U.S. patent application number 13/497704 was filed with the patent office on 2012-12-20 for testing device for identifying antigens and antibodies in biofluids.
This patent application is currently assigned to Monash University. Invention is credited to Gil Garnier, Mohidus Samad Khan, Xu Li, Wei Shen, George Thouas.
Application Number | 20120322086 13/497704 |
Document ID | / |
Family ID | 43795228 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120322086 |
Kind Code |
A1 |
Garnier; Gil ; et
al. |
December 20, 2012 |
TESTING DEVICE FOR IDENTIFYING ANTIGENS AND ANTIBODIES IN
BIOFLUIDS
Abstract
A testing device for identifying an antigen or antibody within a
biofluid sample including: a substrate having a hydrophilic surface
thereon; the surface including a collection zone, and at least one
detection zone extending therefrom; wherein the biofluid sample can
be mixed with a specific antigen or antibody, and deposited on the
collection zone and transferred by capillary action to the
detection zone; the antigen or antibody in the biofluid sample
reacting with an appropriate said antibody or antigen thereby
resulting in a visual indication within the detection zone.
Inventors: |
Garnier; Gil; (Victoria,
AU) ; Shen; Wei; (Victoria, AU) ; Khan;
Mohidus Samad; (Montreal, CA) ; Li; Xu;
(Melbourne, AU) ; Thouas; George; (Melbourne,
AU) |
Assignee: |
Monash University
Victoria
AU
|
Family ID: |
43795228 |
Appl. No.: |
13/497704 |
Filed: |
September 24, 2010 |
PCT Filed: |
September 24, 2010 |
PCT NO: |
PCT/AU2010/001255 |
371 Date: |
May 31, 2012 |
Current U.S.
Class: |
435/7.25 ;
422/69; 435/287.2; 436/501 |
Current CPC
Class: |
Y02A 50/58 20180101;
G01N 30/90 20130101; G01N 2021/825 20130101; G01N 33/80 20130101;
G01N 21/82 20130101; G01N 2021/757 20130101; G01N 2021/752
20130101; G01N 33/558 20130101 |
Class at
Publication: |
435/7.25 ;
436/501; 435/287.2; 422/69 |
International
Class: |
C12M 1/34 20060101
C12M001/34; G01N 21/82 20060101 G01N021/82 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 24, 2009 |
AU |
2009904643 |
Claims
1. A testing device for identifying an antigen or antibody within a
biofluid sample including: a substrate having a hydrophilic surface
thereon; the surface including a collection zone, and at least one
detection zone extending therefrom; wherein the biofluid sample can
be mixed with a specific antigen or antibody, and deposited on the
collection zone and transferred by capillary action to the
detection zone; the antigen or antibody in the biofluid sample
reacting with an appropriate said antibody or antigen thereby
resulting in a visual indication within the detection zone.
2. A testing device for identifying an antigen or antibody within a
biofluid sample including: a substrate having a hydrophilic surface
thereon; the surface including a collection zone, and at least one
detection zone extending therefrom; the detection zone having an
antibody or antigen immobilised therein; wherein the biofluid
sample can be deposited on the collection zone and transferred by
capillary action to the detection zone; the antigen or antibody in
the biofluid sample reacting with an appropriate said antibody or
antigen within the detection zone thereby resulting in a visual
indication therein.
3. A testing device according to claim 1, wherein the substrate is
formed from paper or other cellulosic materials.
4. A testing device according to claim 1, wherein the substrate
includes a chromographic layer thereon or other wettable porous
media.
5. A testing device according to claim 1, wherein the substrate
surface has a hydrophilic microfluidic channel pattern thereon
defining the collection zone and the detection zone.
6. A testing device according to claim 5, including a plurality of
detection zones extending from the collection zone.
7. A testing device according to claim 1, wherein biofluid sample
being tested is blood, and the visual indication is due to an
agglutination of the blood upon reaction with a specific antibody
resulting in reduced wicking and/or separation of the blood in the
detection zone.
8. A testing device according to claim 7, wherein the testing
device includes at least one valve for controlling the wicking of
biofluid to the detection zone from the collection zone.
9. A testing device according to claim 7, wherein the testing
device includes a switch for controlling the contact of the
biofluid with the antibody or antigen.
10. A method for identifying an antigen or antibody within a
biofluid sample, including: mixing the biofluid sample with a
specific antigen or antibody; depositing the mixed biofluid sample
on a collection zone of a testing device including a substrate
having a hydrophilic surface thereon, the surface including said
collection zone and at least one detection zone extending
therefrom, the biofluid sample being transferred by capillary
action to the detection zone; and identifying the antigen or
antibody by a resultant visual indication within the detection zone
arising where the antigen or antibody in the biofluid sample reacts
with an appropriate said antibody or antigen.
11. A method for identifying an antigen or antibody within a
biofluid sample, including: depositing the biofluid sample on a
collection zone of a testing device including a substrate having a
hydrophilic surface thereon, the surface including said collection
zone and at least one detection zone extending therefrom, the
detection zone having an antibody or antigen immobilised therein,
the biofluid sample being transferred by capillary action to the
detection zone; and identifying the antigen or antibody by a
resultant visual indication arising where the antibody or antigen
in the biofluid sample reacts with an appropriate said antigen or
antibody within the detection zone.
12. A method according to claim 10, including mixing nanoparticles
within the biofluids sample to facilitate said reaction in the
detection zone.
13. A method according to claim 10, wherein biofluid sample being
tested is blood, and the visual indication is due to an
agglutination of the blood upon reaction with a specific antibody
resulting in reduced wicking and/or separation of the blood in the
detection zone.
14. A method according to claim 13, including detecting blood type
from the visual indication.
15. A method according to claim 13 including detecting illness from
the visual indication.
16. A testing device according to claim 2, wherein the substrate is
formed from paper or other cellulosic materials.
17. A testing device according to claim 2, wherein the substrate
includes a chromographic layer thereon or other wettable porous
media.
18. A testing device according to claim 2, wherein the substrate
surface has a hydrophilic microfluidic channel pattern thereon
defining the collection zone and the detection zone.
19. A testing device according to claim 18, including a plurality
of detection zones extending from the collection zone.
20. A testing device according to claim 2, wherein biofluid sample
being tested is blood, and the visual indication is due to an
agglutination of the blood upon reaction with a specific antibody
resulting in reduced wicking and/or separation of the blood in the
detection zone.
21. A testing device according to claim 20, wherein the testing
device includes at least one valve for controlling the wicking of
biofluid to the detection zone from the collection zone.
22. A testing device according to claim 20, wherein the testing
device includes a switch for controlling the contact of the
biofluid with the antibody or antigen.
23. A method according to claim 11, including mixing nanoparticles
within the biofluids sample to facilitate said reaction in the
detection zone.
24. A method according to claim 11, wherein biofluid sample being
tested is blood, and the visual indication is due to an
agglutination of the blood upon reaction with a specific antibody
resulting in reduced wicking and/or separation of the blood in the
detection zone.
25. A method according to claim 24, including detecting blood type
from the visual indication.
26. A method according to claim 24 including detecting illness from
the visual indication.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to the identification of
antigens and antibodies within a biofluid. While the invention will
be described with specific reference to its use in determining a
person's blood type, it is to be appreciated that other
applications of the invention are also envisaged.
BACKGROUND TO THE INVENTION
[0002] Blood is essential for sustaining living tissue, with the
most important roles of supplying oxygen and other soluble
nutrients, immune protection and metabolic turnover. While it is a
tissue in its own right, blood in a chemical sense can be
considered as a stable, highly packed colloid suspension made of
red blood cells (erythrocytes, 4-6 million/mL, 6-8 .mu.m), white
cells (leukocytes, 4000-6000/mL, 10-21 .mu.m), platelets
(150,000-400,000/mL, 2-5 .mu.m) carried within a fluid solution
(serum) containing a host of biomolecules (eg albumins, fatty
acids, hormones), metabolites and electrolytes. A subset of these
biomolecules, such as the binding proteins responsible for tissue
immunity (antigens) and blood type, are directly adsorbed onto the
surface of blood cells. Common portable testing methods for blood
include analysis of glucose content, cholesterol, metabolic panel
(sodium, potassium, bicarbonate, blood urea nitrogen, magnesium,
creatine, calcium, triglycerides), microbial and disease markers
and protein molecular profile (liver, prostate). Surprisingly, and
in spite of its vital importance, there are no convenient low cost
disposable tests available for "on the spot" analysis of blood
type. Blood samples are typically outsourced to an analytical
laboratory. Reliable low cost tests which are able to
instantaneously and reliably provide critical blood analysis
without the requirement of sophisticated laboratory analytical
instrumentation such as chromatographic and spectroscopic methods,
would be invaluable for improving human health in developing
countries, where economic resources are limited. Blood analysis is
also important in non-human applications, such as veterinary
medicine, where there is a demand for low cost and versatile
devices suitable for field use.
DESCRIPTION OF INVENTION
[0003] With this in mind, according to one aspect of the present
invention, there is provided a testing device for identifying an
antigen or antibody within a biofluid sample including;
[0004] a substrate having a hydrophilic surface thereon;
[0005] the surface including a collection zone, and at least one
detection zone extending therefrom;
[0006] wherein the biofluid sample can be mixed with a specific
antigen or antibody, and deposited on the collection zone and
transferred by capillary action to the detection zone;
[0007] the antigen or antibody in the biofluid sample reacting with
an appropriate said antibody or antigen thereby resulting in a
visual indication within the detection zone.
[0008] According to another aspect of the present invention there
is provided a testing device for identifying an antigen or antibody
within a biofluid sample including;
[0009] a substrate having a hydrophilic surface thereon;
[0010] the surface including a collection zone, and at least one
detection zone extending therefrom;
[0011] the detection zone having an antibody or antigen immobilised
therein;
[0012] wherein the biofluid sample can be deposited on the
collection zone and transferred by capillary action to the
detection zone;
[0013] the antigen or antibody in the biofluid sample reacting with
an appropriate said antibody or antigen within the detection zone
thereby resulting in a visual indication therein.
[0014] The substrate may be formed from paper or other cellulosic
materials. Alternatively, the substrate may include a chromographic
layer thereon or may be a wettable porous medium.
[0015] The biofluid sample being tested may preferably be blood,
and the visual indication may be due to an agglutination of the
blood upon reaction with a specific antibody resulting in reduced
wicking and/or separation of the blood in the detection zone.
[0016] The substrate surface may have a hydrophilic microfluidic
channel pattern thereon defining the collection zone and the
detection zone. Preferably, a plurality of detection zones may
extend from the collection zone.
[0017] It has been found that red blood cell agglutination,
triggered by specific antigen interaction, drastically decreases
blood wicking and dispersion on paper or chromatographic media. The
agglutination process also considerably enhances the
chromatographic separation (elution) of the individual blood
components, especially the red blood cells from the serum. The
testing device according to the present invention can allow direct
analysis of blood cells because of this visual indication. This can
be performed instantaneously, either with a detection/reporting
system built-in to the device or in conjunction with other off-line
analytical equipment. The testing device may also allow for the
identification and quantification of specific biomolecules (eg
antigens and antibodies) based on induced coagulation, followed by
the wicking and elution (separation) of the soluble protein
fraction from the blood sample onto the porous substrate. The blood
colloids, whose coagulation directly affects their
wicking/separation, can either be present in the fluid of interest,
such as the red blood cells of blood, or introduced as
nanoparticles (gold, silver, micro-silica, zeolite, titanium
dioxide and the like). In the latter case, the nanoparticle is
typically covered with the specific counter-biomolecule or molecule
of interest used as sensitive reporter component. The colloid
particles may be of a size ranging from 1 nm to 100 .mu.m and may
be introduced into the biofluid being analysed.
[0018] In the application of the present invention for determining
the type of blood group, the present invention may determine the
antigens present within a blood sample, the antigens determining
whether the blood type is type A, B, O, AB and Rhesus+/-.
Antibodies A, B and D (Rhesus) are deposited into separate
detection zones. It may also be preferable to include an untreated
control zone in one of the detection zones. A drop of blood is then
deposited on the central collection zone, the blood sample being
transferred by capillary action to each of the detection zones.
When the blood sample contacts an appropriate antibody, the
reaction of the red blood cells antigen with its corresponding
antibody results in agglutination or coagulation of the red cells.
This agglutination results in a drop in velocity of the movement of
the blood sample along the microfluidic channel providing the
detection zone and separation of the red blood cells from the
serum. The velocity of the blood samples travelling along other
detection zones with non-specific antibodies is unaffected. This
visual contrast facilitates easy and rapid identification of the
blood type of the blood sample.
[0019] The applicant has developed a low cost paper based
microfluidic system which is described in International patent
application no. PCT/AU2009/000889 details of which are incorporated
herein by reference. The microfluidic systems described in this
application utilise a paper based substrate, with the described
fabrication methods producing hydrophilic microfluidic channels on
the paper based substrates. It should be noted that the term paper
is used in this application to refer to all cellulosic materials
including woven fabrics and non-woven cellulosic material as well
as paper. The microfluidic systems described in these applications
can be readily adapted for the purpose of the present
invention.
[0020] The testing device according to the present invention may
also be used to detect illness as a result of blood cell
malfunction on the blood cells being of abnormal shape as is the
case with malaria. Alternatively, the testing device according to
the present invention may be used to detect illness by identifying
the presence of an antigen, antibody, virus (such as HIV,
influenza) or protein.
[0021] In the International application, the microfluidic channels
are fabricated by printing a hydrophobic agent on the substrate
surface to define a peripheral edge of the microfluidic channels.
According to the present invention, the antibody or antigen may
also be printed within the microfluidic channels. The technology
used, namely ink jet printing technology, may also be used to print
the antigen or antibody within the microfluidic channels.
[0022] According to a further aspect of the present invention,
there is provided a method for identifying an antigen or antibody
within a biofluid sample, including:
[0023] mixing the biofluid sample with a specific antigen or
antibody,
[0024] depositing the mixed biofluid sample on a collection zone of
a testing device including a substrate having a hydrophilic surface
thereon, the surface including said collection zone and at least
one detection zone extending therefrom, the biofluid sample being
transferred by capillary action to the detection zone; and
[0025] identifying the antigen or antibody by a resultant visual
indication within the detection zone arising where the antigen or
antibody in the biofluid sample reacts with an appropriate said
antibody or antigen.
[0026] According to yet another aspect of the present invention,
there is provided a method for identifying an antigen or antibody
within a biofluid sample, including:
[0027] depositing the biofluid sample on a collection zone of a
testing device including a substrate having a hydrophilic surface
thereon, the surface including said collection zone and at least
one detection zone extending therefrom, the detection zone having
an antibody or antigen immobilised therein, the biofluid sample
being transferred by capillary action to the detection zone;
and
[0028] identifying the antigen or antibody by a resultant visual
indication arising where the antigen or antibody in the biofluid
sample reacts with an appropriate said antibody or antigen within
the detection zone.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] It will be convenient to further describe the invention with
respect to the accompanying drawings which illustrate preferred
embodiments of the testing device according to the present
invention. Other embodiments of the invention are possible, and
consequently, the particularity of the accompanying drawings is not
to be understood as superseding the preceding description of the
invention.
[0030] In the drawings:
[0031] FIG. 1 shows a testing device according to the present
invention used to determine B+ blood;
[0032] FIG. 2 shows a testing device according to the present
invention used to determine O+ blood;
[0033] FIG. 3 shows the testing device according to the present
invention using AB+ and B+ blood;
[0034] FIG. 4 is the testing device according to the present
invention showing blood wicking and blood separation as a function
of time;
[0035] FIG. 5 shows a testing device according to the present
invention incorporating a valve;
[0036] FIG. 6 A-F shows the operation of a testing device according
to the present invention incorporating valves and switches;
[0037] FIG. 7 are schematic representations of testing devices
according to the present invention adapted for testing different
blood types; and
[0038] FIG. 8 shows the testing device of FIG. 7 showing the
separation of red blood cells from the blood serum.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The applicants have discovered that blood agglutination
mediated by specific antibody-antigen interactions drastically
affects its separation behaviour on contact with paper or any thin
layer chromatographic surface. The invention relies on this
biochemical phenomenon to control the rate of wicking and
separation, which enables (i) identification and quantitative
assessment of a specific antibody/antigen, (ii) blood typing, and
potentially (iii) identification of blood-borne pathogens as a
disease diagnostic. The present invention is intended predominantly
for applications in human and veterinary medicine and
biotechnology.
[0040] Standard techniques for detection of blood agglutination are
traditionally manual, involve dispensing of antibodies on a glass
slide and microscopic visualization. However, the visualisation of
agglutination is often subjective, and its automation requires a
bank of sophisticated analytical equipment. The present invention
provides a single-step blood test that simplifies and circumvents
these difficulties.
[0041] One application of the invention involves a two step process
in which the blood sample is first coagulated/agglutinated by
combining it with the specific antibody/antigen of interest,
followed by its deposition on the analytical substrate (eg
Non-woven paper or porous mesh) on which the sample wicking and
separation by elution/chromatography is measured, directly or
indirectly. These two mixing steps can be enhanced and more
accurately performed by mixing on paper substrates using built-in
valving and channelling control features.
[0042] A second application of the invention involves a single-step
process in which the biofluid/sample is deposited directly on the
substrate/device which has previously been treated with the
specific antigen/antibody. For this process, the analyte samples
simultaneously coagulate and elude on the same substrate. The
measured elution velocity and the extent of sample separation are
directly related to the extent of coagulation, enabling the
concentration of the biomolecule to be detected and quantified at
the same time.
[0043] Both applications of the invention can be applied to a test
device made of paper or any non-woven chromatographic surface,
which are relatively cost effective. These substrates are also able
to be modified with the use of advanced printing techniques to
create microfluidic features composed of hydrophobic materials, as
previously described in the applicant's International application
no. PCT/AU2009/000889. Combined with methods of direct antibody
deposition using printing, manufacture and placement of antibody
reagent can enable very accurate spatial control of blood flow
within the paper substrate.
EXAMPLES
Example 1
Sequential Agglutination/Coagulation of Blood Followed by Wicking
on Paper: B+ (Two Step Process) (See FIG. 1)
[0044] Antibody A and B (Epiclone.TM. Anti-A, Anti-B, and Anti-D;
CSL, Australia) solutions were used. Anti-A and Anti-B come as blue
and yellow colour reagents, respectively. `B+` blood was used in
this study. The blood sample was supplied into plastic vials with
anti-coagulant. `B+` blood was separately mixed with pure Anti-A
and Anti-B (as received) to prepare 100 .mu.L solution. Paper
strips (70 mm.times.2 mm) were made from Whatman#4 filter paper on
which 2 mm unit marks were printed. The paper strips were soaked
into phosphate buffer saline (PBS). Excess PBS was removed from the
paper strips using standard blotting papers (Drink Coster Blotting,
280 GSM). The paper strips were then placed on Reflex Paper (80
GSM). 20 .mu.L of every mixed solution was dispensed at the centre
of paper strip using a calibrated micro-pipette. Pictures were
taken after 4 minutes wicking.
[0045] It can be seen that:
[0046] B+ blood mixed with the solution of antibody A wicked and
did not separate upon mixing and paper elution/wicking.
[0047] B+ blood mixed with the solution of antibody B wicked and
STRONGLY separated (red cells from serum) and showed wicking.
[0048] B+ blood mixed with the solution of antibody D (Rhesus+)
wicked and STRONGLY separated (red cells from serum).
[0049] A blood sample agglutinated/coagulated upon contact with its
specific antibodies separated/eluded upon contact with paper (here
Blood B+ with Anti-B and Anti-D antibodies).
[0050] A blood sample upon contact with non-specific antibody (here
Blood B+ with Anti-A) does not agglutinate and does not
separate/elute upon contact with paper.
[0051] This dramatic difference in elution/separation of
blood/antibody mixing can be used to communicate specific
agglutination and therefore can be used to identify blood
typing.
Example 2
Sequential Agglutination/Coagulation of Blood Followed by Wicking
on Paper: O+ (Two Step Process) (See FIG. 2)
[0052] Antibody A and B (Epiclone.TM. Anti-A, Anti-B and Anti-D;
CSL, Australia) solutions were used. Anti-A and Anti-B come as blue
and yellow colour reagents, respectively. `O+` blood was used in
this study. The blood sample was supplied into plastic vials with
anti-coagulant. `O+` blood was separately mixed with Anti-A and
Anti-B to prepare 100 .mu.L solution. Paper strips (70 mm.times.2
mm) were made from Whatman#4 filter paper on which 2 mm unit marks
were printed. The paper strips were soaked into phosphate buffer
saline (PBS). Excess PBS was removed from the paper strips using
standard blotting papers (Drink Coster Blotting, 280 GSM). The
paper strips were then placed on Reflex Paper (80 GSM). 20 .mu.L of
every mixed solution was dispensed at the centre of paper strip
using a calibrated micro-pipette. Pictures were taken after 4
minutes wicking.
[0053] It can be seen that:
[0054] O+ blood mixed with the solution of antibody A wicked and
did not separate upon mixing and paper elution/wicking.
[0055] O+ blood mixed with the solution of antibody B wicked and
did not separate (red cells from serum) and showed wicking.
[0056] O+ blood mixed with the solution of antibody D (Rhesus+)
wicked and STRONGLY separated (red cells from serum).
[0057] A blood sample agglutinated/coagulated upon contact with its
specific antibodies separated/eluded upon contact with paper (here
Blood O+ with Anti-D antibodies).
[0058] A blood sample upon contact with non-specific antibody (here
Blood O+ with Anti-A and Anti-B) does not agglutinate and does not
separate/elute upon contact with paper.
[0059] This dramatic difference in elution/separation of
blood/antibody mixing can be used to communicate specific
agglutination and therefore can be used to identify blood
typing.
Example 3
Simultaneous Agglutination/Coagulation of Blood Followed by Wicking
on Paper: Effect of Antigen Concentration (One Step Process) (See
FIG. 3)
[0060] In another embodiment of the invention, the paper is first
treated with specific antibodies, dried or conditioned before been
exposed to a sample of pure blood. This example provides a single
step treatment in which the only requirement is to deposit a drop
of blood on the paper. This example also illustrates the effect of
diluting the antibody solution on the wicking and separation
performance of blood on paper. Antibody dilution affects the ratio
blood (with its antigen) antibody.
[0061] Antibody A and B (Epiclone.TM. Anti-A and Anti-B; CSL,
Australia) solutions were used. Anti-A and Anti-B come as blue and
yellow colour reagents, respectively. "AB+" and `B+` blood were
used in this study. The blood sample was supplied into plastic
vials with anti-coagulant. Paper strips (70 mm.times.2 mm) were
made from Whatman#4 filter paper on which 2 mm unit marks were
printed. Paper strips were soaked into antibody solutions of
different concentrations (Anti-A@1.0.times., 0.8.times.,
0.6.times., 0.4.times., 0.2.times. and 0.0.times.); phosphate
buffer saline (PBS) was used as diluent. Excess antibody was
removed from the paper strips with blotting papers. The antibody
(Anti-A) active paper strips were then placed on Reflex Paper.
Blood drops of 20 .mu.L were dispensed at the centre of paper strip
using a calibrated micro-pipette. The wicking distance was measured
from centre to either direction. Pictures were taken after 10
minutes.
[0062] The results are shown in FIG. 3. It can be seen that:
[0063] Blood separates upon wicking with its specific antibody
treated paper.
[0064] Blood separation is a non-linear function of the antibody
concentration on the treated paper. The higher the antibody
concentration, the more abrupt is the cell separation from the
serum.
[0065] There is an optimum concentration to maximize
wicking/separation/visualization.
[0066] Coagulation of red cell upon contact with its specific
antibody drastically reduces its wicking/diffusion speed on the
chromatographic surface, which promotes separation of cells from
the serum. This drastic reduction and differentiation of elution
speeds can serve as direct indicator of the type of blood.
Example 4
Effect of Time on the Wicking/Separation of Blood on Bioactive
Antibody Paper (see FIG. 4)
[0067] In another embodiment of the invention, the paper is first
treated with specific antibodies, dried or conditioned before been
exposed to a sample of pure blood. This example illustrates the
effect of contact time blood-antibody treated paper on the wicking
and separation performance of blood on paper.
[0068] Antibody A and B (Epiclone.TM. Anti-A; CSL, Australia)
solutions were used. Anti-A comes as a blue colour reagent. "AB+"
blood was used in this study. The blood sample was supplied into
plastic vials with anti-coagulant. Paper strips (70 mm.times.2 mm)
were made from Whatman#4 filter paper on which 2 mm unit marks were
printed. Paper strips were soaked into antibody solutions
(Anti-A@); phosphate buffer saline (PBS) was used as diluent.
Excess antibody was removed from the paper strips with blotting
papers. The antibody (Anti-A) active paper strips were then placed
on Reflex Paper. Blood drops of 20 .mu.L were dispensed at the
centre of paper strip using a calibrated micro-pipette. The wicking
distance was measured from centre to either direction. Pictures
were taken after different intervals of time.
[0069] It can be seen that:
[0070] blood wicking/separation levels off after about 4
minutes.
[0071] There is a minimum time of contact of antibody-blood
required to allow proper blood coagulation/agglutination and
wicking/separation.
[0072] There is an optimum time of contact of blood-antibody-paper.
Too short, the blood does not properly coagulate; too long, the
separation of red cell and serum can loose some of its
sharpness.
Example 5
Paper Microfluidic System to Control Flow, Reaction and Dilution
(See FIG. 5)
[0073] In the embodiment of the invention, paper-based microfluidic
reactors can be used to conduct blood type tests. Specific
antibodies are printed into the reactor designed on paper. Then
blood cell suspension is introduced into the same reactor. The
required period of time is allowed so that the antibodies and cell
suspension can contact and mix. After a preset period of time, the
valve of the reactor is closed to facilitate penetration of blood
across the valve. If only the penetration of serum is observed, the
test is positive because of agglutination of blood during the
mixing time. If the penetration of blood is observed, the test is
negative. Thus paper-based microfluidic reactor can provide a rapid
visual test of blood type.
Example 6
Microfluidic System with Valves (See FIG. 6)
[0074] Paper microfluidic devices can be designed to increase the
ratio of blood/antibody and to provide the required time delay to
allow blood and antibody interactions before the test. This example
shows that all these steps can be performed using a paper device.
FIG. 6 shows the design of the paper device. (A) A filter paper
sheet is printed and cut as shown, and specific antibodies are
either printed or deposited in the circled region. A paper switch
is made on the right hand side of the device. (B) Blood sample is
introduced onto the indicated region. (C) The cut paper is folded
towards the blood sample as shown. (D) Blood sample is allowed to
stay in contact with the antibody loaded paper for a set time. (E)
After a short period of contact time, the switch is closed as
shown. If the test is positive, blood will agglomerate and only
serum will wick out along the switch. (F) After a short period of
contact, the switch is closed as shown. If the test is negative,
blood will not agglomerate and will wick out along the switch.
Example 7
Paper Microfluidic System for Blood Typing (See FIG. 7)
[0075] In another embodiment of the invention, a microfluidic
system is printed on paper or a chromatographic medium and
antibodies A, B and D (Rhesus) are printed into each of the 3
detection arms. Blood typing is analysed by placing a blood droplet
in the middle reservoir and reading the results. All the different
combinations of blood type and their representations are
represented in FIG. 7.
Example 8
Chromatographic Separation of RBC/Blood Serum on Paper (See FIG.
8)
[0076] FIG. 8 illustrates blood group detection using
chromatographic separation of red blood cells (RBC) and blood serum
on antibody active paper surface; (a) schematic of chromatographic
separation on paper bioassay; (b) and (c)(I) are trial 1 and 2
using A+ blood sample, respectively; (b)(II), (c)(II) are the
converted images of (b)(I), (c)(I) (RGB colour to BRG colour),
respectively, for better resolution.
[0077] Modifications and variations as would be deemed obvious to
the person skilled in the art are included within the ambit of the
present invention as claimed in the appended claims.
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