.g.
glass, plastic, etc) might be used to detect the presence of breath
ethanol of an alcohol-user by blowing air into the test tube.
Determination in saliva or whole serum by application of a drop of
sample onto a ceria-alcohol oxidase modified surface (e.g.
ceria-based paper as described above or a cotton swab) is also
possible. When present in the sample, ethanol diffuses thorough the
ceria-enzyme composite layer where the alcohol oxidase catalyzes
the conversion of ethanol to hydrogen peroxide. The hydrogen
peroxide that is formed will interact with the ceria nanoparticles,
changing the color. The color change and the intensity of the color
is an indication of the level of ethanol present.
[0066] A Device for Estimating Ethanol Production in Anaerobic
Fermentation Processes.
[0067] A test device as the one described above is used in a
fermentation reactor or anaerobic bioreactor to determine the
amount of ethanol produced in the fermentation process.
[0068] A Colorimetric Ceria Based Biosensor for Determining
Glucose.
[0069] Glucose oxidase-modified ceria supports are dipped in
samples containing glucose. Color develops within several minutes.
The change is visible with the naked eye (FIG. 4). The amount of
glucose is estimated by comparing the color formed with that
obtained with standard solutions of glucose. The enzymatic
biosensor is used to detect glucose in the 0.5-500 mM concentration
range with naked eye. For more precise quantification, the supports
can be photographed or scanned with a flat-bed office scanner.
Images are analyzed using imaging software (ImageJ, Adobe, etc) and
compared. This biosensor is for use in clinical or home settings.
For example, the device is suitable for analyzing glucose in body
fluids (whole blood, saliva) and in food products (e.g. juice, tea,
etc). The assay shows sensitivity for detection of physiological
glucose concentrations, it is robust, inexpensive and performs
successfully in human serum samples. One application for the
detection of physiological levels of glucose in human serum sample
is illustrated in FIG. 6.
[0070] A Colorimetric Ceria Based Biosensor for Determining
Cholesterol.
[0071] Another example is a specific ceria based colorimetric
biosensor for cholesterol detection. Here, cholesterol-oxidase is
immobilized in close contact with ceria. When cholesterol is
present, cholesterol-oxidase will convert cholesterol to hydrogen
peroxide that will then be detected by the color change of
ceria.
[0072] A Colorimetric Ceria Based Biosensor for Determining
Lactate.
[0073] In another example of a specific ceria based device is a
biosensor with lactate oxidase immobilized on a basic
ceria-modified sensor, producing hydrogen peroxide in the presence
of lactate in solution. The hydrogen peroxide will then be detected
by the ceria-based colorimetric component. The same principle can
be applied to glutamate using glutamate oxidase. Both clinical
(e.g. cerebrospinal fluid) and food samples (e.g. the level of
glutamate, used as additive in food) can be analyzed.
[0074] A Ceria Based Patch for Food Packaging to Estimate Changes
in the Redox Status Related to Food Freshness.
[0075] A ceria based sensor patch is attached to the interior of
food packaging to determine the release of free radicals or release
of hydrogen peroxide. The patch contains ceria when used to
estimate the peroxide content. In a modified version, the device
contains xanthine oxidase when used to determine the production of
free radicals. The patch is conveniently located in the food
package in a clear transparent window. If food is degraded, the
color of the patch changes to yellow-reddish depending on the
extent of degradation and the amount of peroxide or superoxide
radicals released. This patch is for use in food packages in
stores, deposits or at home. The device is suitable for monitoring
the freshness of packaged food that releases hydrogen peroxide or
free radicals, when degraded. In addition, ceria by itself has
antioxidant activity and thus a device based on this principle can
provide, in addition to detection, removal capacity of the
antioxidant formed. The invention includes the use of such patches
(or other supports) for both detection and antioxidant action
(inactivation of free radicals) purposes.
[0076] A Test Device for Determining the Amount of Enzyme
Inhibitors in a Sample.
[0077] This application is an example of analysis of chemicals that
inhibit the desired chemical or enzymatic reaction in the test
strip by preventing the formation of the color. Exemplified here is
the determination of antioxidant substances which inactivate free
radicals formed in an enzymatic reaction between xanthine (or
hypoxanthine) and xanthine oxidase. Superoxide radicals generated
in the presence of xanthine oxidase change the color of the
colorimetric ceria based component. In the presence of
antioxidants, the antioxidants will inactivate the free radicals as
they are formed, preventing the color change, to an extent
proportional to their relative concentration.
[0078] A Ceria Based Oxidase Enzyme Component Used in Anaerobic
Conditions.
[0079] A composite comprising of ceria is used to provide oxygen in
anaerobic environments (FIG. 5).
[0080] A Ceria Based Composite Material for Biofuel Cells.
[0081] A ceria based composite is used to provide oxygen in biofuel
cells. A homogenous composite comprising of ceria or doped ceria,
oxidase enzymes such as laccase, glucose oxidase, etc. in
conjunction or not with conductive materials, is used as anode or
cathode coating material in biofuel cells.
[0082] A Ceria Based Composite Material for Implantable Biosensors
(e.g. Implantable Glucose, Glutamate, Lactate Sensors).
[0083] A ceria based composite is used to provide oxygen in an
implantable sensor based on the use of oxidase enzymes (e.g.
glucose oxidase, lactate oxidase, glutamate oxidase). A homogenous
composite comprising of ceria (or doped or mixed ceria based
oxides), and an oxidase enzyme such as glucose oxidase in
conjunction or not with conductive materials such as carbon or
conductive polymers (e.g. polyaniline), is used as electrode
coating of an implantable device to be used in vivo.
[0084] Ceria Based Contact Lenses for the Detection of Glucose in
Tears.
[0085] A ceria based composite with glucose oxidase enzyme is used
to coat the surface of contact lenses to determine glucose
concentrations in tears. This is a type of wearable, non-invasive
biosensor that can be used by individuals with diabetes. All
components are biocompatible and the ceria nanoparticles have
antioxidant properties: thus it could also serve a therapeutic
function in the healing of eye wounds, be reducing levels of
reactive oxygen species, further preventing scarring and damage to
the area.
[0086] The foregoing is provided for the purpose of illustrating,
explaining and describing embodiments of the present invention.
Further modifications and adaptations to these embodiments will be
apparent to those skilled in the art and may be made without
departing from the spirit of the invention or the scope of the
following claims.
* * * * *