U.S. patent application number 14/870111 was filed with the patent office on 2016-02-25 for biosensors utilizing ink jet-printed biomolecule compatible sol gel inks and uses thereof.
This patent application is currently assigned to MCMASTER UNIVERSITY. The applicant listed for this patent is John D. Brennan, Carlos Filipe, Zakir Hossain, Julie Lebert, Roger Luckham, Robert Pelton, Anne Marie Smith. Invention is credited to John D. Brennan, Carlos Filipe, Zakir Hossain, Julie Lebert, Roger Luckham, Robert Pelton, Anne Marie Smith.
Application Number | 20160054310 14/870111 |
Document ID | / |
Family ID | 43222096 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160054310 |
Kind Code |
A1 |
Brennan; John D. ; et
al. |
February 25, 2016 |
BIOSENSORS UTILIZING INK JET-PRINTED BIOMOLECULE COMPATIBLE SOL GEL
INKS AND USES THEREOF
Abstract
Novel solid-phase biosensors that utilize ink jet printing of
biocompatible sol-gel based inks to create sensor strips are
reported herein. Biomolecules and other reagents useful in
bioassays to detect, for example, pathogenic microorganisms or
toxic substances, are immobilized on a substrate, which can be
paper based, by layering these substances between two layers of
biomolecule compatible sol gel. The sol gel precursor solutions and
solutions of the assay reagents are printed from separate nozzles
in a layered approach which avoids clogging of the nozzles by the
pre-mature gelling of the sol gel precursor solution. In certain
embodiments of the application, a capture agent is used to
concentrate a compound to be detected in specific areas on the
substrate to facilitate detection.
Inventors: |
Brennan; John D.; (Dundas,
CA) ; Filipe; Carlos; (Ancaster, CA) ; Pelton;
Robert; (Dundas, CA) ; Hossain; Zakir;
(Hamilton, CA) ; Luckham; Roger; (Hamilton,
CA) ; Smith; Anne Marie; (Ancaster, CA) ;
Lebert; Julie; (London, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Brennan; John D.
Filipe; Carlos
Pelton; Robert
Hossain; Zakir
Luckham; Roger
Smith; Anne Marie
Lebert; Julie |
Dundas
Ancaster
Dundas
Hamilton
Hamilton
Ancaster
London |
|
CA
CA
CA
CA
CA
CA
CA |
|
|
Assignee: |
MCMASTER UNIVERSITY
Hamilton
CA
|
Family ID: |
43222096 |
Appl. No.: |
14/870111 |
Filed: |
September 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13322951 |
Feb 13, 2012 |
9157109 |
|
|
PCT/CA2010/000802 |
May 31, 2010 |
|
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14870111 |
|
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|
61182389 |
May 29, 2009 |
|
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Current U.S.
Class: |
435/20 ; 427/258;
427/379; 435/287.9; 435/34 |
Current CPC
Class: |
C12Q 1/46 20130101; G01N
33/569 20130101; B05D 7/584 20130101; G01N 33/552 20130101; G01N
33/548 20130101; G01N 2333/924 20130101; G01N 2333/938 20130101;
C12Q 1/001 20130101; G01N 33/5436 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/569 20060101 G01N033/569; B05D 7/00 20060101
B05D007/00; C12Q 1/46 20060101 C12Q001/46 |
Claims
1. A biosensor comprising: (a) a substrate; (b) at least one
reaction zone immobilized on the substrate, the reaction zone
comprising, in order, beginning adjacent to the substrate: (i) a
first biomolecule compatible sol gel layer; (ii) a recognition
element layer; and (iii) a second biomolecule compatible sol gel
layer; and (c) a detection means, wherein the first and second
biomolecule compatible sol gel layers and the recognition element
layer are immobilized on the substrate using ink jet printing.
2. A biosensor comprising: (a) a substrate having a first and
second end; (b) at least one reaction zone immobilized on the
substrate, the reaction zone comprising, in order, beginning
adjacent to the substrate: (i) a first biomolecule compatible sol
gel layer; (ii) a recognition element layer; and (iii) a second
biomolecule compatible sol gel layer, wherein the first and second
biomolecule compatible sol gel layers and the recognition element
layer are immobilized on the substrate using ink jet printing; and
(c) a detection means, wherein immersion of the first end of the
substrate in a solution comprising or suspected of comprising an
analyte results in lateral flow of the solution from the first end
of the substrate to the second end by capillary action and flow
through the at least one reaction zone results in reaction of the
analyte with the recognition element, the reaction being detected
by the detection means.
3. The biosensor of claim 1, wherein substrate comprises a
substantially planar surface, and is made of a material that
supports lateral flow of a solution.
4. The biosensor of claim 3, wherein the substrate is made from
paper or paper-based material.
5. The biosensor of claim 1, wherein the detection means comprises
a compound to be detected by colormetric methods.
6. The biosensor of claim 5, wherein the compound to be detected is
generated in one of the reaction zones and this reaction zone
further comprises a capture agent.
7. The biosensor of claim 6 wherein the capture agent is a chemical
compound having affinity for the compound to be detected.
8. The biosensor of claim 7, wherein the capture agent is printed
as a layer under the first sol gel layer, adjacent to the
substrate.
9. The biosensor of claim 6, wherein the capture agent is a
physical barrier printed on the substrate around the reaction zone
generating the product to be detected.
10. The biosensor of claim 1, wherein the biomolecule compatible
sol gel is prepared from a sodium silicate precursor solution.
11. The biosensor of claim 1, wherein the ink jet printing is
piezoelectric ink jet printing.
12. A biosensor for the detection of microorganisms having an
intrinsic or recombinant .beta.-glucuronidase or
.beta.-galactosidase enzyme comprising: (a) a substrate having a
first and second end; (b) a first reaction zone immobilized on the
substrate comprising in order, beginning adjacent to the substrate:
(i) a first biomolecule compatible sol gel layer; (ii) an oxidizing
agent; and (iii) a second biomolecule compatible sol gel layer; (c)
a second reaction zone immobilized on the substrate comprising in
order, beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) a chromogenic substrate for the
enzyme and (iii) a second biomolecule compatible sol gel layer;
wherein immersion of the first end of the substrate in a solution
comprising or suspected of comprising the microorganisms, and that
has been treated to lyse the microorganisms, results in lateral
flow of the solution from the first end of the substrate to the
second end of the substrate by capillary action, the flow passing
through the first reaction zone prior to passing through the second
reaction zone, and the chromogenic substrate for the enzymes is one
that, when reacted with the enzyme produces a product that is
oxidized by the oxidizing agent to a colored product that is
detected.
13. A biosensor for the determining AChE activity or for assaying
for AChE modulators comprising: (a) a substrate having a first and
second end; and (b) a first reaction zone immobilized on the
substrate comprising in order, beginning adjacent to the substrate:
(i) a cationic polymer; (ii) a first biomolecule compatible sol gel
layer; (iii) AChE and DTNB; and (iv) a second biomolecule
compatible sol gel layer.
14. A biosensor for the determining AChE activity or for assaying
for AChE modulators comprising: (a) a substrate having a first and
second end; (b) a first reaction zone immobilized on the substrate
comprising in order, beginning adjacent to the substrate: (i) a
first biomolecule compatible sol gel layer; (ii) IPA; and (iii) a
second biomolecule compatible sol gel layer; and (c) a second
reaction zone immobilized on the substrate comprising in order,
beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) AChE; and (iii) a second biomolecule
compatible sol gel layer; wherein the first and second reaction
zones are arranged so that during lateral flow of a solution from
the first end of the substrate to the second end by capillary
action, the solution passes through the first reaction zone prior
to passing through the second reaction zone.
15. A method of detecting one or more analytes in a sample, wherein
the sample comprises or is suspected of comprising the one or more
analytes, the method comprising contacting the sample with a
biosensor of claim 1 and monitoring the detection means for a
positive or negative result, wherein a positive result indicates
the presence of the one or more analytes in the sample.
16. A method for determining if one or more analytes are modulators
of a functional biomolecule comprising: (a) contacting a solution
comprising the one or more analytes with a reaction zone on a
biosensor of claim 1, wherein the reaction zone comprises the
functional biomolecule; (b) contacting the reaction zone with a
substrate for the functional biomolecule; (c) monitoring the
detection means for a positive or negative result; and (d)
comparing the positive or negative result in (c) with a control
biosensor, wherein a positive or negative result in (c) that is
different from the control indicates that the one or more analytes
are modulators of the functional biomolecule.
17. A kit comprising a biosensor of claim 1.
18. A method for preparing the biosensor of claim 1 comprising: (a)
depositing a first biomolecule compatible sol gel precursor
solution in a first reaction zone on a substrate using ink jet
printing and allowing the first sol gel precursor solution to dry;
(b) depositing a solution comprising the recognition element on top
of the first biomolecule compatible sol gel precursor using ink jet
printing and allowing the solution comprising the recognition
element to dry; (c) depositing a second biomolecule compatible sol
gel precursor solution on top of the recognition element using an
ink jet printer and allowing the second sol gel precursor solution
to dry.
19. The method of claim 18 further comprising depositing a solution
comprising a capture agent in the first reaction zone onto the
substrate using ink jet printing prior to depositing the first
biomolecule compatible sol gel precursor solution.
20. The method of claim 18, further comprising depositing a
physical barrier around the first reaction zone.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/322,951, filed Nov. 29, 2011, which claims
the benefit of international application no. PCT/CA2010/00802,
filed May 31, 2010, which claims the benefit of provisional patent
application No. 61/182,389, filed May 29, 2009, the contents of
each of which are herein incorporated by reference.
FIELD OF THE APPLICATION
[0002] The present application is in the field of biosensors, in
particular biosensors comprising reaction zones in which
recognition molecules or other assay components are immobilized
using biomolecule compatible sol gels and the sol gels, recognition
molecules and other assay components are printed on a substrate
using ink-jet printing.
BACKGROUND OF THE APPLICATION
[0003] Recently, paper-based patterned solid-phase sensors (which
are simple, portable, disposable and inexpensive) have been
developed to run multiple bioassays and controls
simultaneously..sup.ivii These portable biosensing papers are
extremely useful in remote settings as well as less industrialized
countries where simple bioassays are essential in the first stages
of detecting disease, and for monitoring environmental and food
based toxins.
[0004] Several conventional deposition techniques such as dip
coating,.sup.viii spin coating,.sup.ix aerosol spraying, and
electrophoretic deposition.sup.xi have previously been used to
deposit bioactive sol-gel derived materials. Among these, dip and
spin coating are not practical for large-scale production. In
addition, they are time consuming and are wasteful when dealing
with expensive bioreagents. Aerosol spraying can be used for
deposition of biomaterials, but is not easily adaptable to
formation of millimeter scale patterns or for precise control of
sol gel deposits. Electrophoretic deposition is normally used for
fabrication of electrodes and the process requires an electrically
conductive surface..sup.xii
[0005] It has been shown that entrapment of biomolecules within
sol-gel derived materials allows proteins to retain their
bioactivity for prolonged periods of time..sup.xiii,xiv
Furthermore, sol-gel based materials have previously been shown to
be amenable to ink jet deposition (although not with
proteins).sup.xv or screen printing with entrapped
enzymes..sup.xvi
[0006] Ink jet printing has been used to dispense, deposit or
pattern, in either 2D or 3D arrangements
cells/tissue,.sup.xviii,xviii DNA,.sup.xix antibodies,.sup.x and
enzymes..sup.ii,xv,xviii
SUMMARY OF THE APPLICATION
[0007] Novel solid-phase biosensors that utilize ink jet printing
of biocompatible sol-gel based inks to create sensor strips have
been developed. In particular, two assays, utilizing two different
colorimetric detection methods to monitor the activity of the
enzyme, acetylcholinesterase (AChE) have been developed, along with
an assay to detect the presence of microorganisms such as E. coli
and total coliform bacteria. The assays all utilize biomolecule
compatible sol gel matrixes that have been printed in specific
configurations onto substrates, in particular substrates that
support lateral flow of solutions. The sol gel matrixes are used to
immobilize certain recognition elements that are appropriate for
the assay to be performed, for example enzymes, functional nucleic
acids or other functional biomolecules, substrates for these
biomolecules and/or compounds used for detection. Advantageously,
the recognition elements are printed in between two layers of the
biomolecule compatible sol gel matrix. In certain embodiments, for
example, when a compound is generated during the assay that is used
for detection purposes, the "sol-gel/recognition element/sol-gel"
configuration further includes a capture means which is used to
restrict movement of the compounds to be detected, improving assay
sensitivity. In the assays reported herein, colorimetric detection
was utilized (although a person skilled in the art would appreciate
that other detection means could also be used) and was achieved
either by eye, using a digital camera and image analysis software,
or using an office scanner, avoiding the need for expensive and
sophisticated instrumentation. The biosensors developed herein
could be used either as a dipstick or a lateral flow sensor,
although lateral flow sensors are a particularly advantageous
embodiment. The use of sol gel based entrapment produced sensors
that retained activity and gave reproducible results after storage
at 4.degree. C. for at least 60 days, making these systems suitable
for storage and use in the field.
[0008] Accordingly, the present application includes a biosensor
comprising: [0009] (a) a substrate; [0010] (b) at least one
reaction zone immobilized on the substrate, the reaction zone
comprising, in order, beginning adjacent to the substrate: (i) a
first biomolecule compatible sol gel layer; (ii) a recognition
element layer; and (iii) a second biomolecule compatible sol gel
layer; and [0011] (c) a detection means, wherein the first and
second biomolecule compatible sol gel layers and the recognition
element layer are immobilized on the substrate using ink jet
printing.
[0012] The present application also includes a biosensor
comprising: [0013] (a) a substrate having a first and second end;
[0014] (b) at least one reaction zone immobilized on the substrate,
the reaction zone comprising, in order, beginning adjacent to the
substrate: (i) a first biomolecule compatible sol gel layer; (ii) a
recognition element layer; and (iii) a second biomolecule
compatible sol gel layer, wherein the first and second biomolecule
compatible sol gel layers and the recognition element layer are
immobilized on the substrate using ink jet printing; and [0015] (c)
a detection means, wherein immersion of the first end of the
substrate in a solution comprising or suspected of comprising an
analyte results in lateral flow of the solution from the first end
of the substrate to the second end by capillary action and flow
through the at least one reaction zone results in reaction of the
analyte with the recognition element, the reaction being detected
by the detection means.
[0016] In an embodiment of the application, certain reaction zones
further include an additional layer comprising a capture agent. The
reaction zones that benefit from the presence of a capture agent
are those that produce a product to be detected, this product being
comprised in the detection means. The capture agent serves to
restrict movement of the product, thereby concentrating the product
in the reaction zone to facilitate detection. When the capture
agent is a chemical compound, it is an embodiment of the
application that the capture agent is printed as a layer under the
first sol gel layer (i.e. adjacent to the substrate). The capture
agent, in alternate embodiments, is a physical barrier printed on
the substrate around the reaction zones that produce a product to
be detected.
[0017] The present application also includes a biosensor for the
detection of microorganisms having an intrinsic or recombinant
.beta.-glucuronidase or .beta.-galactosidase enzyme comprising:
[0018] (a) a substrate having a first and second end; [0019] (b) a
first reaction zone immobilized on the substrate comprising in
order, beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) an oxidizing agent; and (iii) a
second biomolecule compatible sal gel layer; [0020] (c) a second
reaction zone immobilized on the substrate comprising in order,
beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) a chromogenic substrate for the
enzyme and (iii) a second biomolecule compatible sol gel layer;
wherein immersion of the first end of the substrate in a solution
comprising or suspected of comprising the microorganisms and that
has been treated to lyse the microorganisms, results in lateral
flow of the solution from the first end of the substrate to the
second end by capillary action, the flow passing through the first
reaction zone prior to passing through the second reaction
zone.
[0021] In an embodiment, the first and second biomolecule
compatible sol gel layers, the oxidizing agent and the chromogenic
substrate for the substrate are immobilized on the substrate using
ink jet printing of solutions comprising these substances, or in
the case of the sol gels, precursors for these substances.
[0022] The chromogenic substrate for the enzymes is one that, when
reacted with the enzyme produces a product that is oxidized by the
oxidizing agent to a colored product that is detected. In an
embodiment, the chromogenic substrate for .beta.-glucuronidase is
5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-GLUC) and the
chromogenic substrate for .beta.-galactosidase is
bromo-chloro-indolyl-galactopyranoside (X-GAL).
[0023] The present application also includes a biosensor for the
determining AChE activity or for assaying for AChE modulators
comprising: [0024] (a) a substrate having a first and second end;
and [0025] (b) a first reaction zone immobilized on the substrate
comprising in order, beginning adjacent to the substrate: (i) a
cationic polymer; (ii) a first biomolecule compatible sol gel
layer; (iii) AChE and DTNB; and (iv) a second biomolecule
compatible sol gel layer.
[0026] The present application includes an alternate biosensor for
the determining AChE activity or for assaying for AChE modulators
comprising: [0027] (a) a substrate having a first and second end;
[0028] (b) a first reaction zone immobilized on the substrate
comprising in order, beginning adjacent to the substrate: (i) a
first biomolecule compatible sol gel layer; (ii) IPA; and (iii) a
second biomolecule compatible sol gel layer; and [0029] (c) a
second reaction zone immobilized on the substrate comprising in
order, beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) AChE; and (iii) a second biomolecule
compatible sol gel layer; wherein the first and second reaction
zones are arranged so that during lateral flow of a solution from
the first end of the substrate to the second end by capillary
action, the solution passes through the first reaction zone prior
to passing through the second reaction zone.
[0030] In an embodiment, the first and second biomolecule
compatible sol gel layers, the cationic polymer, the AChE, the DTNB
and the IPA are immobilized on the substrate using ink jet printing
of solutions comprising these substances, or in the case of the sol
gels, precursors for these substances.
[0031] The present application also includes assay methods that
utilize the biosensor of the present application. In an embodiment,
the assay is a method of detecting one or more analytes in a
sample, wherein the sample comprises or is suspected of comprising
the one or more analytes, the method comprising contacting the
sample with the biosensor of the application and monitoring the
detection means for a positive or negative result, wherein a
positive result indicates the presence of the one or more analytes
in the sample. In an embodiment of the application, the detection
means is a colormetric method and the positive result is a presence
of a color change on the biosensor.
[0032] In another embodiment, the present application also includes
a method for determining if one or more analytes are modulators of
a functional biomolecule comprising: [0033] (a) contacting a
solution comprising the one or more analytes with a reaction zone
on a biosensor of the application, wherein the reaction zone
comprises the functional biomolecule; [0034] (b) contacting the
reaction zone with a substrate for the functional biomolecule;
[0035] (c) monitoring the detection means for a positive or
negative result; and [0036] (d) comparing the positive or negative
result in (c) with a control biosensor, wherein a positive or
negative result in (c) that is different from the control indicates
that the one or more analytes are modulators of the functional
biomolecule.
[0037] In an embodiment of the application, the detection means is
a colormetric method and the presence of a color change on the
biosensor that is different from that on the control biosensor
indicates that the one or more analytes are modulators of the
functional biomolecule.
[0038] The present application further includes kits comprising the
biosensors of the application. In an embodiment, the kit includes
the biosensor and any further reagents for performing an assay
using the biosensor. In a further embodiment, the kit includes
instructions for using the biosensor in the assay and any controls
needed to perform the assay. The controls may be on the biosensor
itself, or alternatively, on a separate substrate. In a further
embodiment the kit includes all of the components required to
perform any of the assay methods of the present application.
[0039] The present application also includes a method for preparing
a biosensor of the application comprising: [0040] (a) depositing a
first biomolecule compatible sol gel precursor solution in a first
reaction zone on a substrate using ink jet printing and allowing
the first sol gel precursor solution to dry; [0041] (b) depositing
a solution comprising the recognition element on top of the first
biomolecule compatible sol gel precursor using ink jet printing and
allowing the solution comprising the recognition element to dry;
[0042] (c) depositing a second biomolecule compatible sol gel
precursor solution on top of the recognition element using an ink
jet printer and allowing the second sol gel precursor solution to
dry.
[0043] In an embodiment, the method comprises depositing a solution
comprising a capture agent in the first reaction zone onto the
substrate using ink jet printing prior to depositing the first
biomolecule compatible sol gel precursor solution or it further
comprises depositing a physical barrier around the first reaction
zone.
[0044] Other features and advantages of the present application
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples while indicating preferred embodiments of the
application are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
application will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The application will now be described in greater detail with
reference to the drawings in which:
[0046] FIG. 1 is a schematic representation of the detection
principle of the Ellman assay. Acetylcholinesterase (AChE)
hydrolyzes the acetylthiocholine (ATCh) and forms thiocholine
(TCh), which then reacts with dithiobisnitrobenzoate (DTNB) to
generate 5-thio-2-nitrobenzoate (TNB, an anion), which is yellow in
color.
[0047] FIG. 2 shows the topography of ink jet sprayed PVAm, and
AChE (50 U/mL) and DTNB (500 .mu.M) doped sodium silicate (SS) thin
films on paper. (A) Profilometry images of paper that is coated
with or without PVAm only (a), and both PVAm and the
silica/AChE+DTNB/silica layers (b). (B) SEM images of unmodified
(a), modified with PVAm only (b), and modified with PVAm, and AChE
and DTNB doped silica matrix on paper (c). Unmodified paper surface
was rough, while the modified surfaces were relatively smooth.
[0048] FIG. 3 shows an illustration of ink jet printing sequence of
PVAm, sodium silicate (SS) based sol-gel derived silica matrix, and
the tris buffer (100 mM, pH 8.0) containing enzyme
acetylcholinesterase (AChE) and dithiobisnitrobenzoate (DTNB)
layers on paper for development of a portable solid-phase
biosensor.
[0049] FIG. 4 shows the dose-dependent effects of acetylthiocholine
(ATCh) in the presence (a) and absence (b) of AChE (50 U/mL) doped
silica matrix on paper, in which PVAm (0.5 wt. %) was printed prior
to printing of the silica and AChE layers. Inset is the color
intensity (CI) generated at each ATCh concentration. All points are
means.+-.s.d. of five independent experiments for each
concentration.
[0050] FIG. 5 shows the effects of cationic PVAm on entrapment as
well as preservation the anionic TNB in lateral flow-based paper
chromatographic system. (a) The values of retardation factor (Rf)
for anionic TNB in Milli-Q water in the presence of indicated PVAm
levels. (b) Colour intensity (CI) due to elution of ATCh (300
.mu.M, final conc.) in the lateral flow based platform. The areas
within the dashed boxes were printed without (control) and with
PVAm (0.5 wt. %) followed by printing of AChE (50 U/mL). PVAm
concentrates the reaction product, the yellow TNB anion, while in
the control experiment, the yellow TNB anion is dispersed over a
large area. (c) Cardboard dipstick with ink jet printed PVAm or
control (no PVam) and silica/AChE/DTNB/Silica layers after being
immersed in ATCh solution. Both materials show an initial color
response, but only the PVAm (0.5 wt %)-treated paper retains the
reaction product (a yellow TNB.sup.-) for a period of 3 weeks,
while the PVAm untreated paper failed to retain the color.
[0051] FIG. 6 demonstrates the dose-dependent inhibition of
acetylcholinesterase (AChE) by various concentrations of paraoxon
(a). Insets are the color intensity (CI) at each paraoxon
concentration and dose-dependent inhibition responses with the
lower levels of paraoxon. (b) Semi log plot of data in panel (a).
PVAm and AChE (50 U/mL) and DTNB doped silica layers were printed
on paper before conducting experiments. All points are
means.+-.s.d. of five measurements for each concentration.
[0052] FIG. 7 shows: (a) Dose-dependent inhibition effects of
aflatoxin B1 on AChE activity. Insets are the color intensity (CI)
at each aflatoxin B1 concentration and dose-dependent inhibition
responses with the lower levels of aflatoxin B1; (b) Semi log plot
of the data shown in Panel (a). Data are means.+-.s.d. of five
independent measurements for each concentration.
[0053] FIG. 8 shows a schematic diagram of the detection principle
of the Indophenyl Acetate (IPA)-based colorimetric assay.
Acetylcholinesterase (AChE) hydrolyzes the red-yellow colored
substrate IPA at a basic condition (pH 8.0) and forms indophenoxide
anion, which is blue-purple in color. (b) Schematic illustration
for the development of the reagentless bioactive paper-based
lateral flow sensor in which AChE and IPA were entrapped in the two
dashed boxes regions of Whatman 1 paper strip (1.times.10 cm)
following the sequences of PVAm/silica/AChE/silica and
silica/IPA/silica, respectively by using either the ink jet
printing or over spotting. The sensor then can be used two
different ways: (1) directly (normal lateral flow-based
chromatography) without incubating the contaminated sample, and (2)
inverted lateral flow-based chromatography with incubation the
sample.
[0054] FIG. 9 shows the effects of cationic PVAm on entrapment of
indophenoxide anion in lateral flow-based paper chromatographic
system. Colour intensity (CI) due to elution of IPA (3 mM, final
conc.) in the lateral flow based platform. The areas within the
dashed boxes were printed/over spotted without (control) and with
PVAm (0.5 wt. %) followed by printing/overspotting of AChE (50
U/mL). PVAm concentrates the reaction product, the blue
indophenoxide anion, while in the control experiment; the blue
indophenoxide anion is dispersed over a large area. (b) Proof of
concept for the development of reagentless bioactive paper-based
lateral flow platform, in which the sensor was dipped into
dH.sub.2O to bring up the IPA reagent into sensing region for the
generation of blue color.
[0055] FIG. 10 shows the optimization of [AChE] for the development
of paper-based lateral flow sensor. (a) Color intensity at
different [AChE] in the presence of IPA (3 mM) on paper (b) Effects
of [IPA] in the presence of AChE (500 U/mL) doped SS sol-gel matrix
on paper. All points are means.+-.s.d. of four measurements for
each concentration
[0056] FIG. 11 shows the dose-dependent inhibition of
acetylcholinesterase (AChE) by various concentrations of carbamate
(A) and organophosphate (B) pesticides. A-(a) and A-(c) show the
dose-dependent inhibition responses of bendiocarb and carbaryl,
respectively. A-(b) and A(d) show the semi log plots of data in
panels A-(a) and A-(c), respectively. B-(a) and B-(c) show the
dose-dependent inhibition responses of paraoxon and malathion,
respectively. B-(b) and B-(d) show the semi log plots of data in
panels B-(a) and B-(c), respectively. All points are means.+-.s.d.
of four measurements for each concentration
[0057] FIG. 12 shows: (a) Matrix effect in the analysis of paraoxon
in milk and apple juice samples. Color intensity decreased with the
increased standard paraoxon concentration in milk and apple juice;
(b) Real life application of paraoxon, where different
concentration of paraoxon solution was sprayed on apple and head
lettuce, respectively. After air dry, the deposited paraoxon
samples were collected and tested using our developed reagentless
sensor.
[0058] FIG. 13 shows (a) Schematic diagram of the detection
principle of the 3-D-glucoronide (X-Gluc)-based colorimetric assay.
The chromogenic substrate, X-GLUC is hydrolyzed by GUS to form a
dark blue indigo dye. (b) Schematic illustration for the
development of the bioactive paper-based lateral flow E. coli
sensor in which X-Gluc and FeCl.sub.3 were entrapped in the two
dashed boxes regions on a Whatman #1 paper strip (1.times.10 cm)
following the sequences of PVAm/silica/X-Gluc/silica and
silica/FeCl.sub.3/silica, respectively by using either the ink jet
printing or over spotting. A hydrophobic barrier using either MTMS
or wax was introduced over the top of the sensing zone to prevent
leaching color and/or increase signal intensity. The sensor is then
dipped into pre-lysed contaminated sample (cell lysate).
[0059] FIG. 14 shows the effects of X-Gluc, pH, oxidizing agent
(e.g., FeCl.sub.3), and drying time for the development of the
paper-based lateral flow E. coli sensor. Color intensity at
different (a) [X-Gluc], (b) pH, and (c) [FeCl.sub.3] in the
presence of GUS (final concentration 1 U/mL) on paper. Curve A,
drying time 5 min; curve B, drying time 30 min; and curve C, drying
time 60 min. All points are means.+-.s.d. of four independent
experiments for each concentration/level and all paper assays used
0.5 wt % PVAm as a capture agent.
[0060] FIG. 15 shows detection of E. coli BL21 using both
non-patterned and patterned paper strips. (a), (b) Color intensity
with varying E. coli concentrations using non-patterned test trips.
Insets are the color intensity (CI) generated at each cell
concentration and the images were taken using office scanner (a),
and the camera (b) for the same experiment. (c) Different
concentrations of E. coli were detected using patterned paper
sensor. All points are means.+-.s.d. of four independent
experiments for each concentration.
[0061] FIG. 16 shows detection of E. coli from coculture. B.
subtiliss (.about.4.1.times.10.sup.6 CFU/mL) and E. coli BL21
(4.1.times.10.sup.6 CFU/mL) are mixed together, lyzed (by using
B-PER Direct lysing reagent) and tested both in solution assay (a)
and by using the paper strips (b). In the case of bacillus alone-no
color was observed in solution while in the case of co-culture,
color was observed in both the assay systems.
[0062] FIG. 17 shows detection of cell in the food samples, in
which both 1% milk (a) and orange juice (b) were artificially
contaminated with E. coli BL21 (4.1.times.10.sup.5 CFU/mL),
respectively. The contaminated samples were then treated with B-PER
Direct lysing reagent and tested using the bioactive paper sensor.
In the negative control experiments, substrate (X-Gluc) was
absent.
DETAILED DESCRIPTION OF THE APPLICATION
Definitions
[0063] The following definitions, unless otherwise stated, apply to
all aspects and embodiments of the present application, including
each independent embodiment described under separate headings
hereinbelow.
[0064] By "biomolecule-compatible" it is meant that the silica sol
gel either stabilizes proteins, and/or other biomolecules against
denaturation or does not facilitate denaturation.
[0065] The term "biomolecule" as used herein means any of a wide
variety of proteins, enzymes, organic and inorganic chemicals,
other sensitive biopolymers including DNA and RNA, and complex
systems including whole or portions of plants, animals,
microorganisms and cells.
[0066] The term "substrate" as used herein refers to any solid
support to which biomolecule compatible sol gel matrixes or other
chemical entities can be adhered. In an embodiment of the
application, the substrate comprises a substantially planar
surface, and is made of any material that supports lateral flow of
a solution. When the solution is aqueous based, the substrate is
hydrophilic in nature. Conversely, when the solution is
non-aqueous, the substrate is hydrophobic in nature. For aqueous
solutions, therefore, the substrate may be made from, for example,
a paper based material. For non-aqueous solutions, the substrate
may be made from materials that are naturally hydrophobic, or that
have been treated, for example by derivatization with hydrophobic
groups, to make them hydrophobic. In further embodiments, the
substrate is made from paper, glass, plastic, polymers, metals,
ceramics, alloys or composites. In another embodiment, the
substrate is made from paper or a paper-based material. In still
other embodiments, the substrate is in the shape of a rectangular
test strip, with the first and second ends being opposed to each
other.
[0067] The term "paper" or "paper-based material" as used herein
refers to a commodity of thin material produced by the amalgamation
of fibers, typically plant fibers composed of cellulose, which are
subsequently held together by hydrogen bonding. While the fibers
used are usually natural in origin, a wide variety of synthetic
fibers, such as polypropylene and polyethylene, may be incorporated
into paper as a way of imparting desirable physical properties. The
most common source of these kinds of fibers is wood pulp from
pulpwood trees. Other plant fiber materials, including those of
cotton, hemp, linen and rice, may also be used. The paper may be
hydrophilic or hydrophobic, may have a surface coating, may
incorporate fillers that provide desirable physical properties and
may be previously modified prior to coating with the ink jet
deposited sol-gel materials, by, for example, precoating with a
hydrophilic, hydrophobic or charged polymer layer of organic or
inorganic origin.
[0068] As used herein, the term "immobilized" of "entrapped" or
synonyms thereof, means that movement of the referenced component
of the biosensor, is restricted. Immobilization can be accomplished
by physical means such as barriers, electrostatic interactions,
hydrogen-bonding, bioaffinity, covalent interactions or
combinations thereof.
[0069] The term "recognition element" refers to a chemical agent or
a combination of chemical agents that specifically reacts with,
interacts with or binds to the analyte and is immobilized on the
biosensor, between two layers of biomolecule compatible sol gel. In
an embodiment, the recognition element comprises a functional
biomolecule that acts on a substrate that is the analyte or,
conversely, the recognition element comprises a substrate for a
functional biomolecule that is present in the analyte.
[0070] The term "functional biomolecule" refers to molecules,
typically found in biological systems, that act or interact with
substrates to modify the substrate in some detectable way. Examples
of functional biomolecules include enzymes, DNA aptamers, RNA
aptamers, PNA aptamers, DNA enzymes, RNA enzymes, DNA aptazymes and
RNA aptazymes, and combinations thereof.
[0071] The term "capture agent" as used herein refers to a means
for immobilizing or restricting the movement of a component of the
biosensor. In particular, the capture agent restricts the movement
of a compound to be detected, for example by colorimetric
detection. By restricting the movement of a compound to be
detected, this compound is more concentrated in a localized area
which facilitates detection. In an embodiment, the capture agent is
(bio)chemical agent that has affinity for the compound to be
detected. For example, if the compound to be detected is an
ionically charged compound, the capture agent is a chemical agent
having an ionic charge that is opposite to the compound. Selection
of suitable chemical capture agents would be within the abilities
of a person skilled in the art based on the identity of the
compound to be detected. In an embodiment, the compound to be
detected is comprised in the detection means. In another
embodiment, the chemical capture agents are comprised as a layer
located below the first sol gel layer in a reaction zone where the
compound to be detected is generated by reaction of the analyte
with the recognition element. In another embodiment, the chemical
capture agent is an ionic polymer that is printed onto the
substrate below the first sol gel layer or, alternatively, is
associated with the sol gel matrix. For example, when the compound
to be detected is a cationic compound, such as, thionitrobenzoate
(TNB) or indophenoxide anion, or a compound that possesses a
certain anionic charge, such as ClBr-Indigo dye, a suitable
chemical capture agent is a cationic polymer such as polyvinylamine
(PVAm). In other embodiments of the application, the capture agent
is selected from any of a wide variety of small molecules,
proteins, peptides, enzymes and other sensitive (bio)polymers
including DNA and RNA, and complex systems including whole plants,
animals, microorganisms and cells, or portions thereof. Suitable
capture agents, including antibodies, other proteins, DNA, DNA
aptamers, RNA, RNA aptamers, complexing agents such as EDTA,
charged polymers such as polyvinylamine, or molecularly imprinted
polymers etc., are well known to those skilled in the art.
Particular examples of capture agents are, but not limited to,
charged polymers: poly(vinylamine), poly(allylamine),
poly(ethyleneimine), polylysine, polyarginine, poly(acrylic acid),
and poly(glutamic acid). The capture agents may also be
heteropolymers, block co-polymers, or other macromolecules and may
be further modified, for example with biotin, so that they can
interact effectively with streptavidin.
[0072] In yet another embodiment of the application, the capture
agent is a physical barrier that is printed onto the substrate
either before or after the printing of the one or more reaction
zones. For example, the barrier can be a wax ink that is printed on
the substrate in an area that will result in entrapment of the
compound to be detected in a specified area around the reaction
zone.
[0073] The term "analyte" as used herein means any agent,
including, but not limited to, small inorganic and organic
molecules, biopolymers such as carbohydrates, lipids, DNA, RNA,
peptides proteins, cells and micorganisms, for which one would like
to sense or detect using a biosensor of the present application.
The analyte may be isolated from a natural source or be synthetic.
The term analyte also includes mixtures of compounds or agents such
as, but not limited to, combinatorial libraries and samples from an
organism or a natural environment.
[0074] The term "sample(s)" as used herein means refers to any
material that one wishes to assay using the biosensor of the
application. The sample may be from any source, for example, any
biological (for example human or animal medical samples),
environmental (for example water or soil) or natural (for example
plants) source, or from any manufactured or synthetic source (for
example foods and drinks). It is most convenient for the sample to
be a liquid or dissolved in a suitable solvent to make a solution.
For quantitative assays, the amount of sample in the solution
should be known. The sample is one that comprises or is suspected
of comprising one or more analytes.
[0075] The term "detection means" as used herein refers to a means
to detect the presence of an analyte. Detection can be performed
using any available method, including, for example, colorimetric,
electrochemical and/or spectroscopic methods. Conveniently,
detection is performed using colorimetric methods including both
visual and analytical, using digital imagery. The detection means
can simply be detection of the direct product formed, for example,
by reaction or interaction of a functional biomolecule with a
substrate (with either the functional biomolecule or the substrate
being the analyte), if the product being formed possesses a color
(or any signal) that is intense enough to be detected and that is
distinct from the color (or signal) of any of the starting
reagents. In this embodiment, the detection means is not a separate
component of the biosensor, but is instead formed during the assay
and therefore is an inherent part of the biosensor. In a further
embodiment the detection means comprises the compound to be
detected. In a further embodiment, the detection means comprises a
separate entity that reacts or interacts with the direct product
formed by reaction of, for example, a functional biomolecule with a
substrate (with either the functional biomolecule or the substrate
being the analyte), the reaction with the separate entity resulting
in a distinct detectable signal. When the detection means comprises
a separate entity, it can be located in its own reaction zone or
combined with the recognition element.
[0076] The term "organic polyol" as used herein refers to an
organic compound having more than one hydroxy or "OH" group. In an
embodiment of the present application, the organic polyol is
selected from sugar alcohols, sugar acids, saccharides,
oligosaccharides and polysaccharides. Simple saccharides are also
known as carbohydrates or sugars. Carbohydrates may be defined as
polyhydroxy aldehydes or ketones or substances that hydrolyse to
yield such compounds. The polyol may be a monosaccharide, the
simplest of the sugars or a carbohydrate. The monosaccharide may be
any aldo- or keto-triose, pentose, hexose or heptose, in either the
open-chained or cyclic form. Examples of monosaccharides that may
be used in the present application include, but are not limited to
allose, altrose, glucose, mannose, gulose, idose, galactose,
talose, ribose, arabinose, xylose, lyxose, threose, erythrose,
glyceraldehydes, sorbose, fructose, dextrose, levulose and
sorbitol. The polyol may also be a disaccharide, for example, but
not limited to sucrose, maltose, trehalose, cellobiose or lactose.
Polyols also include polysaccharides, for example, but not limited
to dextran, (500-50,000 MW), amylose and pectin and the like. Other
organic polyols that may be used include, but are not limited to
glycerol, propylene glycol and trimethylene glycol.
[0077] The term "aryloxy" as used herein means phenoxy or
naphthyloxy wherein, the phenyl and naphthyl groups may be
optionally substituted with 1-5 groups, specifically 1-3 groups,
independently selected from the group consisting of halo (fluoro,
bromo, chloro or iodo), C.sub.1-6alkyl, C.sub.1-6 alkoxy, OH,
NH.sub.2, N(C.sub.1-6alkyl).sub.2, NHC.sub.1-6alkyl.
C(O)C.sub.1-6alkyl. C(O)NH.sub.2, C(O)NHC.sub.1-6alkyl,
OC(O)C.sub.1-6alkyl, OC(O)OC.sub.1-6alkyl, NHC(O)NHC.sub.1-6alkyl,
phenyl and the like.
[0078] The term "arylalkyleneoxy" as used herein means
aryl-(C.sub.1-4-oxy wherein aryl has the same meaning as in
"aryloxy". Specifically, "arylalkyleneoxy" is a benzyl or
naphthylmethyl group (i.e. aryl-CH.sub.2--O).
[0079] By "normal sol-gel conditions" it is meant the conditions
used herein to effect hydrolysis and condensation of the sol gel
precursors, such as organic polyol derived silanes. This includes,
in aqueous solution, at a pH in the range of 4-11.5, specifically
in the range 5-10, and temperatures in the range of 0-80.degree.
C., and specifically in the range 0-40.degree. C., and optionally
with sonication and/or in the presence of catalysts known to those
skilled in the art, including acids, amines, dialkyltin esters,
titanates, etc.
[0080] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural references unless
the content clearly dictates otherwise. Thus for example, a
composition containing "a chimeric peptide" includes one such
peptide or a mixture of two or more peptides.
[0081] The term "suitable" as used herein means that the selection
of the particular conditions would depend on the specific method to
be performed, but the selection would be well within the skill of a
person trained in the art. All method or process steps described
herein are to be conducted under conditions sufficient to provide
the desired result. Unless otherwise indicated, a person skilled in
the art would understand that all method conditions, including, for
example, solvent, time, temperature, pressure, reactant ratio and
whether or not the method should be performed under an anhydrous or
inert atmosphere, can be varied to optimize the desired result and
it is within their skill to do so.
[0082] In understanding the scope of the present application, the
term "comprising" and its derivatives, as used herein, are intended
to be open ended terms that specify the presence of the stated
features, elements, components, groups, integers, and/or steps, but
do not exclude the presence of other unstated features, elements,
components, groups, integers and/or steps. The foregoing also
applies to words having similar meanings such as the terms,
"including", "having" and their derivatives. The term "consisting"
and its derivatives, as used herein, are intended to be closed
terms that specify the presence of the stated features, elements,
components, groups, integers, and/or steps, but exclude the
presence of other unstated features, elements, components, groups,
integers and/or steps. Finally, terms of degree such as
"substantially", "about" and "approximately" as used herein mean a
reasonable amount of deviation of the modified term such that the
end result is not significantly changed. These terms of degree
should be construed as including a deviation of at least .+-.5% of
the modified term if this deviation would not negate the meaning of
the word it modifies.
Biosensors of the Application
[0083] The automated deposition and permanent immobilization of
biorecognition molecules on solid surfaces is a step in the
development of bioactive paper-based sensors. To achieve this goal
it is desirable to develop immobilization methods that are
compatible with automated coating and/or printing and with
biomolecules and which retain the reactive agents at the surface of
the substrate. In the present application, the use of biocompatible
sol-gel derived materials with ink jet printing methods has been
explored for this purpose.
[0084] To evaluate the potential of ink jet deposition for
fabrication of bioactive sensors, the detection of organophosphates
via inhibition of immobilized acetylcholinesterase (AChE) was used
herein as one model system. Organophosphates (e.g., paraoxon) and
mycotoxins (e.g., aflatoxin B1) are classified as extremely
hazardous compounds due to their potent toxicity to the human
nervous system..sup.xx,xxi Organophosphate compounds are widely
used as agricultural pesticides, insecticides and chemical warfare
agents. These compounds are very stable and can rapidly diffuse
into ground water reservoirs and thus exhibit a threat of
contamination. Mycotoxins, particularly aflatoxin B1 (AfB1), are
carcinogenic contaminants of food and animal feeds and as such are
used as biochemical markers for food spoilage. In one model system
reported in the present application, a signal generation method,
utilizing the Ellman.sup.xxii colorimetric assay (FIG. 1) was
developed. Advantageously, to allow permanent capture of the highly
colored 5-thio-2-nitrobenzoate (TNB.sup.-) anion, a cationic
capture region was incorporated onto paper substrates via ink jet
printing of polyvinylamine (PVAm). The binding of toxins (e.g.,
paraoxon, AfB1) to acetylcholinesterase (AChE) reduces AChE
activity, and the residual activity is monitored based on the
yellow color intensity that was produced. Following this simple and
reliable assay mechanism, it was shown that it is possible to
detect AChE inhibitors in a rapid and cost effective manner using
either a dipstick or lateral flow biosensing format. This report on
the utilization of ink jet printing in the development of sol-gel
based paper biosensors provides a new platform for fabrication of
bioactive paper strips for detection of drugs or environmental
pollutants that affect both animals and humans. A second model
system was based on an indophenyl acetate (IPA)-based colorimetric
assay. AChE hydrolyzes the red-yellow colored substrate, IPA, to
the blue-purple indophenoxide anion (IDO.sup.-) which is then
trapped over a finite region by the cationic polymer, polyvinyl
amine (PVAm). The absence or decrease in blue-purple color, over
this region, is indicative of the presence of AChE inhibitors. This
paper-based sensor does not require any further reagents for proper
functioning, as all reagents are deposited onto the paper surface
with good long-term stability. A third model biosensor was
developed for the detection of any microorganism having a
.beta.-glucuronidase (GUS) enzyme, including, for example, E. coli.
In this biosensor, the chromogenic substrate
5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-GLUC) was
immobilized in one reaction zone and an oxidizing reagent
(FeCl.sub.3) was immobilized in a separate reaction zone. In this
assay, a solution comprising, or suspected of comprising, a
microorganism having GUS, was treated to lyse the microorganism and
a lateral flow-based biosensor was placed into the solution to
allow lateral flow of the solution up the sensor by capillary
action. The flow of the solution passes through the oxidizing agent
zone first and the X-GLUC zone second. When the microorganism
passes into the X-GLUC zone, the GUS enzyme, if present, will
hydrolyse the X-GLUC to form a halogenated indoxyl intermediate
which then dimerizes (via oxidation) to form a dark blue indigo
dye. The use of the oxidizing agent in this assay is unique and
advantageously provided faster response times. If the microorganism
was present in the solution, a dark blue color from the indigo dye
was detected in the X-GLUC reaction zone. To prevent leaching the
color, either the capture agent, PVAm, was incorporated into the
X-GLUC reaction zone, or hydrophobic barriers were pre-printed on
to the biosensor in an area around the reaction zone.
[0085] All biosensors reported herein can be prepared by printing
of biomolecule compatible sol gels onto the substrates.
Conveniently, ink jet printing methods were used, in particular
piezoelectric ink jet printing, to print layers of biomolecule
compatible sol gels onto specific reaction zones on the substrates.
The location of the reaction zones depended on the specific
arrangements required for the assay in question. Advantageously,
the assays were based on lateral flow of a solution up the
substrate by capillary action, passing through the one or more
reaction zones, wherein the solution comprised, or was suspected of
comprising, an analyte to be detected. The biosensors were also
amenable to dip-stick type assay formats.
[0086] Due to the rapid gelation of the sol gel precursor solutions
when combined with many of the biomolecule or other reagent
solutions, it was found to be most convenient to immobilize the
reagents on the substrate using a layered approach to avoid
clogging of the printing nozzles. Therefore, a first sol gel layer
was printed on a reaction zone, followed by printing of a
recognition element layer on top of the first sol gel layer and a
second sol gel layer printed on top of the recognition element
layer. Such an arrangement prevented reaction, and possible
deactivation, of the recognition element by the support (or any
other entity located under the first sol gel layer), prevented
absorption of the recognition element into the support and prevent
leaching of the recognition element from the sensor.
[0087] In reaction zones where a product is generated and is to be
detected, it was advantageous to include a capture means to
concentrate the detectable product in a distinct area. The capture
means included a chemical agent having an affinity for the product
and/or a physical barrier around the reaction zone. When the
capture means was a chemical agent, it was convenient to print the
agent onto the substrate below the first sol gel layer.
[0088] Therefore the present application includes a biosensor
comprising: [0089] (a) a substrate; [0090] (b) at least one
reaction zone immobilized on the substrate, the reaction zone
comprising, in order, beginning adjacent to the substrate: (i) a
first biomolecule compatible sol gel layer; (ii) a recognition
element layer; and (iii) a second biomolecule compatible sol gel
layer; and [0091] (c) a detection means,
[0092] wherein the first and second biomolecule compatible sol gel
layers and the recognition element layer are immobilized on the
substrate using ink jet printing.
[0093] The present application also includes a biosensor
comprising: [0094] (a) a substrate having a first and second end;
[0095] (b) at least one reaction zone immobilized on the substrate,
the reaction zone comprising, in order, beginning adjacent to the
substrate: (i) a first biomolecule compatible sol gel layer; (ii) a
recognition element layer; and (iii) a second biomolecule
compatible sol gel layer, wherein the first and second biomolecule
compatible sol gel layers and the recognition element layer are
immobilized on the substrate using ink jet printing; and [0096] (c)
a detection means,
[0097] wherein immersion of the first end of the substrate in a
solution comprising or suspected of comprising an analyte results
in lateral flow of the solution from the first end of the substrate
to the second end by capillary action and flow through the at least
one reaction zone results in reaction of the analyte with the
recognition element, the reaction being detected by the detection
means.
[0098] The ink jet printing technique is simple, rapid, scalable,
compatible with paper substrates and amenable to precise pattern
formation. One factor to consider for ink jet printing is the
formulation of the bioink and its rheological properties, in
particular the viscosity and surface tension. Several additives can
be introduced in the ink formulations to optimize the physical
properties and to make them stable and ejectable. There are
specific challenges associated with the formulation of biomolecule
containing bioinks for reliable ink jet printing. Printing of
biocompatible sol-gel derived inks is an even larger challenge
since short gelation times of silica sols can cause gelation and
clogging of the ink jet nozzles. At physiological pH, where most
enzymes thrive, gelation of most biocompatible sol-gel precursors
occurs within a minute to a few hours depending on buffer strength
and type or additives being used..sup.xxiii For this reason, a
multi-stage ink jet deposition method is reported here, wherein the
silica sol and the buffered reagents are deposited from separate
ink jet cartridges and thus do not interact prior to deposition on
the paper surface, avoiding gelation in the in jet nozzle.
[0099] In an embodiment of the application, certain reaction zones
further include an additional layer comprising a capture agent. The
reaction zones that benefit from the presence of a capture agent
are those that produce a product to be detected. The capture agent
serves to restrict movement of the product, thereby concentrating
the product in the reaction zone to facilitate detection. When the
capture agent is a chemical compound, it is an embodiment of the
application that the capture agent is printed as a layer under the
first sol gel layer (i.e. adjacent to the substrate).
[0100] In an embodiment of the application, the biomolecule
compatible silica sols and recognition elements are immobilized on
the substrate by printing aqueous solutions of these entities that
optionally include one or more additives. In an embodiment, the
additives are used to optimize the rheological properties to allow
reproducible jetting onto the substrates. Physico-chemical
properties such as surface tension and viscosity are examples of
parameters that can be optimized to make the solutions (also
referred to as "inks") stable and ejectable. Such additives include
surfactants and viscosity modifiers.
[0101] In an embodiment of the application, additives, such as
surfactants, are included to adjust the surface tension of the inks
to a printable range (for example, about, 30-40 mNm.sup.-1) if
adjustment is required. In an embodiment, the surfactant is a mild
detergent such as Triton X-100. In a further embodiment the
detergent is used in an amount of about 0.05 wt % to about 1 wt %,
or about 0.1 wt %.
[0102] In an embodiment of the application additives are included
to adjust the ink viscosity to a desired value (for example, about
2-10 cP) if adjustment is required. In an embodiment, the
viscosity-modifying additive is glycerol. In a further embodiment,
glycerol is used in an amount of about 20% (v/v) to about 50%
(v/v), or about 30% (v/v)
[0103] In the present application, the biomolecule-compatible sol
gels are is prepared using biomolecule-compatible techniques, i.e.
the preparation involves biomolecule-compatible silica precursors
and reaction conditions that are biomolecule-compatible. In an
embodiment of the application, the biomolecule-compatible sol gel
is prepared from a sodium silicate precursor solution. The
preparation of sodium silicate solutions for use as a sol-gel
precursor is known in the art..sup.38 In still further embodiments,
the sol gel is prepared from organic polyol silane precursors.
Examples of the preparation of biomolecule-compatible sol gels from
organic polyol silane precursors are described in inventor
Brennan's co-pending patent applications entitled "Polyol-Modified
Silanes as Precursors for Silica", U.S. patent application
publication no. US2004/0034203 filed on Jun. 2, 2003; and "Methods
and Compounds for Controlling the Morphology and Shrinkage of
Silica Derived from Polyol-Modified Silanes", U.S. CIP patent
application publication no. US2004/0249082 filed on Apr. 1, 2004,
the contents of which are incorporated herein by reference. In
specific embodiments of the application, the organic polyol silane
precursor is prepared by reacting an alkoxysilane, for example
tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS), with an
organic polyol under conditions that avoids hydrolysis and
condensation of the resulting precursor silane. Such conditions
include reacting the alkoxy silane with the organic polyol under
anhydrous conditions. Accordingly, the organic polyol silane
precursor is desirably non-oligomeric so that optimal control over
phase separation and gelation times is provided to permit greater
control over the morphology of the resulting sol gel materials.
[0104] In embodiments of the present application, the organic
polyol is selected from glycerol, sorbitol, maltose and dextran.
Some representative examples of the resulting polyol silane
precursors suitable for use in the methods of the application
include one or more of diglycerylsilane (DGS), monosorbitylsilane
(MSS), monomaltosylsilane (MMS), dimaltosylsilane (DMS) or
dextran-based silane (DS). In embodiments, the polyol silane
precursor is selected from one or more of DGS and MSS.
[0105] In a particular embodiment of the application, the sol-gel
precursors are combined with an additive which causes spinodal
decomposition (phase transition) before gelation, to provide
macroporous silica matrixes. Macroporous silica can be used to
entrap reagents with large molecular weights, i.e. those molecules
that are large enough to not leach from the sol gel. Methods of
forming macroporous silica, in particular, from polyol-modified
silane precursors are described in inventor Brennan's co-pending
patent application entitled "Methods and Compounds for Controlling
the Morphology and Shrinkage of Silica Derived from Polyol-Modified
Silanes", U.S. CIP patent application publication no.
US2004/0249082 filed on Apr. 1, 2004, the contents of which are
incorporated herein by reference. In particular, the sol-gel
precursor is combined with one or more water soluble polymers which
causes spinodal decomposition (phase transition) before gelation.
The water soluble polymer may be selected from any such compound
and includes, but is not limited to, for example, polyethylene
oxide (PEO); polyethylene glycol (PEG); amino-terminated
polyethylene glycol (PEG-NH.sub.2); amino-terminated polyethylene
oxide (PEO-NH.sub.2); polypropylene glycol (PPG); polypropylene
oxide (PPO); polypropylene glycol bis(2-amino-propyl ether)
(PPG-NH.sub.2); polyalcohols, for example, polyvinyl alcohol;
polysaccharides; poly(vinyl pyridine); polyacids, for example,
poly(acrylic acid); polyacrylamides e.g.
poly(N-isopropylacrylamide) (polyNIPAM); or polyallylamine (PAM),
or mixtures thereof. In embodiment of the application the water
soluble polymer is selected from PEO, PEO-NH.sub.2, PEG,
PPG-NH.sub.2, polyNIPAM and PAM, and mixtures thereof. In further
embodiments of the application, the water soluble polymer is
selected from PEO, PEO-NH.sub.2 and polyNIPAM, and mixtures
thereof. In still further embodiments, the water soluble polymer is
PEO, for example PEO having a molecular weight between about
2000-100000 Da, suitably between about 5000 and 50000 Da, more
suitably between about 8000 and 15000 Da. By "water soluble" it is
meant that the polymer is capable of being formed into an aqueous
solution having a concentration effective to result in phase
separation occurring before gelation. It should be noted that the
terms "oxide" (as in polyethylene oxide) and "glycol" (as in
polyethylene glycol) may be used interchangeably and the use of one
term over the other is not meant to be limiting in any way.
[0106] Sol gels may also be obtained by combining the sol-gel
precursors, in particular organic polyol silane precursors, with
one or more compounds of Formula I:
##STR00001##
wherein R.sup.1, R.sup.2 and R.sup.3 are the same or different and
represent a group that is hydrolyzed under normal sol-gel
conditions to provide Si--OH groups; and R.sup.4 is group selected
from polyol-(linker)-, polymer-(linker).sub.n- and
##STR00002##
where n is 0 or 1. Such compounds are also described in detail in
inventor Brennan's co-pending patent application entitled "Methods
and Compounds for Controlling the Morphology and Shrinkage of
Silica Derived from Polyol-Modified Silanes", U.S. CIP patent
application publication no. US2004/0249082 filed on Apr. 1, 2004,
the contents of which are incorporated herein by reference. In
embodiments of the application, OR.sup.1, OR.sup.2 and/or OR.sup.3
are the same or different and are derived from organic mono-, di-,
or polyols. In embodiments of the present application, the group
OR.sup.1, OR.sup.2 and/or OR.sup.3 are derived from a polyol
selected from glycerol, sorbitol, maltose, trehalose, glucose,
sucrose, amylose, pectin, lactose, fructose, dextrose and dextran
and the like. In further embodiments of the present application,
the organic polyol is selected from glycerol, sorbitol, maltose and
dextran. In other embodiments of the application, OR.sup.1,
OR.sup.2 and OR.sup.3 are the same and are selected from
C.sub.1-4alkoxy, for example, methoxy or ethoxy, aryloxy and
arylalkyleneoxy. In further embodiments of the application,
OR.sup.1, OR.sup.2 and OR.sup.3 are all ethoxy. It will be apparent
to those skilled in the art that other leaving groups such as
chloride or silazane may also be used for the formation of silica
according to the methods described in the application.
[0107] It should be noted that the groups OR.sup.1, OR.sup.2 and
OR.sup.3 are capable of participating directly in the
hydrolysis/polycondensation reaction.
[0108] In particular, these functional groups are alkoxy groups
attached to the silicon atom at oxygen, i.e., "Si--OR", which may
be hydrolyzed to provide "Si--O--H", which can condense with other
"Si--O--H" or "Si--OR" groups to provide "Si--O--Si" linkages and
eventually a three-dimensional network within a gel. Trifunctional
silanes form silsesquioxanes upon hydrolysis and there is a lower
degree of crosslinking in systems derived therefrom, in particular
when compared with systems derived from tetrafunctional silanes.
The remaining group attached to the silicon atom (R.sup.4) is a
group that generally does not participate directly in the
hydrolysis/polycondensation reaction.
[0109] R.sup.4 is a group that is not hydrolyzed under normal
sol-gel conditions and preferably is stabilizing to biological
substances, in particular proteins. In specific embodiments,
R.sup.4 is selected from one of the following groups:
##STR00003##
wherein n is 0-1 and OR.sup.1, OR.sup.2 and OR.sup.3 are as defined
above. The term "polyol" in R.sup.4 has the same definition as
described above for the groups OR.sup.1, OR.sup.2 and OR.sup.3. In
an embodiment of the invention, the polyol is derived from glucose
or maltose. The term "polymer" in R.sup.4 refers to any water
soluble polymer, such as, but not limited to: polyethers, for
example, polyethylene oxide (PEO); amino-terminated polyethylene
oxide (PEO-NH.sub.2); polyethylene glycol (PEG); polyethylene
glycol bis(2-amino-propyl ether) (PEG-NH.sub.2); polypropylene
glycol (PPG); polypropylene oxide (PPO); polypropylene glycol
bis(2-amino-propyl ether) (PPG-NH.sub.2); polyalcohols, for
example, polyvinyl alcohol; polysaccharides; poly(vinyl pyridine);
polyacids, for example, poly(acrylic acid); polyacrylamides e.g.
poly(N-isopropylacrylamide) (polyNIPAM); or polyallylamine (PAM). A
linker group is required (i.e. n=1) when a direct bond between the
silicon atom and the polymer would be hydrolyzed under normal
sol-gel conditions. In embodiments of the invention, the polymer is
a water soluble polyether such as PEO.
[0110] The sugar and polymer residues may be attached to the
silicon atom through any number of linkers. Such linkers may be
based on, for example, alkylene groups (i.e. --(CH.sub.2).sub.m--,
m=1-20, specifically 1-10, more specifically 1-4), alkenylene
groups (i.e. --(CH.dbd.CH).sub.m--, m=1-20, specifically 1-10, more
specifically 1-4), organic ethers, thioethers, amines, esters,
amides, urethanes, carbonates or ureas. A person skilled in the art
would appreciate that they are numerable linkers that could be used
to connect the group, R.sup.4, to the silicon atom.
[0111] Illustrative of compounds of Formula I of the present
application, are two classes of the trifunctional silanes based on
saccharides which are prepared as described in inventor Brennan's
co-pending patent application entitled "Methods and Compounds for
Controlling the Morphology and Shrinkage of Silica Derived from
Polyol-Modified Silanes", PCT patent application WO 04/018360,
filed Aug. 25, 2003 and corresponding U.S. CIP patent application
publication no. US2004/0249082 filed on Apr. 1, 2004, the contents
of which are incorporated herein by reference:
monosaccharide-(compound 1) and disaccharide- (compounds 2 and 3)
based trifunctional silanes are shown in Schemes 1 and 2.
Hydrolysis and condensation of these species along with organic
modified silanes (for example diglycerylsilane) allows the
incorporation of these species into sol gel derived siliceous
materials resulting in materials that have non-hydrolyzable sugar
moieties covalently bound into the silica network. Such materials
permanently incorporate protein stabilizing agents into the silica
and retain water in the silica matrix, avoiding denaturation of the
entrapped protein. Also prepared were polymeric bis(trifunctional
silanes) 5 (see Scheme 3).
[0112] Although in both of the saccharide examples shown in Schemes
1 and 2, many different opportunities for modification with silanes
exist, this scheme shows the modification of the anomeric
hemiacetal centre at the terminus of the saccharidic chains.
Oxidation of any of the sugars converts the anomeric hemiacetal
into the lactone (Scheme 1). This is then opened by an
amino-modified alkoxysilane to produce a sugar-modified coupling
agent..sup.xxiv The key functional group tethering the two groups
in this case is an alkylamide. Examples of specific sugar modified
silanes are shown in Scheme 2.
##STR00004##
##STR00005##
[0113] Illustrative of compounds of Formula I wherein R.sup.4
is
##STR00006##
wherein OR.sup.1, OR.sup.2 and OR.sup.3 are as defined above, are
compounds 5 shown in Scheme 3. Compounds 5 can be prepared, for
example, by reacting poly(ethylene oxide), first with allyl bromide
(or any other suitable allylating reagent), followed by reaction
with a trialkoxy-, triarylalkyleneoxy- or triaryloxysilane, in the
presence of a catalyst, such as a platinum-derived catalyst, as
shown in Scheme 3. When modified PEO polymers are used, for example
the compound of Formula 5, it is an embodiment of the application
that the starting PEO have a MW of greater than about 2000 g/mol.
In this example the linker is an alkylene group, with m=3. Note
some allyl-terminated PEO polymers 4 are commercially available. It
would be apparent to one skilled in the art that other levels of
functionality can also be used to bind these species to the
siliceous matrix, such as:
R.sub.3-kJ.sub.kSi-linker-polymer-linker-SiJ.sub.kR.sub.3-k and
polymer-linker-SiJ.sub.kR.sub.3-k where k=1-3 and J is a group that
can participate in hydrolysis and condensation with the silica
network.
##STR00007##
[0114] In further embodiments of the application, the
biomolecule-compatible sol gel precursor is selected from one or
more of functionalized or non-functionalized alkoxysilanes,
polyolsilanes or sugarsilanes; functionalized or non-functionalized
bis-silanes of the structure (RO).sub.3Si--R'--Si(OR).sub.3, where
R may be ethoxy, methoxy or other alkoxy, polyol or sugar groups
and R' is a functional group containing at least one carbon
(examples may include hydrocarbons, polyethers, amino acids or any
other non-hydrolyzable group that can form a covalent bond to
silicon); functionalized or non-functionalized chlorosilanes; and
sugar, polymer, polyol or amino acid substituted silicates.
[0115] In yet another embodiment of the present application, the
biomolecule compatible sol gel precursor solution comprises an
effective amount of one or more other additives. In embodiments of
the application the other additives are present in an amount to
enhance the mechanical, chemical and/or thermal stability of the
matrix and/or assay components. In an embodiment, the mechanical,
chemical and/or thermal stability is imparted by a combination of
precursors and/or additives, and by choice of aging and drying
methods. Such techniques are known to those skilled in the art. In
further embodiments of the application, the additives are selected
from one or more of humectants and other protein stabilizing agents
(for e.g. osmolytes). Such additives include, for example, one or
more of organic polyols, hydrophilic, hydrophobic, neutral or
charged organic polymers, block or random co-polymers,
polyelectrolytes, sugars (natural or synthetic), and amino acids
(natural and synthetic). In embodiments of the application, the one
or more additives are selected from one or more of glycerol,
sorbitol, sarcosine and polyethylene glycol (PEG). In further
embodiments, the additive is glycerol.
[0116] In a particular embodiment of the application the
biocompatible sol gel is a silica based glass prepared from a
polyol modified silane, for example, diglyceryl silane, or sodium
silicate precursor solution.
[0117] The precursor solution is prepared according to methods
available in the art, for example about 1 g to about 5 g, suitably
about 3.0 g, of sodium silicate is dissolved in about 10 mL of
doubly distilled water (DDH.sub.2O) followed by addition of about 5
g of Dowex cation exchange resin to replace the sodium ions with
protons and stirring until a pH of approximately 4 is reached. The
resulting sol is then filted to remove any fine particulates that
could interfere with ink jetting. Suitably, the organic polyol
silane precursor solution is prepared by dissolving about 0.1 g to
about 2.0 g, suitably about 1.0 g, of polyol silane, such as DGS,
in about 10 mL of ddH.sub.2O, followed by sonication. Again, the
resulting sol is then filted to remove any fine particulates that
could interfere with ink jetting. A person skilled in the art would
appreciate that if larger scale preparations are required, then the
amounts of precursor and water may increase proportionally to
provide precursor solutions of approximately the same
concentration.
[0118] It is an embodiment of the application that the
biomolecule-compatible sol gel layers were printed using a
solution, or an ink, comprising about 30% (v/v) glycerol and about
0.1 wt % Triton-X100 and sodium silicate.
[0119] Depositing or printing of the solutions (reagents, capture
agents or sol gel precursors) on the substrate was performed using
ink jet printing. In an embodiment the ink jet printing is
performed using a piezoelectric ink jet printer equipped with means
to control the location of the inks being printed. Each different
solution is printed using a separate printing cartridge.
[0120] The assays that may be performed using the biosensors of the
present application include any assay based on an interaction
between a functional biomolecule and its corresponding substrate
that is amenable to detection. In an embodiment, the functional
biomolecule is an enzyme. Non-limiting and some of their known
substrates and detection systems are as follows: [0121] (ii)
acetylcholinesterase--acetylthiocholine/dithiobisnitrobenzoate
(DTNB); [0122] (iii) acetylcholinesterase--indophenyl acetate;
[0123] (iv) urokinase plasminogin activator (uPA)--S-2244; [0124]
(v) adenosine triphosphatases (ATPases)/kinases--ATP-.beta.S/DTNB;
[0125] (vi)
.beta.-glucuronidase--5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide
(X-GLUC)/FeCl.sub.3/indigo dye; [0126] (vii)
.beta.-galactosidase--bromo-chloro-indolyl-galactopyranoside
(X-GAL)/indigo dye [0127] (viii) DNA/RNA/PNA aptamers, DNA/RNA
enzymes or a DNA or RNA aptamzyme/signaling method; [0128] (ix)
functional nucleic acid/.PHI.29 DNA polymerase, circular template
and dNTPs/gold nanoparticle labeled linear DNA of the same sequence
as the circular template (or a portion thereof).
Biosensor for Microorganisms
[0129] Herein, a self-contained portable bioactive lab-on-paper
sensor for sensitive visual detection of microorganisms based on
the activity of an enzyme that is unique to the microorganism
activity has been prepared. For example, the enzyme
.beta.-glucuronidase is endogenous to E. coli BL21 and K12, as well
as salmonella, and .beta.-galactosidase is endogenous to E. coli
H7:O157 and these enzymes can be used as detection means for these
microorganisms. In one example, the assay system was composed of a
test strip, in which a chromogenic substrate for the
.beta.-glucuronidase, X-GLUC, and an oxidizing agent, such as
FeCl.sub.3, were entrapped using sol-gel derived silica inks in two
different zones. Detection was achieved by eye, using a digital
camera, or by an office scanner and image analysis software,
avoiding the need for instrumentation or trained personnel. The
assay provided good detection limits (.about.4.times.10.sup.3
CFU/mL) and rapid response times (.about.5 min) and remained stable
and reproducible after storage in room temperature for at least 60
days, making the system suitable for storage and use in the field.
Patterned paper sensors showed a higher sensitivity (LOD>2 fold)
than that of non-patterned sensors. The assay system showed a
negligible matrix effect with artificially E. coli contaminated
milk and orange juice samples, provided that pH was adjusted to a
suitable range close to pH 8.0. Based on the data, it is concluded
that this novel paper strip biosensor provides a fast and
convenient method for the visual detection of microorganisms
comprising a .beta.-glucuronidase, such as E. coli, which could be
employed for first level screening of a variety of environmental
and food samples, thus it could be a component of a simple and
inexpensive field kit. Similar test strips could be prepared for
other microorganisms.
[0130] The present application therefore includes a biosensor for
the detection of microorganisms having an intrinsic or recombinant
.beta.-glucuronidase or .beta.-galactosidase enzyme comprising:
[0131] (a) a substrate having a first and second end; [0132] (b) a
first reaction zone immobilized on the substrate comprising in
order, beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) an oxidizing agent; and (iii) a
second biomolecule compatible sol gel layer; [0133] (c) a second
reaction zone immobilized on the substrate comprising in order,
beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) a chromogenic substrate for the
enzyme and (iii) a second biomolecule compatible sol gel layer;
wherein immersion of the first end of the substrate in a solution
comprising or suspected of comprising the microorganisms, and that
has been treated to lyse the microorganisms, results in lateral
flow of the solution from the first end of the substrate to the
second end of the substrate by capillary action, the flow passing
through the first reaction zone prior to passing through the second
reaction zone.
[0134] In an embodiment, the first and second biomolecule
compatible sol gel layers, the oxidizing agent and the a
chromogenic substrate for the substrate are immobilized on the
substrate using ink jet printing of solutions comprising these
substances, or in the case of the sol gels, precursors for these
substances.
[0135] The chromogenic substrate for the enzymes is one that, when
reacted with the enzyme produces a product that is oxidized by the
oxidizing agent to a colored product that is detected. In an
embodiment, the chromogenic substrate for 3-glucuronidase is
5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronide (X-GLUC) and the
chromogenic substrate for .beta.-galactosidase is
bromo-chloro-indolyl-galactopyranoside (X-GAL). Other chromogenic
substrates include, for example, 5-bromo-3-indolyl
.beta.-D-galactopyranoside (Bluo-Gla), 5-bromo-6-chloro-3-indolyl
.beta.-D-galactopryaniside (Magenta-Gal), 6-chloro-3-indolyl
.beta.-D-galactopyranoside (Salmon-Gal), 2-nitrophenyl
.beta.-D-galactopyranoside (ONPG) and 4-nitro
.beta.-D-galactopyranoside (PNPG).
[0136] It is well known to those skilled in the art that magnetic
immunobeads or other known methods can be used to selectively
pre-concentrate analytes present in complex mixtures prior to a
range of different assays. Therefore the solution comprising the
microorganism may be a sample taken directly from, for example the
environment, a patient or food, or the sample can be pre-treated to
concentrate the microorganism or to remove undesired materials.
[0137] Examples of lytic reagents include, for example, lytic
bacteriophage, lysozyme or detergents. Contacting the resulting
lysed solution with the biosensor will result in reaction of the
functional biomolecule with the immobilized substrate, the reaction
being detected by the detection means if the microorganism is
present in the sample.
[0138] In an alternate embodiment, the lytic reagent is immobilized
in a reaction zone on the biosensor, which is deposited by ink-jet
printing at a location such that the solution passes through this
zone prior passing through the substrate zone by lateral flow.
Therefore the present application further includes a biosensor for
the detection of microorganisms having an intrinsic or recombinant
.beta.-glucuronidase or .beta.-galactosidase enzyme comprising:
[0139] (a) a substrate having a first and second end; [0140] (b) a
first reaction zone immobilized on the substrate comprising in
order, beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) a lytic reagent; and (iii) a second
biomolecule compatible sol gel layer; [0141] (c) a second reaction
zone immobilized on the substrate comprising in order, beginning
adjacent to the substrate: (i) a first biomolecule compatible sol
gel layer; (ii) an oxidizing agent; and (iii) a second biomolecule
compatible sol gel layer; [0142] (d) a third reaction zone
immobilized on the substrate comprising in order, beginning
adjacent to the substrate: (i) a first biomolecule compatible sol
gel layer; (ii) a chromogenic substrate for the enzyme and (iii) a
second biomolecule compatible sol gel layer;
[0143] wherein immersion of the first end of the substrate in a
solution comprising or suspected of comprising the microorganisms,
results in lateral flow of the solution from the first end of the
substrate to the second end of the substrate by capillary action,
the flow passing through the first reaction zone prior to passing
through the second reaction zone prior to passing through the third
reaction zone.
[0144] In an embodiment, the first and second biomolecule
compatible sol gel layers, the oxidizing agent, the chromogenic
substrate for the substrate and the lytic reagent are immobilized
on the substrate using ink jet printing of solutions comprising
these substances, or in the case of the sol gels, precursors for
these substances.
AChE Biosensors
[0145] The present application includes a biosensor for the
determining AChE activity or for assaying for AChE modulators
comprising: [0146] (a) a substrate having a first and second end;
and [0147] (b) a first reaction zone immobilized on the substrate
comprising in order, beginning adjacent to the substrate: (i) a
cationic polymer; (ii) a first biomolecule compatible sol gel
layer; (iii) AChE and DTNB; and (iv) a second biomolecule
compatible sol gel layer.
[0148] The present application includes an alternate biosensor for
the determining AChE activity or for assaying for AChE modulators
comprising: [0149] (a) a substrate having a first and second end;
[0150] (b) a first reaction zone immobilized on the substrate
comprising in order, beginning adjacent to the substrate: (i) a
first biomolecule compatible sol gel layer; (ii) IPA; and (iii) a
second biomolecule compatible sol gel layer; and [0151] (c) a
second reaction zone immobilized on the substrate comprising in
order, beginning adjacent to the substrate: (i) a first biomolecule
compatible sol gel layer; (ii) AChE; and (iii) a second biomolecule
compatible sol gel layer; wherein the first and second reaction
zones are arranged so that during lateral flow of a solution from
the first end of the substrate to the second end by capillary
action, the solution passes through the first reaction zone prior
to passing through the second reaction zone.
[0152] In an embodiment, the first and second biomolecule
compatible sol gel layers, the cationic polymer, the AChE, the DTNB
and the IPA are immobilized on the substrate using ink jet printing
of solutions comprising these substances, or in the case of the sol
gels, precursors for these substances.
Assays of the Application
[0153] The present application also includes assay methods that
utilize the biosensor of the present application. In an embodiment,
the assay is a method of detecting one or more analytes in a
sample, wherein the sample comprises or is suspected of comprising
the one or more analytes, the method comprising contacting the
sample with the biosensor of the application and monitoring the
detection means for a positive or negative result, wherein a
positive result indicates the presence of the one or more analytes
in the sample. In an embodiment of the application, the detection
means is a colormetric method and the positive result is a presence
of a color change on the biosensor.
[0154] A non-limiting example of such an assay is the testing of a
sample, such as a food or environmental sample (such as water) for
the presence of one or more pathogenic microorganisms. In this
example, the biosensor will have immobilized in one of the reaction
zones, a substrate for a functional biomolecule, such as an enzyme,
that is representative of the microorganism. The microorganism is
optionally preconcentrated by methods known to those skilled in the
art (e.g., magnetic bead based preconcentration or filter based
preconcentration). A solution is prepared containing the sample and
the solution is treated with lytic reagents that will break apart
the microorganism, releasing its internal contents which include
the functional biomolecule. Examples of lytic reagents include, for
example, lytic bacteriophage, lysozyme or detergents. Contacting
the resulting lysed solution with the biosensor will result in
reaction of the functional biomolecule with the immobilized
substrate, the reaction being detected by the detection means if
the microorganism is present in the sample. In a specific
embodiment the application, the functional biomolecule is the
enzyme .beta.-glucuronidase or .beta.-galactosidase, which are
found only E. coli and coliform bacteria. In an alternate
embodiment, the lytic reagent is immobilized in a reaction zone on
the biosensor, which is deposited by ink-jet printing and the
sample solution passes through this zone prior passing through the
substrate zone by lateral flow.
[0155] Further non-limiting examples, include biosensors comprising
reagents that allow for detection of specific enzymes that may be
biomarkers associated with disease. Such enzymes may be present in
any biological sample, including tissue, blood, urine, tears,
saliva or sweat or within microorganisms. In a specific embodiment,
the enzyme is acetylcholinesterase (AChE), a protease such as
urokinase plasminogen activator (UPa), which is upregulated in
metastatic breast cancer, or kinases such as adenosine
triphosphatase (ATPase), protein kinase A (PKA) or glycogen
synthase kinase-3 (GSK-3), which are upregulated in certain disease
states. In this embodiment a substrate for the enzyme and a
suitable reporter molecule (detection means) are printed onto a
substrate within a suitable matrix using ink-jet methods as
described above. Suitable substrates and reporters include IPA for
AChE, S-2244 for uPA, ATP-.beta.S/DTNB for ATPases and kinases, and
X-GLUC and X-GAL for .beta.-glucuronidase or .beta.-galactosidase,
though other colorimetric reagents suitable for assaying such
enzymes are known to those skilled in the art and are included
within the scope of the application. The biosensor is contacted
with the sample or a solution prepared from the sample and the
analytes in the sample solution are allowed to move up the
biosensor by lateral flow or the biosensor is simply dipped into
the sample or the sample solution. Enzymes that are present within
the sample will contact the reaction zone(s) containing the
substrate and detection means and will produce a change, such as a
color change that can be correlated to the presence and
concentration of the enzyme.
[0156] In another embodiment, lysis of a microorganism can also
release ATP. As an example, of a biosensor for ATP, a reaction zone
may contain ATPase and malachite green. Alternatively, the reaction
zone may contain adenylate kinase to convert AMP+ATP to two
molecules of ADP, while another zone contains polyphosphate kinase
to convert ADP back to ATP (ATP amplification) while a further zone
contains a colorimetric reagent for ATP detection. Another
alternative is to use a colorimetric assay based on oxidation of
Fe(II) to Fe(III), which forms a colored complex with xylenol
orange (XO). In this case, a reaction zone contains adenylate
kinase (AK) and pyruvate kinase (PK) to amplify the amount of ATP
and produce pyruvate. The same zone or another zone can contain
pyruvate oxidase to generate H.sub.2O.sub.2 which oxidizes
entrapped Fe(II) to Fe(III). The Fe(III) can form a complex with
xylenol-orange (XO) so the colour of the dye changes from yellow to
purple. Other colorimetric assays for ATP can also be employed and
are within the scope of the application.
[0157] Alternative arrangements of reaction zones are also
possible, as would be apparent to one skilled in the art. The
present disclosure therefore includes an assay method for the
detection of potentially pathogenic organisms, for example, E.
coli, in, for example blood, tissue, air, water and food samples.
The development of low-cost, portable and technically
straightforward assay technologies is beneficial in a number of
areas, including rapid testing of food or water quality,
point-of-care diagnostics (i.e. field or home setting), or the
rapid detection of bioterror agents. Development of such bioassays
could also be useful for performing routine analysis in
underdeveloped countries, or as an alternative to more expensive
technologies for rapid testing in emergency situations..sup.xxv
[0158] In another embodiment, the present application also includes
a method for determining if one or more analytes are modulators of
a functional biomolecule comprising: [0159] (a) contacting a
solution comprising the one or more analytes with a reaction zone
on a biosensor of the application, wherein the reaction zone
comprises the functional biomolecule; [0160] (b) contacting the
reaction zone with a substrate for the functional biomolecule;
[0161] (c) monitoring the detection means for a positive or
negative result; and [0162] (d) comparing the positive or negative
result in (c) with a control biosensor, wherein a positive or
negative result in (c) that is different from the control indicates
that the one or more analytes are modulators of the functional
biomolecule. In an embodiment of the application, the detection
means is a colormetric method and the presence of a color change on
the biosensor that is different from that on the control biosensor
indicates that the one or more analytes are modulators of the
functional biomolecule.
[0163] Control biosensors are typically identical biosensors
treated in the same way as the biosensor contacted with the
solution comprising the one or more analytes, except that it is not
contacted with the solution. Other controls include biosensors
without the functional biomolecule or substrate.
[0164] In the latter method, when the biosensor is contacted with a
substrate for the functional biomolecule, a product is produced,
the product reacting with a reporter molecule (detection means) to
produce a change, such as a color change, that can be detected. As
a representative, non-limiting example, the enzyme
acetylcholinesterase (AChE) acts on its substrate,
acetylthiocholine (ATCh) in a sample to produce thiocholine which
reduces 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to
thionitrobenzoate. In an embodiment, the yellow colored
thionitrobenzoate can be captured on cationic zone, for example a
poly(vinylamine) (PVA) coated zone, (capture means) of the test
strip to concentrate the color into a defined area. Further, most
kinases will cleave the terminal phosphate off the substrate
ATP.beta.S (ATP with a sulfur on the .beta. phosphorous) to produce
a product that will also reduce DTNB. Also, AChE acts on the
red-yellow subsrate indophenyl acetate (IPA) to produce the
blue-purple product indophenoxide (IDO.sup.-) anion. Again the
IDO.sup.- can be captured and concentrated using a cationic zone.
The present disclosure therefore provides methods for assaying the
activity of functional biomolecules, such as, kinases and ATPases,
using the biosensors of the application. However, in further
embodiments, the biosensor is initially contacted, in the reaction
zone comprising the functional biomolecular, with a solution
comprising one or more analytes that modulate, or are suspected of
modulating, the activity of the functional biomolecule prior to
contacting the reaction zone or with the one or more analytes. In
this embodiment, if there is a change, such as a change in color,
after exposure to the substrate in the presence of analyte(s)
compared to the change, such as change in color, in the absence of
analytes, or compared to biosensor controls not containing the
functional biomolecule, then the activity of the functional
biomolecule has been modulated. For example, if the color is less
intense in the presence of the one or more analytes compared to in
the absence of the one or more analytes, then the one or more
analytes are inhibitors of the functional biomolecule. Conversely,
if the color is more intense in the presence of the one or more
analytes compared to in the absence of the one or more analytes,
then the one or more analytes are promoters or enhancers of the
functional biomolecule (e.g. for AChE, species whose reactions are
catalyzed by a reducing entity to produce products that can
directly reduce the DTNB or co-factors of the enzyme). Contacting
the reaction zone comprising the functional molecule with solutions
of the one or more analytes can be accomplished by for example,
over spotting this zone with the solution, dipping or placing the
second end of the biosensor in the analyte solution and allowing
the solution to travel by capillary flow just into the reaction
zone. In this latter example the biosensor can be inverted and the
first end placed into a substrate solution to allow the substrate
to travel into the reaction zone by lateral flow.
[0165] In another embodiment reaction zones can be placed on a test
strip to allow for detection of analytes using functional
biomolecules including DNA/RNA/PNA aptamers, DNA/RNA enzymes or a
DNA or RNA aptamzyme, coupled to a signalling method. In an
embodiment, the reaction zones contain reagents to allow detection
of an analyte using colorimetric detection of the products of a
reaction involving rolling circle amplification (RCA) or a primer
that is exposed upon interaction of an aptamer, DNA enzyme or
aptazyme (collectively referred to as a functional nucleic acid)
with a target analyte. In a specific embodiment, the reaction zones
are placed in the following order: (i) functional nucleic acid;
(ii) .PHI.29 DNA polymerase, circular template and dNTPs; (iii)
gold nanoparticle labeled linear DNA of the same sequence as the
circular template (or a portion thereof) each sandwiched between
two layers of biomolecule compatible sol gel. In an embodiment of
the application, the assay is performed by placing the end of a
lateral flow-based biosensor into a test solution. Analytes in the
test solution first reach the functional nucleic acid and cause a
structure-switching event or catalysis of cleavage of a suitable
substrate. This reaction results in the release of a segment of DNA
that flows to the second reaction zone containing the polymerase,
dNTPs and circular template. The segment released is complementary
to a portion of the circular template and thus acts as a primer to
initiate the RCA reaction in the second reaction zone. After a
suitable reaction time, the RCA product is detected by either
moving the RCA product to the gold nanoparticle zone, or inverting
the lateral flow device and flowing the gold nanoparticles into the
zone containing the RCA product. The AuNP-labelled complementary
DNA will initially be in a de-aggregated state and thus will be red
in color. Upon hybridizing with the RCA product the AuNPs will be
in close contact and thus will form blue colored aggregates.
Formation of the blue colored aggregates indicates the presence of
the target analyte in the test solution. In embodiments of the
application, the analytes may be any analyte of interest in the
biomedical, environmental, bioterror or agricultural fields. The
analyte may also be a biomarker associated with a specific disease
state, a microorganism or a metabolite present in a microorganism,
a gene or a gene product. The analyte may be present in any test
solution, including tissue, blood, urine, tears, saliva or sweat,
or in food, water, soil or other samples.
[0166] In an embodiment of the application, when the change is a
color change it may be quantified, for example, using a digital
camera with a macrofocus lens and using standard image analysis
software.
[0167] As stated above, the analyte may be contacted with the
biosensor using either lateral flow of the analyte solution up the
substrate via capillary action, overspotting of analyte solution
onto the biosensor (i.e., with a pipette) or dipping the biosensor
into the analyte solution.
[0168] In other embodiments of the present disclosure, a series of
reaction zones can be placed on a test strip to allow multi-step
reactions to occur as a result of lateral flow of analyte along the
biosensor.
[0169] In an embodiment of the present application, ink-jet
printing is used to print different reaction zones onto a biosensor
in a manner that allows movement of reactants from one area to
another by capillary flow. As a representative, non-limiting,
example, the chromogenic substrate IPA can be printed in one zone
and AChE can be printed in a second zone, while PVAm is printed
either in the same zone as AChE or in a separate region. In an
embodiment of the present disclosure, the IPA, AChE and PVAm zones
are printed such that lateral flow of liquid upon contacting an
analyte test solution will result in liquid reaching the IPA zone
first, transporting the IPA to the AChE zone to undergo reaction
with the enzyme, and the product is then transported to the PVAm
capture zone to allow detection of a color change. In another
embodiment the IPA, AChE and PVAm zones are printed such that
lateral flow of an analyte solution will cause analytes to first
contact the AChE zone when one end of the biosensor is placed in
contact with the analyte solution, allowing for incubation of
analytes with the enzyme. The other end of test strip can then be
placed in the analyte solution to allow lateral flow of liquid into
a reaction zone containing IPA so that the IPA is transported into
the AChE region. Any product that is produced can be captured by a
PVAm layer that is directly below the AChE-containing sol-gel. In
this manner, slow inhibitors of the enzyme can be detected by
measuring the color change in the presence of the analyte solution
and comparing it to the color of controls that have no analyte
compounds present or no enzyme present.
EXAMPLES
[0170] The following non-limiting examples are illustrative of the
present application:
Example 1
Development of Bioactive Paper Sensors Using Piezoelectric Ink Jet
Printing of Sol Gel Derived Bioinks
[0171] Chemicals: Sodium silicate solution (.about.14% NaOH,
.about.27% SiO.sub.2), tetraethylorthosilicate (TEOS, 98%), Dowex
50WX8-100 ion-exchange resin, acetylcholinesterase (AChE, from
electrophorus electricus, EC 3.1.1.7), paraoxon, aflatoxin B1
(AfB1, from aspergillus flavus), 5,5'-dithiobis-(2-nitrobenzoic
acid) (DTNB), carboxymethylcellulose sodium salt (CMC), and Triton
X-100 were obtained from Sigma-Aldrich. Anhydrous glycerol and
acetylthiocholine iodide (ATCh) were purchased from Fluka
BioChemika Ultra (UK). Diglyceryl silane (DGS) was synthesized in
our lab using by transesterification of TEOS with anhydrous
glycerol as described in detail elsewhere..sup.xxvi Polyvinylamine
(PVAm; 1.5 MDa) was obtained from BASF (Mississauga, Canada), as a
gift. Mead brand cardboard paper substrate with a white hydrophobic
clay coating (Manufactured by Hilroy, Toronto, Canada) was
purchased from McMaster University Bookstore. Distilled deionized
water (ddH.sub.2O) was obtained from a Milli-Q Synthesis A10 water
purification system. All other reagents were of analytical
grade.
[0172] Preparation of Solutions: Stock solutions of the ATCh,
paraoxon and AfB1 were made up daily and were not used for more
than 3 h after preparation to minimize the potential for
hydrolysis. Tris buffer (100 mM, pH 8) was used for dilution of
ATCh. A mixture of Tris buffer (50 mM, pH 6.8) and 5% cyclohexane
(Sigma) was used for dilution of paraoxon, while a mixture of Tris
buffer (50 mM, pH 6.8) and 5% methanol (Sigma) was used for
dilution of AfB1. These solvents not only aid in dissolution of the
AChE inhibitors, but also enhance the affinity of
paraoxon.sup.XXVii and aflatoxin.sup.xxviii for binding to AChE.
Furthermore, this level of organic solvent has been shown not to
affect the stability of AChE in any way. Note that experiments
conducted without organic solvent present produced significantly
lower detection limits for paraoxon or AfB1. Therefore, for
practical applications, it is recommended that low levels of
organic solvents be used not only for dissolution purposes but also
to enhance sensitivity. Distilled deionized water (ddH.sub.2O) was
used to dissolve PVAm. All other solutions were prepared using Tris
buffer (100 mM, pH 8) if not otherwise stated. CAUTION: Both AfB1
and paraoxon are extremely toxic. These materials should be handled
with gloves and used in a fumehood.
[0173] Preparation of Sol-Gel Materials:
[0174] Two biocompatible sol-gel precursors, diglyceryl silane
(DGS) and sodium silicate (SS) were used to prepare sols for enzyme
entrapment and printing onto paper. DGS sols were made by mixing 10
mL of ddH.sub.2O with 1 g of finely ground DGS. The mixture was
sonicated on ice bath for 20 min to dissolve the DGS and then
filtered through a 0.22 .mu.m membrane syringe filter to remove any
particulates in the solution.
[0175] SS sols were prepared by mixing 10 mL of ddH.sub.2O with 2.9
g of sodium silicate solution (pH.about.13) followed by addition of
5 g of Dowex cation exchange resin to replace Na.sup.+ with
H.sup.+. The mixture was stirred for 30 seconds to reach a final pH
of .about.4, and then vacuum filtered through a Buchner funnel. The
filtrate was then further filtered through a 0.45 .mu.m membrane
syringe filter. These sols were used to formulate silica-containing
inks as described below.
[0176] Construction of Bioactive Paper-Based Solid-Phase
Sensor:
[0177] The papers were coated with a total of three different
materials in a specific sequence. In general, this involved: 1)
printing of a PVAm underlayer directly onto the paper surface; 2)
printing of a silica sol intermediate layer; 3) printing of a
buffered enzyme solution containing AChE (final conc. 50 U/mL) and
DTNB (final conc. 500 .mu.M); and 4) printing of a silica sol
overlayer, as shown in FIG. 1. Between printing of the different
silica and bioinks, 15-20 min was allowed for air drying. The
different printing solutions (PVAm, sol or enzyme) were modified by
addition of glycerol to control viscosity and Triton X-100 to
control surface tension so as to optimize the printing performance
(ability to jet the inks) as well as the enzyme activity, as
described below. As noted in Table 1, addition of glycerol to PVAm
inks was not necessary as the viscosity of an aqueous solution of
this polymer was on the order of 3 cP. This high viscosity of the
0.5 wt % solution is likely due to the high molecular weight (ca.
1.5 MDa) of the polymer. The solutions were deposited using a
piezoelectric ink jet printer (DMP-2800) from Fujifilm Dimatix, Inc
(Japan) using Drop Manager software (version 1.3.0.7). This system
has a microelectromechanical system (MEMS)-based cartridge-style
printhead that allows filling with desired bioinks (ca. 0.5-2 mL).
Each cartridge has 16 nozzles linearly spaced at 254 microns with
typical drop sizes of 1-10 pL. The instrument is equipped with a
drop imaging system (Drop Watcher) that allows observation and
capture of the events during drop formation on the printhead
nozzles and the trajectory of the drops after ejection. Jetting
conditions are described in Table 1. In all cases the bioactive
inks were printed by applying 16 piezo firings with one printing
cycle per ink in a stepwise fashion as a 0.25.times.0.25 cm square
pattern onto Mead brand cardboard (paper substrate, 10.times.8 cm)
using a separate cartridge for each of the PVAm, silica and enzyme
"inks". For control experiments, a buffer that did not contain AChE
was printed between the silica layers. Other controls involved
printing of AChE+DTNB directly onto the PVAm underlayer without a
silica coating, and printing of AChE+DTNB onto PVAm/silica without
printing a silica overlayer.
[0178] Ink Viscosity and Surface Tension Measurements:
[0179] The dynamic viscosity of the bioink components was measured
using a capillary viscometer (Cannon-Fenske viscometer, size 75,
Vineland, N.J.) at room temperature. The viscometer was calibrated
with MilliQ water (viscosity .about.1 cP) before measuring the
viscosity of the bioinks. Surface tension values were measured
using a Kruss pendant drop apparatus. The shape of the pendant drop
was analyzed using DSA10 shape analysis software by applying the
Laplace equation. Pendant drops were formed by a Kruss needle with
an outer diameter of 1.5 mm, connected to a 1 mL Perfektum glass
syringe from Popper & Sons Inc. MilliQ water with surface
tension of 72.8 mN/m was used to calibrate the needle's inner
diameter. All surface tension values of pendant drops were measured
at 22.degree. C. with temperature controlled by a NESLAB thermostat
system.
[0180] Surface Topography:
[0181] Paper substrates were subjected to optical profilometry and
SEM imaging prior to deposition of any materials, after ink jet
deposition of PVAm and after deposition of both PVAm and the
silica/enzyme/silica layers. Optical profilometry images were
obtained using WYKO NT 1100 Optical Profiling System using the VSI
measurement mode and a magnification of 20.times.. For SEM, samples
were sputter-coated with platinum (layer thickness 15 .ANG.) to
avoid charging effects and were imaged using a JEOL Ltd. (Tokyo,
Japan) JSM-7000F instrument operating at 5 kV and a probe distance
of 5.8 mm. The hydrophilic or hydrophobic nature of surface was
also estimated by measuring the contact angle of ddH.sub.2O drop on
paper using a Kruss pendant drop apparatus.
[0182] Monitoring AChE Activity on Paper:
[0183] Prior to monitoring AChE activity on paper, the activity of
AChE as a function of enzyme concentration was optimized. Different
concentrations of AChE (0.about.200 U/mL) were entrapped in SS+30%
glycerol in a 96 well plate (total volume of 80 .mu.L). A mixture
(20 .mu.L) of DTNB (500 .mu.M) and ATCh (300 .mu.M) was then added
into each well and incubated for 5 min to allow color development.
The absorbance at 412 nm was then measured using a TECAN Safire
microwell plate reader.
[0184] The AChE activity on the bioactive paper strip was evaluated
by measuring the color intensity produced by the enzymatic reaction
using Ellman's method. The performance can be assessed in two ways:
a) by direct addition of substrate solution to sensing area, and b)
by immersion into the substrate solution. The performance of our
sensor was essentially the same for both these cases. However, in
the case of direct analyte addition, only small amounts of reagent
are needed (5 .mu.L) relative to dipstick sensors (.about.2 mL),
reducing cost per assay. For optimization of AChE activity, small
amounts (5 .mu.L) of different concentrations of the substrate ATCh
(0-500 .mu.M) was added directly onto the sensing area of the paper
strip and incubated for 5 min at room temperature to allow the
yellow color to develop. The color intensity of the sensing areas
was quantified by obtaining a digital image (Canon A630, 8.0
MegaPixel operated in automatic mode with no flash and with the
macroimaging setting on) and using ImageJ.TM. software to analyze
the jpeg images. ImageJ.TM. software uses a 256 bit color scale and
for image processing the images were inverted so that areas that
were originally white became black and corresponded to a color
intensity of zero and while areas that were originally black became
white and corresponded to 256. Based on this scale, increases in
the amount of yellow color cause an increase in color intensity of
the sensor strips. To account for variations in color intensity
owing to differences in environmental illumination, a background
subtraction (color intensity of the paper surface closest to the
sensing area) was done for each data point.
[0185] The long-term stability of the enzyme printed on the paper
strip was evaluated over a period of 60 days with the paper strip
stored at 4.degree. C. The assay conditions were the same as those
described above.
[0186] Monitoring the Effect of PVAm:
[0187] In order to investigate the effect of the PVAm underlayer on
the sensor performance, a lateral flow-based paper chromatographic
system was developed. In this case, strips of Whatman No. 1 filter
paper (Sigma-Aldrich) were used in place of the Mead cardboard as
the Whatman paper is more hydrophilic and thus supports capillary
flow of aqueous solutions. The Whatman paper strips (1.times.10 cm)
were printed with aqueous inks containing various levels of PVAm
(0-1 wt. %) and were allowed to air dry for 15 min. The PVAm
treated strips were then immersed into a solution of
5-thio-2-nitrobenzoate (TNB.sup.-, the colored product of the AChE
catalyzed reaction), which was produced enzymatically from ATCh
(final conc. 300 .mu.M), DTNB (final conc. 500 .mu.M), and AChE
(final conc. 50 U/mL) with the sensing area above the liquid level.
The retardation factor (Rf) was calculated based on the ratio of
migration distance of the product (TNB.sup.-) relative to the
migration distance of solvent (Milli-Q water) from this lateral
flow based platform.
[0188] Lateral flow and dipstick assay formats were also developed
to monitor the capability of PVAm to capture and preserve the color
produced from the AChE catalyzed reaction when performed on paper.
In this case, Whatman (for lateral flow) or Mead cardboard
dipsticks (1.times.10 cm) were prepared using ink jet deposited
PVAm (0 or 0.5 wt %), silica (SS+30% glycerol) and AChE+DTNB (50
U/mL+500 .mu.M in 30% glycerol, with 1 wt % Triton X-100 present in
all solutions) as described above, followed by placing the sensor
strip into a solution of substrate (300 .mu.M ATCh) with the
sensing area located above the liquid level (lateral flow) or
within the solution (dipstick) and allowing the color to develop
for 5 min. Following the assay the resulting color intensity
remaining on the paper strip was monitored once a day for up to
three weeks.
[0189] Measurement of AChE Inhibitors Using Bioactive Paper:
[0190] The inhibitory effects of paraoxon and AfB1 on the
solid-phase biosensor were evaluated by measuring the decrease in
the color intensity produced by the Ellman reaction. The
PVAm/silica/AChE+DTNB/silica bioactive paper strip was first
incubated with various concentrations of paraoxon solution (5
.mu.L, 0-100 .mu.M) or AfB1 solution (5 .mu.L, 0-100 .mu.M)
[CAUTION: these assays should be performed in a fumehood using
appropriate protective apparel], for 10 min after which 10 .mu.L of
a solution of 300 .mu.M ATCh was deposited onto the paper strip and
incubated for 5 min. The intensity was determined by analyzing a
digital image with the ImageJ software as described above.
[0191] Results and Discussion
[0192] Bioink Formulation and Jetting:
[0193] Initial attempts at ink jet deposition of sol-gel based
bioinks utilized silica sols to which buffered proteins had been
added. While it was possible to produce protein-doped sols with
relatively long gelation times, it was not possible to deposit such
materials without gelation occurring in the nozzles of the ink jet
cartridge. For this reason all further studies on sol-gel based
inks utilized a multi-layer deposition method wherein the silica
sol and buffered aqueous protein solutions were deposited from
separate cartridges so as to avoid mixing prior to deposition on
the paper substrate.
[0194] Both the silica and aqueous protein "inks" were optimized to
allow reproducible jetting onto paper substrates. Physico-chemical
properties such as surface tension and viscosity are parameters
that effect ink stability and ejectability. Several additives
(e.g., surfactants, viscosity modifiers) are generally incorporated
in the ink formulations to optimize these rheological properties.
However, inappropriate additives may often negatively affect enzyme
activity. Therefore, it is desirable to produce a suitable ink
formulation in which the enzyme is active and at the same time
produces stable and reproducible drops during jetting.
[0195] To adjust the ink surface tension (initially in the range of
60-78 mNm.sup.-1 without surfactant) to the printable range (30-40
mNm.sup.-1), Triton X-100, a mild detergent, was used as a
surfactant. To determine the effect of Triton X-100 on AChE
activity, a solution of AChE (50 U/mL) in Tris buffer containing
0.1 wt. % of Triton X-100 was prepared, and then the enzyme
activity in solution was measured using the Ellman assay. No
significant loss of AChE activity was observed in the presence of
this low level of Triton X-100. Therefore, 0.1 wt. % Triton X-100
was included in all bioink formulations (e.g., AChE, sol-gel
derived silica, PVAm) to get the optimum surface tension for
printing (Table 1).
[0196] In order to adjust the ink viscosity (initially in the range
of 1.01-1.33 cP without viscosity modifiers) to the desired value
(2-10 cP), two well-known viscosity modifiers,
carboxymethylcellulose sodium salt (CMC) and anhydrous glycerol,
were investigated. PEG and PVA, though biocompatible, were not
examined as these species have a tendency to promote macroscopic
phase separation in sol-gel derived silica,.sup.xxix, xxx which
could result in significant protein leaching in this
example..sup.xxxi These materials could be utilized when larger
molecules are to be entrapped and there is less concern about
leaching from the macroscopic silica matrix. CMC, a charged
polymer, and glycerol were both chosen for evaluation.
[0197] Initial studies on the effects of CMC (0.about.0.5 wt. %) on
AChE activity in solution showed that this additive led to a
significant decrease in AChE activity at concentrations above 0.2
wt %. Therefore, CMC was not investigated further as a viscosity
modifier. On the other hand, glycerol did not produce a decrease in
AChE activity, even at levels of 30% v/v. Addition of this level of
glycerol to either the SS derived silica sol or AChE-based inks
also resulted in excellent drop formation and jettability, as shown
in Table 1. However, all inks prepared from DGS derived silica sols
using the conditions utilized herein, while jettable, resulted in
poor adhesion and cracking after drying on the paper substrate.
Therefore SS was used as the precursor of choice for further
studies. The use of DGS may be possible be adjusting conditions
using methods known in the art.
[0198] Based on the excellent ink jetting properties and the high
quality of the resulting deposited materials, all inks were
formulated with 30% (v/v) glycerol and 0.1 wt % Triton-X100 using
sodium silicate as the silica precursor.
[0199] Coating Properties:
[0200] To characterize the coatings on paper substrates, optical
profilometry, contact angles and SEM images were obtained for both
unmodified and modified paper substrates. FIG. 2A shows optical
profilometry images of paper that is coated with PVAm only (FIG.
2A(a)), and with both PVAm and the silica/AChE+DTNB/silica layers
(FIG. 2A(b)). No bioinks were printed on the non-sensing region.
The profilometry results show that the PVAm layer is approximately
4 .mu.m thick, while the sol-gel based coating had an average
thickness of about 24 .mu.m. Similar results were obtained for
layers printed on glass slides, suggesting that the majority of the
sensing layer was present on top of the paper rather than within
the paper. This is further supported by the contact angles for PVAm
coated and PVAm free Mead cardboard and Whatman paper substrates,
which were approximately 112.2.+-.1.1.degree., 50.1.+-.0.3.degree.
and zero, respectively. The results indicate that the PVAm layer
imparts high hydrophobicity to an already coated paper surface, and
thus liquid penetration into the surface is improbable.
[0201] FIG. 2B shows SEM images of the unmodified paper (FIG.
2B(a)), PVAm coated paper (FIG. 2B(b)) and paper that was coated
with both the PVAm and silica/AChE+DTNB/silica layers (FIG. 2B(c)).
The unmodified paper surface is very rough (average roughness of
.+-.972 nm) and heterogeneous, and clearly shows the presence of
significant amounts of fillers (i.e., clay particles) at the
surface of the paper and no evidence for paper fibers at the
surface. This is consistent with the fact that the Mead paper used
in this study had a protective coating. The deposition of PVAm
resulted in a much smoother surface (.+-.554 nm), and suggests that
the cationic polymer likely provides a barrier coating above the
filler layer onto which the silica layer is deposited, consistent
with the data described above. Thus, in addition to acting as an
anion capture agent, PVAm also produces a more uniform surface
which may prevent enzyme leaching into the paper. Deposition of the
silica/AChE+DTNB/silica layer resulted in a relatively homogeneous,
crack-free layer (roughness of .+-.668 nm) which showed no evidence
of large scale macropores (diameter >0.5 .mu.m), consistent with
the inability of glycerol to act as a porogen. Taken together, the
profilometry and SEM images show that the sol-gel based ink layer
is present on top of the paper, rather than penetrating through the
paper. This is advantageous as it should help to retain the
colorimetric signal within a thin layer rather than having it
diffuse throughout the thickness of the paper, making visualization
easier.
[0202] Monitoring AChE Activity and its Storage Stability:
[0203] Prior to developing the dipstick sensor, the activity of
AChE was evaluated as a function of enzyme concentration (0-200
U/mL) via the Ellman assay when entrapped in sol-gel derived
monolithic silica prepared from SS with 30% glycerol. The signal,
measured 5 min after addition of 300 .mu.M ATCh and 500 .mu.M DTNB,
increased linearly over the concentration range from 0-50
UmL.sup.-1 after which the signal showed negative deviation and
reached a plateau at .about.100 UmL.sup.-1. A value of 50 U/mL was
chosen the best compromise between a low enzyme loading, a
sufficiently high signal (>4-fold increase over background) and
good long-term stability. The high activity of entrapped AChE is in
agreement with previous reports showing that the enzyme is active
and stable in sol-gel derived silica materials..sup.xxxii
[0204] Based on the AChE activity data, a solid-phase bioactive
paper based sensor was constructed by multilayer ink jet printing
of several bioinks in the order: PVAm, silica, 50 U/mL AChE+500
.mu.M DTNB, silica using the compositions listed in Table 1 with
30% glycerol added to all inks except PVAm. This resulted in a
layered system as shown in FIG. 3. The effects of adding varying
levels of ATCh over the bioactive paper sensor are shown in FIG. 4a
(a separate paper sensor is used for each ATCh concentration). Here
it is seen that as the concentration of ATCh increases so too does
the color intensity, recorded 5 min after addition of the substrate
over the sensing area. The color intensity saturated at a level of
.about.300 .mu.M ATCh and suggested a K.sub.M of ca. 150 .mu.M,
which is in good agreement with previous literature
reports..sup.xxxiii The color intensity was about four-fold higher
than that of a negative control (absence of ATCh but with AChE
present).
[0205] To ascertain whether or not the supporting silica or PVAm
materials degraded DTNB or ATCh, a similar experiment was conducted
using the same inks as above but with AChE absent from the aqueous
ink layer. As shown in FIG. 4b, the responses remained at the
baseline signal upon addition of varying levels of ATCh and were
similar to signals obtained from control experiments performed in
the absence of substrate. Thus, these results confirm that the
change in the color intensity is due to the AChE catalyzed
hydrolysis of ATCh to TCh which then reduces DTNB to TNB.sup.-.
Furthermore, this result shows that the AChE is able to withstand
the shear forces associated with ink jet deposition and remains
active when deposited between silica layers on a paper
substrate.
[0206] The long-term stability AChE within the layered coating on
the paper substrate was also investigated. Results demonstrated
that the enzyme retained >95% of its initial activity over a
period of at least two months when stored at 4.degree. C.,
indicating that both the enzyme and the DTNB reagent remained
viable during storage. The observed stability of the enzyme when
entrapped in silica shows that the bioactive paper sensor should
have a sufficient shelf-life to allow storage and shipping.
[0207] To further assess the "sandwich architecture", two controls
were done to assess the effects of both the PVAm and the silica
layers on AChE activity and stability. In the first case, the
enzyme was printed directly onto PVAm without silica. In this case,
the enzyme showed activity on day 1, but no activity by day 2,
likely owing to dehydration and/or strong electrostatic
interactions between the enzyme and the cationic polymer which
caused denaturation. In the second case, the enzyme+DTNB solution
was printed onto the silica material that covered the PVAm layer,
but the silica overlayer was not added. Such sensors showed
activity that was comparable to the "sandwich architecture" device
after 2 days, however, after prolonged storage the "non-sandwich"
device produced significantly lower signal as a result of enzyme
inactivation. Additionally, using this approach (i.e. no top silica
layer), it was not possible to employ the sensor for dip-stick
assays as the enzyme readily desorbed from the paper/silica
surface.
[0208] Effects of PVAm on Biosensor Performance:
[0209] Two problems that were initially encountered when carrying
out the Ellman assay using sol-gel entrapped AChE on the
paper-based sensor were dispersion of the colored product over a
large area, leading to lower color intensity, and loss of color
intensity with time. The former issue is expected based on the
ability of the small molecular weight colored product to readily
move through the pores of the silica matrix and thus leach out of
the sensing area. The latter issue appears to be related to a
secondary chemical reaction of the TNB.sup.- with either the silica
or some component in the paper, causing complete loss of color
intensity over a period of a few days (see below). This makes
storage of used sensors for future reference impossible. Therefore,
trapping and preserving the color within a finite region was
desirable to obtain the highest output signals and keep the signal
stable over long periods of time. It was reasoned that the best
method for capturing the anionic colored product was to introduce a
cationic polymer, PVAm, onto the surface of the paper. This polymer
has recently been shown to be useful for enhancing the wet strength
of paper,.sup.xxxiv and thus should be compatible with the
substrate, and binds strongly to silica,.sup.xxxv which should
promote adhesion of the silica overlayer and not interfere with
this coating layer. However, introduction of PVAm directly to a
silica sol causes very rapid gelation owing to base catalyzed
condensation, and hence this polymer was printed on paper prior to
printing of the silica sol to avoid this problem.
[0210] To examine the effect of PVAm on sensor performance, a
series of experiments were performed. Initial studies utilized a
paper chromatographic set-up (lateral flow-based) to assess the
ability of PVAm coatings containing varying levels of the polymer
to retain the anionic product. For this purpose, hydrophilic
Whatman 1 paper strips were printed with solutions containing 0-1
wt % of PVAm and a solution of TNB.sup.- (produced by mixing 300
.mu.M ATCh, 500 .mu.M DTNB and 50 U/mL of AChE in Milli-Q water)
was allowed to travel up the paper via capillary action. FIG. 5a
shows the values of retardation factor (Rf), a measure of the
relative mobility of TNB.sup.-, as a function of PVAm concentration
and demonstrates that the Rf values decreased with increasing
levels of PVAm up to a level of 0.5 wt % of PVAm. Thus, 0.5 wt %
PVAm was utilized in further studies to trap the negatively charged
analyte.
[0211] FIG. 5b shows the colour intensity due to elution of
Ellman's solution using the lateral-flow based paper
chromatographic system. The areas within the dashed boxes were
either treated or not treated (control) with 0.5 wt % PVAm,
deposited via ink jet spraying onto Whatman 1 paper, followed by
printing of the silica/AChE+DTNB/silica layers over the same area.
It was found that PVAm was able to trap and concentrate the
TNB.sup.- reaction product without diminishing the color intensity,
while the unmodified paper (control) failed to trap the yellow
color. As a result, the intensity of the yellow color was much
higher when PVAm was present (0.25.times.1 cm), which should
produce a better detection limit when using the paper to sense AChE
substrate or inhibitors.
[0212] The capability of PVAm to preserve the TNB.sup.- product for
an extended period of time was also investigated. For this, Mead
cardboard dipsticks were coated with the PVAm ink followed by
silica/AChE+DTNB/silica ink layers and the dipsticks were immersed
into a solution of 300 .mu.M ATCh for 1 min. Images were obtained
30 min or 24 h after exposure to ATCh. A control sample was also
tested in which the PVAm underlayer was not present. FIG. 5c shows
images of the dipsticks under the different testing conditions, and
clearly demonstrates that the PVAm is able to retain the yellow
product while untreated paper causes the yellow color to disappear
almost completely after only 24 h. Further testing revealed that
the PVAm layer was capable of retaining the color for at least
three weeks. Thus, the bioactive paper strips can be stored for
future reference.
[0213] Neurotoxin Measurement Based on AChE Inactivation:
[0214] Neurotoxins such as paraoxon and aflatoxin B1 are well known
inactivators of
acetylcholinesterase..sup.xxvii,xxxvi,xxxvii,xxxviii The ability to
detect these compound using the ink jet printed AChE-based paper
sensor was investigated by using an overspotting method wherein
small volumes of reagents were added to the sensing area directly.
In this case, as little as 10 .mu.L of solutions containing various
concentration of paraoxon or AfB1 could be tested by applying them
onto the sensing area of the strip, incubating for 10 min at room
temperature, adding 10 .mu.L of a solution containing 300 .mu.M
ATCh and finally measuring the color intensity after 5 min using a
digital camera and image processing software. FIG. 6a shows the
dose-dependent inhibition effects of paraoxon, while FIG. 6b shows
the semi-logarithmic plot of color intensity, corresponding to the
same experiments, demonstrating an IC.sub.50 in the range of 1
.mu.M and a detection limit (S/N=3) of ca. 100 nM. FIG. 7a and FIG.
7b show the dose-dependent inhibition responses and a
semi-logarithmic plot of color intensity, respectively, for AfB1,
and show that the IC.sub.50 in this case is ca 100 nM, with a LOD
of .about.30 nM. A comparison of the responses obtained at 100 nM
of either paraoxon or AfB1 show that AfB1 is a more potent
inhibitor (.about.45% inhibition vs..about.25% inhibition for
paraoxon), in agreement with previous
studies..sup.xxviii,xxxiii,xxxix
[0215] The insets for FIGS. 6 and 7 show the images of the paper
strips at each inhibitor concentration, and clearly demonstrate
that detection of the inhibitors is possible using the naked eye.
This is an advantageous aspect of the bioactive paper strips, as
this permits the use of the test strips directly in the field and
eliminates the need for sophisticated instrumentation. The presence
of a simple colorimetric readout also enables rapid imaging and
transmission to a central lab for further quantitative analysis
using a simple cell phone camera combined with e-mail or MMS
messaging..sup.iii,v,vii Hence, rapid, on-site qualitative or
quantitative analysis of organophosphates or aflatoxins should be
possible using this bioactive paper platform.
Example 2
Reagentless Bioactive Paper-Based Lateral Flow Sensor for Detection
of Pesticides
[0216] Reagents:
[0217] All chemical from commercial sources were of analytical
grade. Sodium silicate solution (.about.14% NaOH, .about.27%
SiO.sub.2), dowex 50WX8-100 ion-exchange resin,
acetylcholinesterase (AChE, from electrophorus electricus, EC
3.1.1.7), Triton X-100, the pesticides including both
organophosphate (OP) (e.g., paraoxon and malathion), and carbamate
(CM) (e.g., carbaryl and bendiocarb) were obtained from
Sigma-Aldrich (Oakville, ON, Canada). The indophenyl acetate (IPA)
was purchased from Pealtz & Bauer, Inc (USA). Polyvinylamine
(PVAm; 1.5 MDa) was obtained from BASF (Mississauga, Canada), as a
gift. Anhydrous glycerol was purchased from Fluka BioChemika Ultra
(UK). Distilled deionized water was obtained from a Milli-Q
Synthesis A10 water purification system.
[0218] Solutions Preparation:
[0219] Stock solutions of the IPA, bendiocarb, carbaryl, paraoxon
and malathion were made up daily and were not used for more than 3
h after preparation to minimize the potential for hydrolysis. A
mixture of Tris buffer (10 mM, pH 6.8) and 5% methanol (Sigma) was
used for dissolving bendiocarb, carbaryl, malathion, and paraoxon
while a mixture of Tris buffer (10 mM) with pH 8.0 and 5% methanol
(Sigma) was used for dilution of IPA. These solvents not only aid
in dissolution of the AChE inhibitors, but also enhance the
affinity of pesticides for binding to AChE. Furthermore, this level
of organic solvent has been shown not to affect the stability of
AChE in any way. Note that experiments conducted without organic
solvent present produced significantly lower detection limits for
pesticides. Therefore, for practical applications, it is
recommended that low levels of organic solvents be used not only
for dissolution purposes but also to enhance sensitivity. Distilled
deionized water (ddH.sub.2O) was used to dissolve PVAm. All other
solutions were prepared using Tris buffer (10 mM, pH 8) if not
otherwise stated. CAUTION: Both carbamate (e.g., bendiocarb,
carbaryl) and organophospate (e.g., paraoxon, malathion) pesticides
are extremely toxic. These materials should be handled with gloves
and used in a fumehood.
[0220] Sol-Gel Material Preparation:
[0221] A biocompatible sol-gel precursor, sodium silicate (SS) was
used to prepare sols for both enzyme and IPA entrapment and
printing onto paper. SS sols were prepared by mixing 10 mL of
ddH.sub.2O with 2.59 g of sodium silicate solution (pH.about.13)
followed by addition of 5 g of Dowex cation exchange resin to
replace Na.sup.+ with H.sup.+. The mixture was stirred for 30
seconds to reach a final pH of .about.4, and then vacuum filtered
through a Buchner funnel. The filtrate was then further filtered
through a 0.45 .mu.m membrane syringe filter. These sols were used
to formulate silica-containing inks as described below.
[0222] Fabrication of Reagentless Bioactive Paper-Based Lateral
Flow Sensor:
[0223] A section of Whatman #1 paper was cut into small pieces
(1.times.10 cm) in which enzyme, AChE and substrate, IPA were
entrapped in the two different regions (e.g., sensing and substrate
regions) following the sequences of PVAm(0.5 wt %)/silica/AChE (500
U/mL)/silica and silica/IPA (3 mM)/silica, respectively by using
either ink jet printing (using a piezoelectric ink jet printer
(DMP-2800), Fujifilm Dimatix, Inc, Japan) or over spotting, as
shown in FIG. 8(b). However in the case of ink jet printing, all
inks were modified with respect to viscosity and surface tension
similarly as described in Example 1. The sensor was then allowed to
air dry at room temperature. Two control experiments: (1) a buffer
that did not contain AChE was entrapped between the silica layers
in the sensing region, and (2) a buffer that did not contain IPA
was entrapped between the silica layers in the substrate region.
Other controls involved entrapment of AChE directly onto the PVAm
underlayer without a silica coating, and entrapment of AChE onto
PVAm/silica without printing a silica overlayer.
[0224] Optimization of Reagentless Paper-Based Lateral Flow Assay
Platform:
[0225] The lateral flow assay format was optimized with regard to
the PVAm levels, the pH level of Tris buffer (10 mM) that was used
to dissolve the enzyme substrate, concentration of substrate, and
the concentration of the enzyme.
[0226] In Example 1, a lateral flow-based paper chromatographic
system was developed to investigate the effect of the PVAm
underlayer on the solid phase sensor performance where the PVAm
treated strips were immersed into a solution of
5-thio-2-nitrobenzoate (TNB.sup.-, the colored product of the AChE
catalyzed reaction), which was produced enzymatically from ATCh
(final conc. 300 .mu.M), DTNB (final conc. 500 .mu.M), and AChE
(final conc. 50 U/mL) with the sensing area above the liquid level.
The retardation factor (Rf) was calculated based on the ratio of
migration distance of the product (TNB.sup.-) relative to the
migration distance of solvent (Milli-Q water) from this lateral
flow based platform. Therefore, the PVAm level was not optimized
further to entrap as well as preserve the anionic dye
(indophenoxide.sup.-); hence, the previously optimized PVAm level
was taken as an optimum value in this example.
[0227] The effect of pH of the Tris buffer on IPA stability was
investigated in solution. For this, IPA (3 mM) was dissolved with
Tris buffer having different levels of pH (4.about.9.5). 80 .mu.L
of IPA solution and 20 .mu.L of AChE (final conc 500 U/mL) were
then mixed into a 96-well plate and incubated for 5 min to allow
color development. The absorbance at 640 nm was then measured using
a TECAN Safire microwell plate reader.
[0228] Prior to monitoring AChE activity on paper, the activity of
AChE as a function of enzyme concentration and chromogenic AChE
substrate, IPA concentrations were optimized. Different
concentrations of AChE (0.about.1500 U/mL) were entrapped in SS+30%
glycerol in a 96 well plate (total volume of 80 .mu.L). 20 .mu.L of
IPA (0.about.5 mM) was then added into each well and incubated for
5 min to allow color development. The absorbance at 640 nm was then
measured using a TECAN plate reader.
[0229] The AChE activity on the bioactive paper strip was evaluated
by measuring the color intensity produced by the enzymatic
hydrolysis of IPA. The performance of the sensor under optimized
conditions can be assessed in two ways: (1) directly (normal
lateral flow-based chromatography) without incubating the
contaminated sample, and (2) inverted lateral flow-based
chromatography with incubation of the sample. For the latter case,
after incubation 5 min at room temperature, the sensor was inverted
again and immersed into dH.sub.2O for bringing up IPA by lateral
flow action into sensing area of the strip above the liquid
level.
[0230] The color intensity of the sensing areas was quantified by
obtaining a digital image (Canon A630, 8.0 MegaPixel operated in
automatic mode with no flash and with the macroimaging setting on)
and using ImageJ.TM. software to analyze the jpeg images.
ImageJ.TM. software uses a 256 bit color scale and for our image
processing the images were inverted so that white corresponded to a
color intensity of 256 and black corresponded to zero. Based on
this, increases in the amount of blue color cause an increase in
color intensity of our reagentless sensor strips. To account for
variations in color intensity owing to differences in environmental
illumination, a background subtraction (color intensity of the
paper surface closest to the sensing area) was done for each data
point.
[0231] Measurement of Pesticides Using Reagentless Paper-Based
Lateral Flow Platform:
[0232] The inhibitory effects of carbamate (e.g., bendiocarb,
carbaryl) and organophosphate (e.g., paraoxon, malathion)
pesticides on the reagentless paper-based sensor were evaluated by
measuring the decrease in the color intensity produced by enzymatic
hydrolysis of IPA. The sensing area of the bioactive paper strip
was first incubated with various concentrations of either carbamate
or organophosphate solution for 5 min following the inverted
lateral flow procedure as described above. The sensor was then
inverted again and immersed into dH.sub.2O to bring up IPA by
lateral flow action into the sensing area of the strip above the
liquid level. The color intensity was determined by analyzing a
digital image with the ImageJ software as described above.
[0233] Matrix Effect in the Analysis of Paraoxon into Foods:
[0234] The matrix effects in the analysis of paraoxon in drinking
milk (2%, pH 7.2) and apple juice (pH 3.6) samples were
investigated. Several standard paraoxon solutions (0.about.10
.mu.M) were mixed into both milk (10 mL) and apple juice (10 mL)
samples. The developed reagentless sensor was immersed inversely
into each paraoxon containing samples. The sensor was incubated for
5 min and the inhibition of AChE was tested following the inverted
lateral flow procedure as described above. The pH of the apple
juice was adjusted between 7.about.8 using a few drops of 1N NaOH
before doing experiment.
[0235] Analysis of Paraoxon in Real-Life Samples:
[0236] Different concentrations of paraoxon solution (0.about.50
mM) was sprayed on apple and head lettuce. After air dry, the
deposited paraoxon samples were collected using a cotton-swab and
transferred into 2 mL dH.sub.2O; this solution was tested following
the inverted lateral flow procedure as described above with 5 min
incubation using our developed reagentless sensor.
[0237] Storage Stability of the AChE- and IPA-Immobilized
Strip:
[0238] The long-term stability of the enzyme and enzyme substrate,
IPA entrapped on the paper strip was evaluated over a period of 12
weeks with the paper strip stored at 4.degree. C. The performance
of the stored sensor was also examined by assaying for paraoxon.
The assay conditions were the same as those described above.
[0239] Result and Discussion
[0240] Optimization of the Reagentless Lateral-Flow Assay
Format:
[0241] The concept of the reagentless lateral flow assay is shown
in FIG. 8. FIG. 8a shows the IPA reaction, which results in a color
change from yellow to blue. FIG. 8b shows a schematic of the ink
jet layers used in the assay, with AChE or IPA entrapped between
two silica layers in distinct regions of the paper-based device.
Flow of liquid moves the IPA up to the AChE region and produces a
product that can be captured by a PVAm underlayer. The assay can be
done directly or in an "inverted" format. In the first case an
inhibitor is present in the test solution and is flowed through the
IPA region to the AChE region. In the second case, the test
solution is flowed from the opposite end of the device into the
AChE region where it is incubated. After a set amount of time the
other end is placed into the test solution and the IPA is moved
into the AChE region. This allows compounds that are slow
inhibitors of the enzyme to be detected.
[0242] Prior to developing an efficient reagentless lateral flow
based paper sensor with high sensitivity and rapid response, all
the parameters, such as PVAm levels, the pH of the buffer,
concentration of substrate, and the concentration of the enzyme
were optimized.
[0243] Trapping and preserving the color (that is produced during
enzymatic hydrolysis of substrate) within a finite region of the
sensor is advantageous to obtain the highest output signals and
keep the signal stable over long periods of time. In Example 1, a
lateral flow-based paper chromatographic system was developed to
investigate the effect of the PVAm underlayer on the solid phase
sensor performance where the PVAm (0.about.1 wt %) treated strips
were immersed into a solution of 5-thio-2-nitrobenzoate (TNB.sup.-,
the colored product of the AChE catalyzed reaction), which was
produced enzymatically from ATCh (final conc. 300 .mu.M), DTNB
(final conc. 500 .mu.M), and AChE (final conc. 50 U/mL) with the
sensing area above the liquid level. The retardation factor (Rf)
was calculated based on the ratio of migration distance of the
product (TNB.sup.-) relative to the migration distance of solvent
(Milli-Q water) from this lateral flow based platform. The
capability of PVAm to preserve the TNB.sup.- product for an
extended period of time was also investigated. Therefore, the PVAm
level was not optimized further to entrap as well as preserve the
anionic dye (indophenoxide.sup.-) in this Example. Here, it was
determined whether or not the previously optimized PVAm level (0.5
wt %) was enough to trap as well as concentrate the blue color
product (indophenoxide.sup.-). FIG. 9(a) shows the color intensity
(CI) due to elution of IPA (3 mM, final conc.) in the lateral flow
based platform. The areas within the dashed boxes were either
treated or not treated (control) with 0.5 wt % PVAm, deposited via
ink jet spraying/overspotting onto Whatman #1 paper, followed by
printing/overspotting of the silica/AChE(500 U/mL)/silica layers
over the same area. The data indicated that the 0.5 wt % PVAm level
was able to trap and concentrate the indophenoxide.sup.- reaction
product efficiently without diminishing the color intensity, while
the unmodified paper (control) failed to trap the blue color. As a
result, the intensity of the blue color was much higher when PVAm
was present, which should produce a better detection limit when
using the paper to sense AChE substrate or inhibitors. Based on
these data, a reagentless lateral-flow based paper sensor was
constructed. FIG. 9(b) represents a reagentless bioactive paper
platform, in which AChE and IPA were entrapped into two different
regions and then the sensor was dipped into dH.sub.2O to bring up
the IPA reagent by capillary action into sensing region for the
generation of blue color due to enzymatic hydrolysis of substrate.
This result indicates a proof of concept study for the development
of reagentless bioactive lateral-flow paper based sensing
platform.
[0244] Indophenyl acetate (IPA) is a well known pH-sensitive
chromogenic substrate for AChE. Therefore, the pH of the buffer
effects IPA stability as well as the effective enzymatic-substrate
reaction.
[0245] AChE hydrolyzes the substrate IPA at pH 8.0 to produce a
highly blue colored product. Preliminary studies on the effects of
pH (4.about.9.5) of the Tris buffer (10 mM) on IPA stability in
solution showed that at levels slightly lower than pH 8.0 gives
considerably lower absorbance readings for the enzyme-substrate
reaction product, while at slightly higher levels than pH 8.0, auto
hydrolysis of IPA occurred even in the absence of AChE. In
addition, the maximum absorbance of the reaction product was
saturated at pH 8.0. Therefore, pH 8.0 was considered as an optimum
value for the Tris buffer (10 mM), which was used to dissolve IPA
in this study. The observed stability of IPA at buffer pH 8.0 is in
agreement with previous reports.
[0246] Prior to monitoring AChE activity on paper, both the
activity of AChE as a function of enzyme concentration and
chromogenic AChE substrate, IPA concentrations (0-5 mM) were
optimized via the IPA-based colorimetric assay when entrapped in
sol-gel derived monolithic silica prepared from SS with 30%
glycerol in a 96-well plate. With increasing concentration of AChE
solution (0-1500 U/mL), the signal, measured 5 min after addition
of 3 mM IPA, increased linearly over the concentration range from
0-500 UmL.sup.-1 after which the signal showed negative deviation
and reached a plateau at .about.1500 UmL.sup.-1. Similarly, with
increasing concentration of IPA (0.about.5 mM), the color
intensity, measured after addition of 500 U/mL AChE, increased
linearly over the concentration range from 0-3 mM. The
concentrations of AChE and IPA were optimized further over the
reagentless lateral flow based paper sensor. For this, a
reagentless lateral-flow based paper sensor was constructed by
entrapment of AChE and IPA in the two different regions (e.g.,
sensing and substrate regions) following the sequences of PVAm(0.5
wt %)/silica/AChE/silica and silica/IPA/silica, respectively by
using either ink jet printing or over spotting. This resulted in a
layered system as shown in FIG. 8(b). The effects of immobilization
of different concentrations of AChE over the reagentless paper
sensor are shown in FIG. 10(a) (a separate paper sensor is used for
each AChE concentration). Almost similar responses were observed
for both the cases (optimization of AChE in plate reader and on
paper). Therefore, a value of 500 U/mL was chosen the best
compromise between a low enzyme loading, a sufficiently high signal
(>7-fold increase over background) and good long-term stability.
The high activity of entrapped AChE is in agreement with previous
reports showing that the enzyme is active and stable in sol-gel
derived silica materials..sup.xl The effects of entrapment varying
levels of IPA over the bioactive paper sensor are shown in FIG.
10(b) (a separate paper sensor is used for each IPA concentration).
Here it is seen that as the concentration of IPA increases so too
does the color intensity. The color intensity saturated at a level
of .about.3 mM IPA and suggested a K.sub.M of ca. 1.5 mM, which is
similar to that of plate reader. Therefore, the most suitable
concentration of enzyme substrate selected by compromise was 3 mM
for IPA. Moreover, the observed K.sub.M value (1.5 mM) in sol-gel
material for the present study is sufficiently higher than the
literature value (.about.0.70 mM in solution). This high K.sub.M
value is probably due to the diffusion limitation of IPA to come
into contact with AChE, which is entrapped within sol-gel material
(solid phase) instead of solution. The color intensity was about
seven-fold higher than that of a negative control (absence of IPA
but with AChE present).
[0247] To ascertain whether or not the supporting silica or PVAm
materials degraded IPA, a similar experiment was conducted using
the same inks as above but with AChE absent from the aqueous ink
layer of the sensing region. The responses remained at the baseline
signal upon addition of varying levels of IPA and were similar to
signals obtained from control experiments performed in the absence
of substrate. Thus, these results confirm that the change in the
color intensity is due to the enzyme catalyzed hydrolysis of IPA to
idophenoxide anion (blue color product). Furthermore, this result
shows that both the AChE and IPA remain active when deposited
between silica layers on a paper substrate.
[0248] Performance of Reagentless Lateral-Flow Assay System: Both
carbamate (e.g., bendiocarb, carbaryl) and organophosphate (e.g.,
paraoxon, malathion) pesticides are well known inactivators of
acetylcholinesterase..sup.xxvii,xxxvi,xxxvii,xxxviii The ability as
well as the performance to detect these compounds using the
developed reagentless lateral-flow based paper sensor was
investigated by two ways: a) by dipping the test strip directly
into solutions containing various concentrations of pesticides
without incubating at room temperature, and b) by immersion of the
test strip inversely into solutions containing various
concentrations of pesticides and allowing them to rise up into the
sensing area. The sensor was incubated for 5 min and the inhibition
of AChE was tested following the inverted lateral flow procedure as
described in above. A decrease in color formation (due to enzymatic
hydrolysis of IPA at pH 8.0 to produce a highly blue colored
product) indicates the presence of an inhibitory substance. It was
found that the detection limit of these pesticides was considerably
lower for the first case in comparison with the second one.
Therefore, inverted lateral flow system with incubation was used
for all subsequent experiments to assess pesticide detection in
this study.
[0249] FIGS. 11 A-(a) and A-(c) show the dose-dependent inhibition
responses of two carbamate pesticides such as bendiocarb and
carbaryl, respectively, while FIGS. 11 A-(b) and A(d) show the semi
log plots of data in panels A-(a) and A-(c), respectively. The data
suggest that increasing concentrations of both bendiocarb and
carbaryl progressively inhibit the activity of AChE. The apparent
saturated inhibition concentrations for bendiocarb and carbaryl
were found to be around 1 .mu.M and 5 .mu.M, respectively. On the
basis of these results, the calculated IC.sub.50 values for
bendiocarb and carbaryl to inhibit the activity of AChE were 20 nM
and 50 nM, respectively. In addition, the detection limits (S/N=3)
of bendiocarb and carbaryl were found to be ca. 10 and 100 nM,
respectively. The results demonstrate that bendiocarb is a more
potent pesticide for blocking AChE active sites than carbaryl. Our
observations are in agreement with results of previous studies in
which the same experiments except for the detection method were
carried out. FIGS. 11 B-(a) and B-(c) show the dose-dependent
inhibition effects of paraoxon and malathion, respectively. FIGS.
11 B-(b) and B-(d) represent the semi log plots of color intensity,
corresponding to the same experiments in panels B-(a) and B-(c),
respectively. The IC.sub.50 values and LOD for both paraoxon and
malathion were approximately 10 nM and 10 .mu.M, respectively. The
data are consistent with the results previously reported.
[0250] The insets for FIG. 11 shows the images of the paper strips
at each inhibitor concentration, and clearly demonstrate that
detection of the inhibitors is possible using the naked eye. This
is an advantageous aspect of the bioactive paper strips, as this
permits the use of the test strips directly in the field and
eliminates the need for sophisticated instrumentation. The presence
of a simple colorimetric readout also enables rapid imaging and
transmission to a central lab for further quantitative analysis
using a simple cell phone camera combined with e-mail or MMS
messaging..sup.iii,v,vii Hence, rapid, on-site qualitative or
quantitative analysis of organophosphates or carbamates be possible
using this reagentless lateral-flow bioactive paper platform.
[0251] Matrix Effect in the Analysis of Paraoxon in Food Samples:
The matrix effects in the analysis of paraoxon in drinking milk
(2%, pH 7.2) and apple juice (pH 3.6) samples were investigated.
For this, several standard paraoxon solutions (0.about.10 .mu.M)
were mixed into both milk and apple juice samples. In order to get
optimum enzymatic reaction product (indophenoxide.sup.-) as well as
the blue color, pH of the apple juice was adjusted to within the
range of 7-8 using a few drops of NaOH (1N). FIG. 12(a) shows that
the color intensity decreased almost similarly with the increased
paraoxon concentration in both milk and apple juice samples. The
detection limit for paraoxon was estimated to be .about.10 nm for
all the samples. The milk and apple juice samples show very small
matrix effects, which would make the recovery determination of
pesticide from these fortified samples possible.
[0252] It is well known that organic solvents (e.g., 5% methanol or
cyclohexane) not only aid in dissolution of the AChE inhibitors,
but also enhances the affinity for binding to AChE. However, in
order to increase the possible practical application of this paper
test, where most certainly 100% aqueous sample solutions would be
applied, no organic cosolvent (cyclohexane or methanol) was used in
either milk or apple juice samples except 5% organic solvent (e.g.,
methanol or cyclohexane) was used to make the stock solution of
pesticides. The results obtained by using the reagentless paper
sensor suggest that the optimized bioactive paper platform is very
effective, sensitive and can play a very important role in both
quantitative as well as qualitative measurement of pesticides.
[0253] Paraoxon Analysis in Real-Life Sample:
[0254] In order to investigate pesticide (e.g., paraoxon) effects
in real life samples, different concentrations of paraoxon solution
(0.about.50 mM) were sprayed on apple and head lettuce. After air
drying, the deposited paraoxon samples were collected using a
cotton-swab and tested using the reagentless lateral flow sensor
strip. The results of the sensor strip assays are presented as a
bar graph in FIG. 12(b), where .about.50% inhibition was observed
when 1 mM paraoxon was sprayed, while almost complete inhibition
was observed when 50 mM paraoxon was sprayed. Beam and Hankinson
reported that pesticides remain stable for at least eight days with
negligible loss. Usually a highly concentrated pesticide (10M) is
sprayed in the fields. Therefore it is dangerous to intake any
vegetable or fruits directly from fields where pesticides have been
sprayed. Thus the present system could be a good pivotal tool for
assessment of low concentrations of class specific OP or CM
pesticides, affecting both humans and animals.
[0255] Storage Stability of the Sensor Platform:
[0256] The long-term stability AChE and its substrate IPA within
the layered coating on the paper substrate were investigated.
Results demonstrated that the enzyme retained >95% of its
initial activity over a period of at least two months when stored
at 4.degree. C., indicating that both the enzyme and the IPA
reagent remained viable during storage. The observed stability of
the enzyme when entrapped in silica is in agreement with previous
reports.sup.xxxii and shows that the reagentless bioactive paper
sensor should have a sufficient shelf-life to allow storage and
shipping.
Example 3
Development of a Bioactive Lab-on-Paper Sensor for the Detection of
E. coli Based on .beta.-Glucuronidase Activity
[0257] Chemicals and Solutions: p-Glucuronidase (GUS, type VII-A,
from E. coli, EC 3.2.1.31), indoxyl-.beta.-D-glucuronide
cyclohexylammonium salt (IBDG), 5-bromo-4-chloro-3-indolyl
.beta.-D-glucuronide sodium salt (X-Gluc),
p-nitrophenyl-.beta.-D-glucuronide (PNPG), sodium silicate solution
(.about.14% NaOH, .about.27% SiO.sub.2), Dowex 50WX8-100
ion-exchange resin, methyltrimethoxysilane (MTMS), polyacrylic acid
(PAA, MW.about.1.25 MDa), 2,3-dichloro-5,6-dicyanobenzoquainone
(DDQ), m-chloroperbenzoic acid (MCPBA), 2-iodoxybenzoic acid (IBX),
H.sub.2O.sub.2, FeCl.sub.3. 6H.sub.2O, Triton X-100, Tween-20, and
B-lysing agent were purchased from Sigma-Aldrich. Polyvinylamine
(PVAm; 1.5 MDa) was obtained from BASF (Mississauga, Canada), as a
gift. B-PER Direct bacterial protein extraction reagent was
obtained from Thermo Scientific. Fe.sub.2O.sub.3 beads and E. coli
polyclonal antibody were purchased from BioClone Inc. and Abcam,
respectively. Distilled deionized water (ddH.sub.2O) was obtained
from a Milli-Q Synthesis A10 water purification system. All other
reagents were of analytical grade.
[0258] Stock solutions of enzyme, GUS and substrates (e.g., X-Gluc,
PNPG, IBDG) were made up using phosphate buffer supplemented with
0.5 wt. % BSA (75 mM, pH 8). These solutions can be used up to
three months under appropriate storage conditions (-20.degree. C.).
Both PVAm (0.5 wt. %) and PAA (0.025 wt. %) solutions were prepared
by dissolving in distilled deionized H.sub.2O.
Methyltrimethoxysilane (MTMS) was hydrolyzed by mixing 98% MTMS
with 0.1N HCl in a 5:1 volume ratio. This mixture was sonicated for
20 minutes on ice to promote ether hydrolysis. All other solutions
were prepared using phosphate buffer (75 mM, pH 8) if not otherwise
stated. CAUTION: All oxidizing agents are toxic. These materials
should be handled with gloves and used in fumehood.
[0259] Organisms and Plate Counting:
[0260] Two non-pathogenic bacteria (E. coli BL21 and B. Subtilis)
strains were used in this study. Standard LB media (total vol. 25
mL with ampicillin 100 .mu.g/mL and chloramphenicol 33 .mu.g/mL)
was used for both E. coli BL21 and B. Subtilis culture. Tryptic soy
broth (TSB, total vol. 5 mL) is used for E. coli 0157:H7 culture.
The cultures were grown overnight at 37.degree. C. with shaking at
125 rpm.
[0261] To count the numbers of CFU/mL in bacterial suspensions,
cultures are serially diluted with sterile water, and 10 .mu.L of
selected dilution was spread evenly over the surface of the warm LB
(for E. coli BL21 and B. Subtilis). TSB agar plates are used for E.
coli 0157:H7. Plates were incubated at 37.degree. C. for 24 h and
count the colonies. Dilutions showing between 30 and 100 colonies
were used for calculation of CFU/mL. [CAUTION: E. coli BL21 and B.
Subtilis are non-pathogenic and should be handled following Level 1
biosafety procedures, while E. coli 0157:H7 is pathogenic and
should be handled in a Level 2 biohazard hood using BSL-2 safety
procedures].
[0262] Sol-Gel Material Preparation:
[0263] A biocompatible sol-gel precursor, sodium silicate (SS) was
used to prepare sols for both substrates and oxidizing agents
entrapment onto paper. SS sols were prepared by mixing 10 mL of
ddH.sub.2O with 2.6 g of sodium silicate solution (pH.about.13)
followed by addition of 5 g of Dowex cation exchange resin to
replace Na.sup.+ with H.sup.+. The mixture was stirred for 30
seconds to reach a final pH of .about.4, and then vacuum filtered
through a Buchner funnel. The filtrate was then further filtered
through a 0.45 .mu.m membrane syringe filter. These sols were used
to prepare silica-containing inks as described below.
[0264] Construction of Bioactive Paper-Based Lateral Flow E. Coli
Test Strips:
[0265] A section of Whatman #1 paper was cut into small pieces
(1.times.8 cm) on which substrate, X-GLUC and an oxidizing agent,
FeCl.sub.3 were entrapped using sol-gel derived silica inks in two
different zones (e.g., substrate/sensing and oxidizing agent
zones). To prevent leaching of the colored product, a hydrophobic
barrier was also introduced on the top of sensing zone using either
wax (by a wax printer) or MTMS (by a piezoelectric ink jet printer,
DMP-2800, Fujifilm Dimatix, Inc, Japan). The sensing region was
prepared by depositing PVAm (0.5 wt %)/silica/X-GLUC/silica layers
in the order described, while the oxidizing agent region was
prepared by depositing silica/FeCl.sub.3/silica layers using either
ink jet printing or deposition via micropipette (for
proof-of-concept studies and assay optimization), as shown in FIG.
13b. In the case of ink jet printing, all inks were modified with
respect to viscosity and surface tension, and printing was done as
reported in Examples 1 and 2. After printing, the sensor was
allowed to dry for at least 1 h in air at room temperature. For
control experiments, a buffer that did not contain X-GLUC was
entrapped between the silica layers in the sensing region.
[0266] Optimization of Paper-Based Later Flow Test Strips Assay
System:
[0267] The test strips assay format was optimized with regard to
the type and concentration of capture agents, pH, type and
concentration of substrate, the kind and concentration of oxidizing
agents, and the time for color development.
[0268] In order to monitor the capability of capture agents to
capture and preserve the color produced from GUS catalyzed reaction
when performed on paper, two potential capture agents including PAA
(anionic polymer) and PVAm (cationic polymer) were used. In this
case, Whatman #1 paper strips (1.times.10 cm) were treated with
both PAA (0 or 0.025 wt. %), and PVAm (0 or 0.5 wt %) using either
ink jet deposition or over spotting and were allowed to air dry for
15 min. The capture agents treated strips were then immersed into a
solution of blue ClBr-indigo dye (which is produced via reaction of
GUS, 5 U/mL and X-Gluc, 4 mM) and allowing the dye to move-up via
lateral flow for 10 min. Following the assay the resulting color
intensity remaining on the paper strip was monitored once a week
for up to 8 weeks.
[0269] The effect of pH on enzyme assay was initially investigated
in solution. For this, X-Gluc was dissolved with potassium
phosphate buffer (75 mM) with 0.5% BSA having different pH values
(6-9). 80 .mu.L of X-Gluc (Final concentration of 3 mM) solution
and 20 .mu.L of GUS (final concentration of 1 U/mL) were mixed into
a 96-well plate. A kinetic study at 610 nm was then performed using
a TECAN Safire microwell plate reader for up to 60 min. The effect
of pH on enzyme assay with different incubation time on paper was
also studied. For this, PVAm/Silica/X-Gluc/Silica layers were
printed or over spotted at a width of 0.5 cm across the Whatman #1
filter paper 5 cm from the bottom of the paper (X-Gluc
concentration of 3 mM), while silica/FeCl.sub.3/silica layers were
printed or over spotted in a 0.5 cm wide area across the paper
strip 4 cm from the bottom of the paper (FeCl.sub.3 concentration
of 2 mM) (same as FIG. 13b). The sensor was then immersed into GUS
solution (final concentration of 1 U/mL) having different pH values
(6-8.5) and was removed as soon as the solution has reached to the
sensing region via lateral flow. GUS hydrolyzes the chromogenic
substrate X-Gluc to produce a transition from a colorless substrate
to deep blue colored product. The color intensity was then
monitored with different incubation time (5, 30, and 60 min).
[0270] The chromogenic substrates (e.g., X-Gluc, PNPG, and IBDG)
were tested initially in silica monoliths present in 96 well plates
and later on paper. For this, all of these substrates were
dissolved separately with sodium phosphate buffer (75 mM, pH 8).
Different concentrations (final concentration of 0-10 mM) of each
of them were entrapped in SS+30% glycerol in a 96 well plate (total
volume of 80 .mu.L). 20 .mu.L of GUS (final concentration of 1
U/mL) was then added into each well and incubated for 30 min to
allow color development. The absorbance at 610, 400, and 380 nm was
then measured using a TECAN M1000 plate reader. A digital camera
was also used to take a photograph of the plate. Within these three
substrates, only the concentration of X-Gluc was further optimized
on paper assay due to formation of relatively high visual color
intensity produced via enzymatic hydrolysis of X-Gluc. For this,
different concentration of X-Gluc (0-10 mM), and FeCl.sub.3 (2 mM)
were entrapped on paper as described previous section (FIG. 13b).
The sensor was then immersed into GUS solution (1 U/mL) and removed
as soon as the Gus solution has reached the sensing region through
FeCl.sub.3 zone followed by a certain incubation time (5, 30, and
60 min) at room temperature. Note that the speed of reaction
between GUS and X-Gluc is somewhat slow (about 1 hr), while the
reaction in the presence of the oxidizing agent, FeCl.sub.3 is far
more rapid, leading to color formation in 5 min.
[0271] Five different oxidizing agents including,
2,3-dichloro-5,6-dicyanobenzoquainone (DDQ), m-chloroperbenzoic
acid (MCPBA), 2-iodoxybenzoic acid (IBX), H.sub.2O.sub.2,
FeCl.sub.3 were used to determine the most effective oxidizing
agent to speed up the enzyme (GUS)-substrate (X-Gluc) reaction.
Experiments were conducted firstly in 96-well plate, in which GUS
(final concentration of 1 U/mL) was entrapped in silica gel. The
substrate, X-Gluc (4 mM) and oxidizing agents (1 mM each) were then
added to each well (designated for the individual oxidizing agent),
and incubated for 30 min. The concentration gradient (0-30 mM) of
FeCl.sub.3 was also observed in 96-well plate. The absorbance at
610 was measured using a TECAN M1000 plate reader for each
experiment. A digital camera was also used to take a photograph of
the plate. The effect of FeCl.sub.3 activity was then optimized on
the bioactive paper strip by measuring the color intensity produced
by the enzymatic hydrolysis of X-Gluc. Various concentrations of
FeCl.sub.3 (0-10 mM), and X-Gluc (3 mM) were entrapped on paper
using biocompatible silica material in order to make the sensor
strips, as described in the previous section. The performance of
the sensor was then assayed by dipping it into the GUS solution (1
U/mL) to allow the enzyme solution to reach the sensing zone
through FeCl.sub.3 zone via lateral flow. The color development
times tested were 5, 30, and 60 min.
[0272] The detection as well as the color intensity of the sensing
areas was monitored by naked eye, by obtaining a digital image
(Canon A630, 8.0 MegaPixel camera operated in automatic mode with
no flash and with the macroimaging setting on) or office scanner,
and using ImageJ.TM. software to analyze the jpeg images as
described in Examples 1 and 2.
[0273] Performance of E. coli Test Strips:
[0274] Different concentrations of bacteria cells (e.g., E. coli
BL21 or B. Subtilis) suspensions ranging from 0-1.times.10.sup.7
CFU/mL were made for this study. Three different set of experiments
were conducted for each of the bacterial strains. 2 mL of bacterial
cell suspension was mixed with 200 .mu.L of B-PER DIRECT bacteria
lysing reagent by pipetting up and down and incubated for 15 min at
room temperature. The bacteria sample or lysate was then assayed
using the paper sensor via lateral flow technique as outlined
above. When the lysate reached to the sensing zone of the sensor,
lateral flow was stopped and the sensor was allowed to dry in air.
A colorless-to-deep blue color change could be observed within a
few min due to enzymatic hydrolysis of substrate, X-Gluc. The level
of E. coli in samples was detected by measuring the color intensity
with incubation time at 5, 30, or 60 min. The color intensity was
determined by analyzing a digital image with the ImageJ.TM.
software as described above.
[0275] Preparation of Antibody-Conjugated Magnetic Beads (MBs) for
E. coli Capture:
[0276] Conjugation of antibody (goat pAb to E. coli, Abcam) to MBs
(hydrazide modified, 1 .mu.m, 1.7.times.10.sup.8 beads/mg, Bioclone
Inc.) was conducted according to the instructions provided by the
manufacturer. Briefly, the conjugation protocol could be performed
in two steps: 1) Oxidation of Antibody. A measured amount of pAb
was added to 1 mL of sodium acetate buffer (0.1 M, pH 5.6). A mild
oxidizing agent, sodium meta-periodate (NaIO.sub.4, final
concentration of 10 mM) was added into pAb solution and incubated
the sample in the dark room at room temperature for at least 30 min
with gentle rotation. The unreacted NaIO.sub.4 was then removed
using a Nanosep Centrifugal Device (consists of a sample reservoir
with encapsulated membrane with pore size, 30K and a filtrate
receiver) followed by centrifugation at 14 000 g for 5 min, and the
oxidized pAb was then dissolved with 500 .mu.L sodium acetate
buffer (0.1 M, pH 5.6). 2) Coupling of Antibody to Magbeads. A
solution of completely suspended MBs (100 uL, 4-8.times.10.sup.8
beads/mL) was transferred to a microcentrifuge tube and placed into
a magnetic separator for 2-3 min. The supernatant was discarded and
the bead pellet was redispersed in 500 .mu.L of sodium acetate
buffer (0.1 M, pH 5.6) after washing with the same buffer 3 times.
This MBs solution was then mixed with 500 .mu.L of oxidized
antibody (from step 1). The mixture was then allowed to keep
shaking for at least 6 h at room temperature. The loading of pAb
onto MBs was determined by measuring fluorescence intensities of
the supernatant (intrinsic tryptopphan fluorescence measurements at
Ex 284 nm and Em 342 nm using TECAN Infinite M1000) every 2 h
followed by magnetic separation. Various amounts of pAbs were added
to 500 .mu.L of washed Magbeads (same as above) to optimize the Ab
loading on MBs. The equation (1) was used for a rough quantitative
measurement of how much pAb was bound to the beads.
% Ab bound=[1-(FI.sub.a/FI.sub.b)]*100 (1)
[0277] where,
FI.sub.b=Fluorescence intensity of Ab solution before its binding
to beads, and FI.sub.a=Fluorescence intensity of Ab solution
(supernatant) after its binding to beads
[0278] After successful binding, antibody conjugated MBs were
washed with sodium acetate buffer (0.1 M, pH 5.6) 3 times and then
with PBS buffer (pH 7.4) 5 times followed by a magnetic separation.
The antibody conjugated MBs were resuspended with 1 mL PBS (10 mM,
pH 7.4), which could be stored at 4.degree. C. for more than 1
month.
[0279] Preconcentration of E. Coli Using MB-Ab:
[0280] MB-Ab (200 .mu.L, 1.4-2.4.times.10.sup.8 beads) was
dispersed in 10 mL of E. coli containing PBS solution
(10.sup.2-10.sup.6 CFU/mL). The mixture was incubated at room
temperature under shaking (220 rpm) for 1 h. After incubation, the
MB-Ab-E. coli complex was collected using a magnetic separator and
the supernatant was transferred into a new tube for quantification
of captured E. coli by the plate counting method. A capture
efficiency of MB-Ab system was estimated by counting colonies after
addition of MB-Ab in sample tubes containing various numbers of E.
coli. The amount of MB-Ab was also optimized by varying the volume
of MB-Ab (20-500 .mu.L) with constant cell number (10.sup.4
CFU/mL). MB-Ab-E. coli complex was washed 5 times in 10 mM PBS (pH
7.4) and then resuspended in 1.8 mL of phosphate buffer (pH 8). 200
.mu.L of B-PER DIRECT bacteria lysing reagent was mixed and
incubated for 15 min at room temperature. Finally, the sensor
strips was immersed into the cell lysate solution and the level of
E. coli in samples was detected by measuring the color intensity
through image analysis as described above.
[0281] Detection of E. coli in Food, Beverage, and Environmental
Samples:
[0282] Apple juice, milk (1%), and water samples were used for this
study. All samples (10 mL each) were contaminated artificially with
a known number of E. coli cells (10.sup.4 CFU/mL). Prior to
analysis, the pH of the apple juice was adjusted to between 7 and 8
by adding a few drops of 1 N NaOH. Milk and water samples were
tested with no additional processing. Cells were pre-concentrated
using MB-Ab as described above and resuspending them with 2 mL
fresh juice, milk or water instead of phosphate buffer (pH 8) in
order to make the sensor more applicable for testing the real life
samples. The samples were then tested using the sensor strips as
outlined above. The color intensity was determined by analyzing a
digital image with the ImageJ software. Uninnoculated apple juice,
milk, and water were used as controls and also analyzed to
determine the presence of E. coli using the sensor strips.
[0283] Interference and Specificity Test:
[0284] Though bacteria with no GUS activities do not interfere with
the signal, interference by non-target bacteria with high GUS
activities cannot be neglected. To demonstrate the interference
study (only with GUS negative bacterial strain), E. coli BL21 and
B. Subtilis were grown individually. 2 mL of E. coli suspension
(10.sup.6 CFU/mL), 2 mL of B. Subtilis (10.sup.6 CFU/mL), and a
mixture of both bacterial suspensions (1 mL each, 10.sup.6 CFU/mL
each) were lysed using B-PER DIRECT bacteria lysing agent
separately. The sensor was used to test the GUS activities followed
by lysis the cells as outlined in the previous section and the
color intensities produced (from individual and mixture cells
lysate) were compared.
[0285] To test the specificity of non-pathogenic bacteria, goat pAb
(specific to E. coli) was used in the immunomagnetic separation of
E. coli (in the same manner as described above) from a mixture of
E. coli and B. Subtilis. In the case of pathogenic bacteria,
anti-E. coli O157:H7 antibody-coated MBs (purchased from Invitogen)
is used for separation of E. coli 0157:H7 specifically from a
mixture of cells. To determine the detection limit, triplicate
analysis of controls/blanks were performed for all the
experiments.
[0286] Storage Stability of the Test Strips:
[0287] The long-term stability of the test strips (at room
temperature) was examined every week up to 8 weeks by immersing the
test strip into E. coli (10.sup.5 CFU/mL) lysate solution. The
assay conditions were the same as those described above.
RESULTS AND DISCUSSION
[0288] Optimization of Paper-Based Later Flow Test Strips Assay
Format: Assay time, sensitivity, selectivity and reproducibility
are factors to consider for rapid detection of E. coli in food,
medical, environmental or other samples. Therefore, in order to
develop an efficient paper-based E. coli sensor, parameters such as
capture agents (type and concentration), substrate (type and
concentration), pH of the buffer, oxidizing agents (kind and
concentration), and the time for color development were
optimized.
[0289] It was assumed that the dark blue color product, ClBr-indigo
dye (which is produced via GUS catalyzed hydrolysis of X-Gluc) is a
neutral material. Therefore, in order to monitor the capability of
capture agents to capture and preserve the color product on paper,
two potential capture agents including anionic (Polyacrylic acid,
PAA) and cationic (Polyvinylamine, PVAm) polymers were used in a
lateral flow-based paper chromatographic system. The results
indicated that a 0.5 wt % PVAm level was able to trap and
concentrate the indigo dye efficiently, while the PAA treated and
unmodified paper (control) failed to trap the blue color product
indicating that the ClBr-indigo dye is anioinic. This level of PVAm
also preserved the blue color product after storage for more than
two months. Thus this level of PVAm was used in all further
paper-based assays in this Example.
[0290] To develop an efficient E. coli test trip assay platform, it
is desirable that a GUS chromogenic substrate suitable for visual
detection is used. Initially the substrate was selected from three
different chromogenic substrates, such as
indoxyle-.beta.-D-glucuronide (IBDG),
para-nitro-.beta.-D-glucuronide (PNPG) and
5-bromo-4-chloro-3-indolyl .beta.-D-glucuronide (X-Gluc) based on
their capability for visual color formation via GUS catalyzed
hydrolysis of these substrates in solution for 30 min. Results
showed that IBDG and PNPG produced a relatively faint color (blue
and yellow, respectively), while X-Gluc produced a distinct deep
blue color. Therefore, the X-Gluc was selected as the most suitable
substrate and the concentration of X-Gluc was further optimized on
paper for the development of the colorimetric paper-based E. coli
assay platform. For this, an ideal lateral-flow paper based E. coli
sensor was constructed following the procedure as described above.
The effects of [X-Gluc] on signal levels with different drying time
at room temperature are shown in FIG. 14a (a separate paper sensor
is used for each X-Gluc concentration). Here it is seen that with
increasing concentration of X-Gluc, the color intensity increased
with saturation at [X-Gluc].about.4 mM. Based on this, 4 mM X-Gluc
was chosen as the optimal substrate concentration. Using the
paper-based assay, the K.sub.M for X-Gluc was found to be
.about.0.7 mM, which is in good agreement previous literature
reports. For the prescribed times tested for color development,
increasing time caused an increase of the color intensity. The most
suitable time for color development was around 1 h though color can
be detected at an earlier time.
[0291] It is well known that the rates of oxidation of indoxyls
(intermediate product produced due to GUS catalyzed hydrolysis of
X-Gluc) to indigo dyes are relatively slow at room
temperature..sup.xli The rate of indoxyl oxidation is also known to
be pH and oxidizing agent-dependent and therefore, optimization of
pH and oxidizing agent are the other parameters for increasing the
sensor performance as well as reducing the assay time. Therefore
the absorbance of the GUS catalyzed hydrolysis of X-Gluc was
determined as a function of time and pH in solution. It was found
that the absorbance (due to rate of oxidation of indoxyle)
increased with increasing pH (over the range ph 6.0 to 8.0) and
time. A slight variation from pH 8.0 gives considerably lower
absorbance readings. The most suitable time for color development
was around 1 h. The data also suggested that pH values above pH 8.5
were not compatible with maintenance of GUS activity, while maximum
signal was obtained at pH 8 with 1 h incubation time when measuring
absorbance at 610 nm. The effect of variation of pH and time for
color development were further optimized in paper-based assays. For
this, substrate, X-Gluc and an oxidizing agent, FeCl.sub.3 were
entrapped at two different zones (e.g., lower FeCl.sub.3 region and
upper X-Gluc region) of the paper-based sensor strip (FIG. 13b).
The sensor was then immersed into GUS solution (final concentration
of 1 U/mL) having different pH values (6-8.5) and was removed as
soon as the solution had reached to the sensing region via lateral
flow. The produced blue color intensity was then measured with
different drying time (5, 30, and 60 min). FIG. 14b shows that the
color intensity increases with pH over the range pH 6.0 to 8.0 and
above pH 8.5, color intensity is decreased owing to possibility of
degradation of enzyme at this higher pH level. The data also
demonstrated that color intensity increases with time and 5 min was
the minimum length of time to allow measurement of color. The
maximum signal was obtained at pH 8 (similar to that of solution
assay) with 1 h drying time therefore, pH 8 and 1 h drying time was
used for all further paper-based assays.
[0292] A 96-well format was used to select the appropriate
oxidizing agent for oxidation of indoxyl to blue color product,
ClBr-indigo dye rapidly. A comparison of the blue color development
(visual detection) in silica monoliths was performed for 30 min
using five different oxidizing agents (e.g., FeCl.sub.3, DDQ,
MCPBA, IBX, and H.sub.2O.sub.2). Among these oxidizing agents
tested, FeCl.sub.3 was selected as the most suitable one based on
the strongest visual color intensity produced. The poorest
performance for blue color development was observed in the presence
of DDQ. The effect of adding varying levels of FeCl.sub.3 over the
bioactive paper strip are shown in FIG. 14c (a separate paper
sensor is used for each FeCl.sub.3 concentration). Prior to this,
the paper-based E. coli sensor was made following the protocol
mentioned above. Here it is seen that as the concentration of
FeCl.sub.3 increases so too does the color intensity, recorded 5,
30 and 60 min after reaching the GUS solution (1 U/mL) over the
substrate area via lateral flow chromatographic technique. The
color intensity saturated at a level of .about.1 mM FeCl.sub.3. The
color intensity was about four-fold higher (after 5 min drying
time) than that of a negative control (absence of FeCl.sub.3).
[0293] Also assessed was whether or not the supporting silica
material, paper substrate or PVAm layers degraded X-Gluc, therefore
similar experiments were conducted to those described above with no
GUS present in the solution. The signals remained at the baseline
level upon addition of varying levels of X-Gluc and were similar to
signals obtained from control experiments performed in the absence
of substrate. Thus, the present results confirm that the change in
the color intensity is due to the enzyme catalyzed hydrolysis of
X-Gluc to ClBr-Indigo dye (blue color product). Furthermore, this
result shows that both the X-Gluc and FeCl.sub.3 remain stable when
deposited between silica layers on a paper substrate.
[0294] Analytical Performance of E. coli Test Strips:
[0295] To evaluate the analytical performance of the developed
paper-based E. coli test strips, different known concentrations of
bacteria cell (over the range 0-9.times.10.sup.6 CFU/mL)
suspensions were lyzed using B-PER direct bacteria lysing reagent
in order to release the intracellular GUS enzyme into solution. The
cell lysate was then tested by dipping the test strip directly into
it. Colorless-to-deep blue color changes could be observed within a
few minutes due to GUS catalyzed hydrolysis of substrate, X-Gluc.
The developed blue color intensity corresponds to the level of E.
coli in samples. In parts a and b of FIG. 15 represent the semi-log
plot of blue color intensity (due to different E. coli
concentrations) obtained using an office scanner and a digital
camera, respectively, and through digital image analysis of the
same data. An increase in blue color intensity was observed with
increasing concentration of E. coli BL21. However, color intensity
was relatively low in FIG. 15b compared to FIG. 15a. The reason for
this is that environmental illumination interferes with the signal
when the pictures were taken using a camera. The visual results
indicated that the sensor is able to detect E. coli at a level of
ca. 1.times.10.sup.6 and ca. 9.times.10.sup.3 CFU/mL within 5 min
and after a 1 h drying time, respectively. This is because the
produced enzyme concentration is limited from the lower number of
cells. The limit of detection was defined as the cell concentration
producing a signal equal to or higher than the average signal
produced by the blank plus three standard deviations was
9.times.10.sup.3 CFU/mL. The cell sensing system proved to be
precise, with within-assay coefficients of variation less than
5%.
[0296] Prior to performing the cell lysis experiment, an
appropriate bacteria lysis reagent was selected from 4 different
lysis reagents such as Tween.TM. 20, Triton X100, CellLytic.TM.
B.cell lysis reagent, and B-PER direct protein extraction reagent.
For this, E. coli BL21 cell suspension (1.times.10.sup.6 cfu/mL in
75 mM phosphate buffer, pH 8) was mixed separately with all of
these lysis reagents for 15 min at room temperature. X-Gluc (final
concentration, 4 mM) and FeCl.sub.3 (1 mM) were then added and
incubated 60 min for color development. The visual results
indicated that B-PER direct protein extraction reagent is more
efficient to lyse the cell and had less inhibitory effects on GUS
enzyme in comparison to that of other lysis reagents. Therefore,
B-PER lysis reagent was used for all subsequent paper-based
assays.
[0297] It was also shown that patterned paper can concentrate the
color to a narrow zone/band, which in turn increases the sensor
sensitivity. For this, two different patterns (e.g., open or closed
microfluidic channel type) were created using Microsoft
PowerPoint.TM. with black lines at 2 pt thickness. The patterns
were printed on Whatman 1 paper using wax ink and then placed in an
oven at 100.degree. C. for 5 min to melt the wax and impregnate the
fibers. The paper was then treated with 0.5 wt % PVAm and entrapped
X-GLUC and FeCl.sub.3 in the indicated regions using sol-gel
derived silica matrix. Both patterns were dipped in GUS solution (1
U/mL) to test their utility. Results showed that the microfluidic
closed channel based patterned paper provided a total intensity
that was 1.7-fold higher than the others. Therefore, this patterned
paper sensor was used for the E. coli assay, which is shown in FIG.
15c, where color intensity increases with E. coli concentration
(separate paper sensor was used for each concentration). The
results demonstrated that the paper strip biosensor was able to
detect low E. coli concentration (LOD) down to 4.2.times.10.sup.3
CFU/mL upon color development for 60 min indicating that the
pattern paper sensor is more sensitive and has a better LOD (>2
fold) for E. coli (4.2.times.10.sup.3 CFU/mL) than for the
non-patterned paper sensor (LOD=8.9.times.10.sup.3 CFU/mL), likely
due to the ability to concentrate the color into a narrow area.
Overall, the data clearly show that the detection of the E. coli is
possible with the naked eye, thereby avoiding the need for
expensive and sophisticated instruments or electric power. Such a
platform also makes it possible to perform remote sensing as
qualitative estimations can be made on-site or images can be sent
via camera phone to a central laboratory for quantitative
assessment though image analysis.
[0298] Interferences on Assay Performance:
[0299] In order to demonstrate the interferences on signal by other
non-producing GUS cells, initially E. coli BL21 and B. Subtilis
(4.1.times.10.sup.6 CFU/mL each) were mixed, lyzed, and tested in
solution. In the case of B. subtilis alone, no color was observed,
while for the mixed-culture, deep blue color was observed (FIG.
16a). After the solution assay, the paper sensor was then used to
test the GUS activities for different concentrations of mixed cell
lysates following the protocol as outlined in the previous section.
FIG. 16b shows that there was a negligible effect on the general
shape of the response vs. concentration or the limit of detection,
with the detection limit for E. coli being 4.1.times.10.sup.3
CFU/mL after a 1 hr drying time. While it is true that other GUS
producing cells would be expected to interfere, it is likely that
more than 95% of E. coli strains are able to produce GUS and E.
coli is a common indicator of fecal contamination and pollution
(e.g., coliforms and fecal coliform etc). At the very least, the
sensor is able to detect any GUS producing cells but does not
provide identification of the specific cell strain that is
detected.
[0300] E. Coli Assay in Beverage Samples:
[0301] Milk (1%) and orange juice were artificially contaminated
with E. coli (ca. 4.times.10.sup.5 CFU/mL) to assess the effect of
such sample matrices on assay performance and detection limits.
Prior to the assay, the pH of the samples was adjusted to the range
of 7-8, which retained the activity of the GUS enzyme in the
samples. As shown in FIG. 17, the signal variation (for both milk
and orange juice samples) is almost similar to that typically
obtained (see FIG. 15c), which indicates that the food samples
matrices had a negligible effect on the limit of detection. The
above findings clearly show that the proposed assay platform can be
used for rapid and convenient visual monitoring of beverage and
environmental samples, thus providing to be a valuable portable
tool for on-site analysis.
[0302] Storage Stability of the Sensor:
[0303] The long-term stability of the sensor strips was also
investigated. The results demonstrated that the strips could be
stored for more than two months at room temperature without
noticeable decrease in the sensing GUS activity. It is important to
note that no biomolecules are required for constructing the novel
E. coli sensor of the present application.
Example 4
Test Kit Based on Magnetic Preconcentration and Bioactive Paper
Strips
[0304] A test kit that combines magnetic preconcentration and
bioactive paper strips should allow selective detection of
pathogenic bacteria in food and water with no need for culturing or
instrumentation, making it suitable for remote and resource limited
locations that may lack electricity. The test strip is printed with
a chromogenic substrate region and a capture/preconcentration
region to allow ultrasensitive detection of bacteria based on the
ability of specific bacterial enzymes to convert the colorless
substrate to a highly colored product (like that described in
Example 3).
[0305] An advantage of the above approach is the ability to provide
a cost-effective, portable, easy-to-use test strip that can either
be observed directly by eye or recorded by a digital camera. The
combination of magnetic preconcentration and amplified detection of
bacteria should allow detection of as little as 10-20 CFU/mL of
pathogenic bacteria with no need for culturing, and can be produced
as a simple kit, permitting the test to be run even by untrained
personnel. Thus, this example will provide a new test strip
platform technology that directly addresses the need for low-cost
diagnostics that can protect against infectious disease.
[0306] The assay kit is designed to perform three intricately
linked chemical and biochemical processes: 1) specific
preconcentration of one or multiple pathogenic organisms using
magnetic preconcentration, 2) lysing of cells to release
intracellular .beta.-glucoronidase (.beta.-gus) or
.beta.-galactosidase (.beta.-gal) enzymes, and 3) a paper-based
multi-pathogen detection assay utilizing a chromogenic substrate
for .beta.-gus or .beta.-gal, along with a strong oxidizing agent
to accelerate product formation and a capture zone to concentrate
the product into a narrow band for highly sensitive detection.
Together, this combination of processes has the potential to
provide rapid (<5 min) and ultralow detection limits (.about.10
CFU/mL) for a variety of pathogenic bacteria.
[0307] More specifically, the assay utilizes antibody-derivatized
magnetic beads to selectively concentrate one or more pathogenic
bacteria from an initial volume of .about.50 mL to a final volume
of .about.0.5 mL, which results in a 50-100 fold concentration
enhancement depending on capture efficiency of the cells by the
magnetic particles. To the 0.5 mL of sample is added 0.5 mL of a
lysing buffer, which releases either .beta.-gus (endogenous to E.
coli BL21 and K12, as well as salmonella) or .beta.-gal (endogenous
to E. coli H7:O157) in an active form. A paper strip is then
introduced to the solution to allow lateral flow of the sample up
the paper. The paper contains several lanes which contain either:
1) a suitable chromogenic substrate for .beta.-gus; 2) a
chromogenic substrate for .beta.-gal; 3) a chromogenic substrate
for a common bacterial enzyme as a positive control; or 4) no
substrate (negative control). In addition, each lane contains a
strong oxidizing agent, which accelerates the rate of product
formation, and a capture zone (cationic polymer coated region) that
concentrates the anionic product within a narrow zone. All reagent
zones are printed by an ink-jet method using a special sol-gel
derived ink. After a suitable reaction time (ca. 5 min), the
developed color is used to quantify the amount of a selected
pathogen initially present in the test sample.
[0308] The results in Example 3 have demonstrated the ability to 1)
form multi-channel fluidic devices on paper using wax printing; 2)
print all reagent zones onto the paper strips using an ink-jet
method and a sol-gel based ink, 3) detect .beta.-gus enzymes
liberated from lysed E. coli K12 cells using the paper strip. Using
this format, E. coli has been successfully detected with a
detection limit of 10.sup.3 CFU/mL. Preliminary data has also been
obtained on the use of magnetic preconcentration, with
preconcentration levels being .about.50-fold for E. coli K12, and
detection limits being on the order of 20 CFU/mL (starting with a
sample volume of 50 mL).
[0309] While the present application has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the application is not
limited to the disclosed examples. To the contrary, the application
is intended to cover various modifications and equivalent
arrangements included within the spirit and scope of the appended
claims.
[0310] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety. Where a term in the present application
is found to be defined differently in a document incorporated
herein by reference, the definition provided herein is to serve as
the definition for the term.
TABLE-US-00001 TABLE 1 ##STR00008## Note: +, the AChE retains
activity (>95%); **, not applicable
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* * * * *