U.S. patent application number 15/646616 was filed with the patent office on 2017-12-28 for microfluidic flow assay and methods of use.
This patent application is currently assigned to Colorado School of Mines. The applicant listed for this patent is Colorado School of Mines. Invention is credited to Keith B. Neeves, Abimbola Onasoga.
Application Number | 20170370953 15/646616 |
Document ID | / |
Family ID | 50025865 |
Filed Date | 2017-12-28 |
United States Patent
Application |
20170370953 |
Kind Code |
A1 |
Neeves; Keith B. ; et
al. |
December 28, 2017 |
MICROFLUIDIC FLOW ASSAY AND METHODS OF USE
Abstract
A method for evaluating a blood product of an individual are
provided. Specifically, a method to utilize a microfluidic flow
assay, which includes a substrate surface comprising lipid coated
particles and microfluidic channels through which a blood product
can flow. The lipid coated particles comprise functional molecules
that can induce or inhibit the coagulation cascade.
Inventors: |
Neeves; Keith B.; (Denver,
CO) ; Onasoga; Abimbola; (Golden, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Colorado School of Mines |
Golden |
CO |
US |
|
|
Assignee: |
Colorado School of Mines
Golden
CO
|
Family ID: |
50025865 |
Appl. No.: |
15/646616 |
Filed: |
July 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13929141 |
Jun 27, 2013 |
9709579 |
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15646616 |
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61665177 |
Jun 27, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/86 20130101 |
International
Class: |
G01N 33/86 20060101
G01N033/86 |
Claims
1. A method for evaluating a blood product of an individual,
comprising: a. perfusing the individual's blood product over a
microfluidic device under flow conditions to contact the blood
product with a functional molecule of a plurality of coated lipid
particles, wherein the microfluidic device, comprises at least one
microfluidic channel; and at least one substrate surface provided
in the at least one microfluidic channel, wherein the at least one
substrate surface comprises a plurality of lipid coated particles
immobilized on the substrate surface, wherein the plurality of
lipid coated particles comprises at least one functional molecule,
wherein the at least one functional molecule induces coagulation;
and b. detecting one or more coagulation products associated with
the at least one functional molecule of the plurality of the lipid
coated particles.
2. The method of claim 1, wherein the blood product is selected
from the group consisting of whole blood, plasma, platelet rich
plasma, and platelet poor plasma.
3. The method of claim 1, wherein the flow conditions simulate
hemodynamic conditions of the individual.
4. The method of claim 1, wherein the functional molecule is one or
more transmembrane proteins.
5. The method of claim 1, wherein the transmembrane protein is
selected from the group consisting of tissue factor,
thromobomodulin, endothelial cell protein C receptor, glycoprotein
Ilb/IIIa, glycoprotein VI, glycoprotein 1b/IX/V, P-selectin,
glycoprotein IV, CD9, platelet endothelial cell adhesion molecule
(PECAM-1), Ras-related protein 1b (rap1b), c-type lectin-like
receptor 2 (CLEC-2), intracellular adhesion molecule 1 (ICAM-1),
intracellular adhesion molecule 2 (ICAM-2) and combinations
thereof.
6. The method of claim 1, wherein the functional molecule initiates
coagulation.
7. The method of claim 1, wherein the functional molecule inhibits
coagulation.
8. The method of claim 1, wherein the step of detecting comprises
quantifying the one or more coagulation products.
9. The method of claim 1, wherein the one or more coagulation
products consist of proteins selected from the group consisting of
thrombin, fibrin, thrombin-antithrombin complex, fibrinopeptide A,
fibrinopeptide B, D-dimer, prothrombin fragment 1+2, activated
factor X, activated factor V, activated factor VIIIa, activated
factor IXa, activated factor XIa, activated factor XIIa, activated
protein C, activated protein S, and mixtures thereof.
10. The method of claim 1, wherein the one or more coagulation
products are detected by a method selected from the group
consisting of brightfield microscopy, darkfield microscopy,
fluorescence microscopy, multi-photon excitation, second harmonic
generation, third harmonic generation, atomic force microscopy,
scanning electron microscopy, and absorbance.
11. The method of claim 1, wherein the plurality of the lipid
coated particles comprises a plurality of particles having a
hydrophilic surface.
12. The method of claim 1, wherein the plurality of lipid coated
particles comprises one or more phospholipid structures selected
from the group consisting of phosphatidylserine,
phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine,
phophoinositides, phosphosphingolipids, and combinations
thereof.
13. The method of claim 1, wherein the plurality of lipid coating
particles are immobilized on the substrate surface by a method
selected from covalent bonding, electrostatic interactions or
hydrogen bonding.
14. The method of claim 1, wherein the at least one microfluidic
channel is capable of receiving fluid at a first end of the at
least one microfluidic channel and allowing the fluid to flow
through the at least one microfluidic channel to a second end of
the at least one microfluidic channel.
15. The method of claim 1, wherein the at least one microfluidic
channel is split into multiple channels.
16. The method of claim 1, wherein the at least one functional
molecule is a tissue factor or a thrombomodulin.
17. The method of claim 1, wherein a flow rate of the individual's
blood product is between about 50 to 2600 sec.sup.-1.
18. The method of claim 1, wherein a flow rate of the individual's
blood product is between 0 and about 500,000 sec.sup.-1.
19. The method of claim 1, further comprising an agent wherein the
agent is an anticoagulant agent or coagulating agent.
20. The method of claim 1, wherein the individual is a mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. of patent
application Ser. No. 13/929,141, filed on Jun. 27, 2013, which
issued as U.S. Pat. No. 9,709,579 on Jul. 18, 2017, which claims
the benefit of U.S. Provisional Patent Application Ser.
No.61/665,177, filed Jun. 27, 2012. These references are hereby
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The invention relates to a microfluidic-based flow assay and
device for use in analyzing bleeding and anticoagulation disorders,
dosing anticoagulant drugs, tracking the effects of pharmacological
interventions on coagulation, and methods of making the same.
BACKGROUND OF INVENTION
[0003] Maintaining the balance between bleeding and thrombosis
remains one of the greatest challenges facing the biomedical
community. Excessive bleeding is an important medical issue. For
example, post partum bleeding represents a leading cause of
maternal mortality and causes serious morbidity in developing
countries. Individuals with genetic bleeding disorders, such as
hemophilia, have a decreased ability to clot blood because of
deficiencies in certain coagulation factors.
[0004] On the other end of the spectrum, excessive clotting, or
thrombosis, is a major complication of surgery and is integrally
involved in atherosclerosis, obesity, infection, diabetes, cancer,
and autoimmune disorders. Over the last decade, significant
advances have been made in understanding the molecular basis of
bleeding and thrombotic disorders; however, a large portion of the
observed variability remains unknown.
[0005] Parallel with these discoveries, there has been a rapid
development of new drugs such as recombinant proteins for
replacement and interventional therapies. Interestingly, what
remains strikingly deficient in clinical hematology are techniques
to diagnose a very broad range of disorders of both deficient and
excessive clotting as well as to monitor the effects of therapeutic
interventions.
[0006] The formation of a stable fibrin network is necessary for
hemostasis, which requires fibrinogen conversion to fibrin. In
purified systems containing only thrombin and fibrinogen, it has
been shown that fibrin polymerization can only occur in a narrow
set of conditions that are defined by the rate of thrombin
formation and the shear rate (Neeves et al., Biophysical Journal,
2010, 98; 1344-1352). Most coagulation assays do not account for
the interplay between flow and surface reactions, which could
affect clot properties like fiber thickness, fibrin clot height,
fiber alignment with flow, and resistance to lysis. These
properties can be useful in differentiating plasma clots of healthy
individuals from those with thrombotic or haemostatic
disorders.
[0007] Diagnosing the severity of bleeding a disorder is impossible
with current bleeding assays, particularly because most current
bleeding assays test for either platelet function and/or platelet
coagulation using whole blood, however, these assays do not allow
for the use plasma. Additionally, most existing solutions do not
properly create an environment which properly simulates a natural
human wound or point of bleeding. Also, most of these conventional
assays occur under static, or no flow, conditions. Since blood is a
moving fluid, however, there are several advantages to studying it
under flow in bleeding diagnostics.
SUMMARY OF INVENTION
[0008] One embodiment of the invention relates to a microfluidic
device comprising at least one microfluidic channel and at least
one substrate surface in the microfluidic channel. The substrate
surface comprises a plurality of lipid coated particle that are
immobilized on the substrate surface. The lipid coated particles
comprise at least one functional molecule that induces
coagulation.
[0009] In one aspect, the substrate surface is functionalized
glass.
[0010] In another aspect, the plurality of lipid coated particles
comprises a plurality of particles having a hydrophilic surface. In
one aspect, the lipid coated particles comprise one or more
phospholipid structures. The phospholipid structures can be
selected from phosphotidylserine, phosphotidlcholine, phosphatidic
acid, phosphatidylethanolamine, phophoinositides,
phosphosphingolipids, and combinations thereof. In still another
aspect, the plurality of lipid coated particles is immobilized to
the substrate surface by an immobilization method. The
immobilization can be selected from covalent bonding, electrostatic
interactions and hydrogen bonding. In yet another aspect, the
immobilized plurality of lipid coated particles is patterned to the
substrate surface by a patterning method. The patterning method can
be selected from microblotting and microstenciling. In another
aspect, the immobilized and patterned lipid coated particles are
integrated into at least one microfluidic channel.
[0011] In another aspect, the microfluidic device further comprises
hydrodynamic focusing.
[0012] In still another aspect, the functional molecule of the
coated lipid particle of the device is one or more transmembrane
proteins. The transmembrane proteins can be selected from tissue
factor, thromobomodulin, endothelial cell protein C receptor,
glycoprotein IIb/IIIa, glycoprotein VI, glycoprotein Ib/IX/V,
P-selectin, glycoprotein IV, CD9, platelet endothelial cell
adhesion molecule (PECAM-1), Ras-related protein 1b (rap1b), c-type
lectin-like receptor 2 (CLEC-2), intracellular adhesion molecule 1
(ICAM-1), intracellular adhesion molecule 2 (ICAM-2) and
combinations thereof.
[0013] Another embodiment of the invention relates to a
microfluidic device made by a method comprising providing a
substrate, creating at least one surface on the substrate,
immobilizing and patterning a plurality of lipid coated particles
onto the surface of the substrate. The lipid coated particles are
coated with lipid bilayers and comprise a functional molecule that
induces coagulation. The plurality of lipid coated molecules is
integrated into at least one microfluidic channel, which intersects
at least a portion of the substrate surface.
[0014] Another embodiment of the invention relates to a plurality
of lipid coated particles made by a method comprising providing
silica beads, making the silica beads hydrophilic, coating the
surface of the hydrophilic silica beads with lipid bilayers and a
functional molecule. The lipid bilayers comprise one or more
phospholipid structures.
[0015] Yet another embodiment of the invention relates to a kit for
measuring clotting characteristics of a blood product. The kit
comprises a hermetically sealed microfluidic device, the
microfluidic device comprising at least one microfluidic channel
and at least one substrate surface provided in the at least one
microfluidic channel, wherein the at least one substrate surface
comprises a lipid coated particle wherein the lipid coated particle
comprises a functional molecule embedded in the lipid, wherein the
functional molecule induces coagulation.
[0016] A further embodiment of the invention relates to a method
for evaluating a blood product of an individual comprising
perfusing the individual's blood product over a microfluidic device
under flow conditions to contact the blood product with a
functional molecule of a plurality of coated lipid particles,
wherein the microfluidic device, comprises at least one
microfluidic channel; and at least one substrate surface provided
in the at least one microfluidic channel, wherein the at least one
substrate surface comprises a plurality of lipid coated particles
immobilized on the substrate surface, wherein the plurality of
lipid coated particles comprises at least one functional molecule,
wherein the at least one functional molecule induces coagulation;
and detecting one or more coagulation products associated with the
at least one functional molecule of the plurality of the lipid
coated particles. In one aspect, the blood product is selected from
whole blood, plasma, platelet rich plasma, and platelet poor
plasma. In another aspect, the flow conditions simulate hemodynamic
conditions of the individual. In still another aspect of the
method, the functional molecule is one or more transmembrane
proteins. The transmembrane proteins can be selected from tissue
factor, thromobomodulin, endothelial cell protein C receptor,
glycoprotein IIb/IIIa, glycoprotein VI, glycoprotein Ib/IX/V,
P-selectin, glycoprotein IV, CD9, platelet endothelial cell
adhesion molecule (PECAM-1), Ras-related protein 1b (rap1b), c-type
lectin-like receptor 2 (CLEC-2), intracellular adhesion molecule 1
(ICAM-1), intracellular adhesion molecule 2 (ICAM-2) and
combinations thereof. In yet another aspect, the functional
molecule initiates coagulation. In still another aspect, the
functional molecule inhibits coagulation.
[0017] In yet another aspect of the method, the step of detecting
comprises quantifying the coagulation product. The coagulation
product can be selected from thrombin, fibrin,
thrombin-antithrombin complex, fibrinopeptide A, fibrinopeptide B,
D-dimer, prothrombin fragment 1+2, activated factor X, activated
factor V, activated factor VIIIa, activated factor IXa, activated
factor XIa, activated factor XIIa, activated protein C, activated
protein S, and mixtures thereof. In still another aspect of the
method, the coagulation product can be detected by a method
selected from brightfield microscopy, darkfield microscopy,
fluorescence microscopy, multi-photon excitation, second harmonic
generation, third harmonic generation, atomic force microscopy,
scanning electron microscopy, and absorbance.
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 depicts a top view of an exemplary microfluidic
device with at least some embodiments of the present invention.
[0019] FIG. 2 depicts an exploded top view of a portion of an
exemplary microfluidic device with at least some embodiments of the
present invention.
[0020] FIG. 3 depicts an exploded side-view of a portion of an
exemplary microfluidic device with at least some embodiments of the
present invention.
[0021] FIG. 4 depicts the formation of lipid coated particles of
the present invention and patterning on glass slides. Tissue Factor
(TF); L-.alpha.-phosphatidylcholine (PC) and
L-.alpha.-phosphatidylserine (PS); DHPE (Texas red
1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine);
3-[(2-Aminoethylamino) propyl] trimethoxysilane (APTMS);
Polydimethylsiloxane (PDMS).
[0022] FIG. 5A depicts 100 .mu.M patterned Tissue Factor lipid
coated particles. A pluarity of lipid coated particles is found
within each spot.
[0023] FIG. 5B depicts a microfluidic device with hydrodynamic
focusing which is used to force Alexa 488-labelled plasma (lightest
shaded region indicated as the "Blood Product Area") to flow in the
center over the patterned lipid coated particles in the spots,
while bounded on the side by Texas labeled buffer (mid-shaded
regions adjacent to the lightest shaded regions indicated as the
"Buffer Area").
[0024] FIG. 5C depicts fibrin that is formed over the lipid coated
particle spots of FIG. 5B over a 10 minute perfusion of plasma at 0
s, which represents start of perfusion.
[0025] FIG. 5D depicts fibrin that is formed over the lipid coated
particle spots of FIG. 5B over a 10 minute perfusion of plasma at
300 s, which represents 300 seconds after the start of
perfusion.
[0026] FIG. 5E depicts fibrin that is formed over the lipid coated
particle spots of FIG. 5B over a 10 minute perfusion of plasma at
600 s, which represents 600 seconds (or 10 minutes) after the start
of perfusion.
[0027] FIG. 5F depicts thrombin generation over the lipid coated
particle spots of FIG. 5B as measured by a blue signal that is
emitted when the thrombin substrate boc-VPR-AMC is cleaved over a
10 minute perfusion of plasma at 0 s, which represents start of
perfusion.
[0028] FIG. 5G depicts thrombin generation over the lipid coated
particle spots of FIG. 5B as measured by a blue signal that is
emitted when the thrombin substrate boc-VPR-AMC is cleaved over a
10 minute perfusion of plasma at 300 s, which represents 300
seconds after the start of perfusion.
[0029] FIG. 5H depicts thrombin generation over the lipid coated
particle spots of FIG. 5B as measured by a blue signal that is
emitted when the thrombin substrate boc-VPR-AMC is cleaved over a
10 minute perfusion of plasma at 600 s, which represents 600
seconds (or 10 minutes) after the start of perfusion.
[0030] FIG. 6A depicts fibrin generation after normal pooled plasma
(NPP) was perfused over TF lipid coated particles using the
microfluidic device of the present invention, wherein the TF
concentration was 50 molecules/.mu.m.sup.2 at wall shear rates of
50, 100, 250, 500 and 1000 s.sup.-1. Relative Fluorescence Units
(RFUs) were determined in real-time using three metrics to quantify
the dynamics of fibrin generation (i) the lag time to fibrin fiber
generation, (ii) the maximum fibrin density, and (iii) the rate of
fibrin generation.
[0031] FIG. 6B depicts fibrin generation after normal pooled plasma
(NPP) was perfused over TF lipid coated particles using the
microfluidic device of the present invention, wherein the TF
concentration was 5 molecules/.mu.m.sup.2 at wall shear rates of
50, 100, 250, 500 and 1000 s.sup.-1. Relative Fluorescence Units
(RFUs) were determined in real-time using three metrics to quantify
the dynamics of fibrin generation (i) the lag time to fibrin fiber
generation, (ii) the maximum fibrin density, and (iii) the rate of
fibrin generation.
[0032] FIG. 6C depicts fibrin generation after normal pooled plasma
(NPP) was perfused over TF lipid coated particles using the
microfluidic device of the present invention, wherein the TF
concentration was 0.5 molecules/.mu.m.sup.2 at wall shear rates of
50, 100, 250, 500 and 1000 s.sup.-1. Relative Fluorescence Units
(RFUs) were determined in real-time using three metrics to quantify
the dynamics of fibrin generation (i) the lag time to fibrin fiber
generation, (ii) the maximum fibrin density, and (iii) the rate of
fibrin generation.
[0033] FIG. 6D depicts thrombin generation after normal pooled
plasma (NPP) was perfused over TF lipid coated particles using the
microfluidic device of the present invention, wherein the TF
concentration was 50 molecules/.mu.m.sup.2 at wall shear rates of
50, 100, 250, 500 and 1000 s.sup.-1. Relative Fluorescence Units
(RFUs) were determined in real-time using three metrics to quantify
the dynamics of thrombin generation (i) the lag time to thrombin
generation, (ii) the maximum thrombin fluorescence, and (iii) the
rate of thrombin generation.
[0034] FIG. 6E depicts thrombin generation after normal pooled
plasma (NPP) was perfused over TF lipid coated particles using the
microfluidic device of the present invention, wherein the TF
concentration was 5 molecules/.mu.m.sup.2 at wall shear rates of
50, 100, 250, 500 and 1000 s.sup.-1. Relative Fluorescence Units
(RFUs) were determined in real-time using three metrics to quantify
the dynamics of thrombin generation (i) the lag time to thrombin
generation, (ii) the maximum thrombin fluorescence, and (iii) the
rate of thrombin generation.
[0035] FIG. 6F depicts thrombin generation after normal pooled
plasma (NPP) was perfused over TF lipid coated particles using the
microfluidic device of the present invention, wherein the TF
concentration was 0.5 molecules/.mu.m.sup.2 at wall shear rates of
50, 100, 250, 500 and 1000 s.sup.-1. Relative Fluorescence Units
(RFUs) were determined in real-time using three metrics to quantify
the dynamics of thrombin generation (i) the lag time to thrombin
generation, (ii) the maximum thrombin fluorescence, and (iii) the
rate of thrombin generation.
[0036] FIG. 7 shows the results of a D-dimer analysis of the
cumulative fibrin deposited (generated) as described in FIGS. 6A-6C
on all the spots comprising a plurality of the TF lipid coated
particles as measured by D-dimer concentration following plasmin
digestion. The wall shear rates are provided at the top of the
graph.
[0037] FIG. 8A shows the fiber height of the fibrin fibers that
accumulated on the individual spots from the assay described in
FIGS. 6A-6C wherein the fiber height is provided for each of the
wall shear rates of 50, 100, 250, 500 or 1000 s.sup.-1 to show the
shear rate effects with distribution of the fibrin fibers.
[0038] FIG. 8B shows final fluorescence images showing a decrease
in fibrin fiber density and intensity with an increase in shear
rate (scale bars=20 um) at 50 s.sup.-1 from the assay described in
FIGS. 6A-6C.
[0039] FIG. 8C shows final fluorescence images showing a decrease
in fibrin fiber density and intensity with an increase in shear
rate (scale bars=20 um) at 100 s.sup.-1 from the assay described in
FIGS. 6A-6C.
[0040] FIG. 8D shows final fluorescence images showing a decrease
in fibrin fiber density and intensity with an increase in shear
rate (scale bars=20 um) at 250 s.sup.-1 from the assay described in
FIGS. 6A-6C.
[0041] FIG. 8E shows final fluorescence images showing a decrease
in fibrin fiber density and intensity with an increase in shear
rate (scale bars=20 um) at 500 s.sup.-1 from the assay described in
FIGS. 6A-6C.
[0042] FIG. 8F shows final fluorescence images showing a decrease
in fibrin fiber density and intensity with an increase in shear
rate (scale bars=20 um) at 1000 s.sup.-1 from the assay described
in FIGS. 6A-6C.
[0043] FIG. 8G shows scanning electron micrographs of fibrin
diameter decreasing with an increase in shear rate at 50 s.sup.-1
from the assay described in FIGS. 6A-6C.
[0044] FIG. 8H shows scanning electron micrographs of fibrin
diameter decreasing with an increase in shear rate at 100 s.sup.-1
from the assay described in FIGS. 6A-6C.
[0045] FIG. 8I shows scanning electron micrographs of fibrin
diameter decreasing with an increase in shear rate at 250
s.sup.-from the assay described in FIGS. 6A-6C.
[0046] FIG. 8J shows scanning electron micrographs of fibrin
diameter decreasing with an increase in shear rate at 500 s.sup.-1
from the assay described in FIGS. 6A-6C.
[0047] FIG. 8K shows scanning electron micrographs of fibrin
diameter decreasing with an increase in shear rate at 1000 s.sup.-1
from the assay described in FIGS. 6A-6C.
[0048] FIG. 9A shows fibrin generation for factor deficient plasma
at 50 s.sup.-1 and TF concentration of 50 molecules/.mu.m.sup.2
using the assay described in FIGS. 6A-6C. Normal pooled plasma
(NPP); plasma deficient with factor XI (FXI-def); plasma deficient
with factor VIII (FVIII-def); and plasma deficient with factor IX
(FIX-def).
[0049] FIG. 9B shows fibrin generation for factor deficient plasma
at 5 s.sup.-1 and TF concentration of 5 molecules/.mu.m.sup.2 using
the assay described in FIGS. 6A-6C. Normal pooled plasma (NPP);
plasma deficient with factor XI (FXI-def); plasma deficient with
factor VIII (FVIII-def); and plasma deficient with factor IX
(FIX-def).
[0050] FIG. 9C shows thrombin generation for factor deficient
plasma at 50 s.sup.-1 and TF concerntration of 50
molecules/.mu.m.sup.2 using the assay described in FIGS. 6D-6F.
Normal pooled plasma (NPP); plasma deficient with factor XI
(FXI-def); plasma deficient with factor VIII (FVIII-def); and
plasma deficient with factor IX (FIX-def).
[0051] FIG. 9D shows thrombin generation for factor deficient
plasma at 5 s.sup.-1 and TF concentration of 5
molecules/.mu.m.sup.2 using the assay described in FIGS. 6D-6F.
Normal pooled plasma (NPP); plasma deficient with factor XI
(FXI-def); plasma deficient with factor VIII (FVIII-def); and
plasma deficient with factor IX (FIX-def).
DETAILED DESCRIPTION
[0052] This invention generally relates to a microfluidic device
and methods and uses of the device for evaluating and testing a
blood product from an individual as well as for measuring the
clotting characteristics of a blood product from an individual.
This invention describes a flow based assay that allows the use of
a blood product such as plasma for measuring end products of the
coagulation cascade (such as thrombin and fibrin generation). The
advantage of this invention is that it integrates the coagulation
cascade into a fluidic architecture, which allows for measurement
of coagulation products under the hemodynamic conditions found in
the body. Furthermore, because this assay allows the use of plasma,
the plasma samples can be stored for long periods of time before
being tested. This is in contrast to most flow based assays that
use only whole blood, which needs to be used within hours of a
blood draw.
[0053] This invention fills a technology gap for a flow based
plasma assay for measuring coagulation potential.
[0054] There are no known flow based plasma assays for coagulation.
Static plasma assays for measuring coagulation include thrombin
generation (TG), prothrombin time (PT), partial thromboplastin time
(PTT), and turbidity-based assays.
[0055] With reference to FIG. 1 an embodiment of the present
invention is illustrated. This is an exemplary microfluidic device
in accordance with at least some embodiments of the present
invention. More specifically, the microfluidic device may include
one or more fluid receiving passages which allows for fluid to flow
through a microfluidic device; an inlet for a blood product (such
as plasma) which is capable of receiving a blood product (shown as
"2") and an outlet (shown as "3") where blood product can be
collected. The microfluidic device may also include a buffer inlet
for hydrodynamic focusing (shown as "1") which is capable of
receiving buffer. Each shaded circular spot represents a plurality
of lipid coated particles of the invention that is attached to a
substrate surface. The arrows represent the direction of flow of
the blood product and buffer through the channels of the
microfluidic device.
[0056] With reference to FIG. 2 an embodiment of the present
invention is illustrated. This is an exploded top view of an
exemplary microfluidic device as described in FIG. 1 with at least
some embodiments of the present invention. Each shaded circular
spot represents a plurality of lipid coated particles of the
invention that plasma flows over. The buffer sections represent
buffer that is flowed adjacent to the plasma over the spots
demonstrating focusing of the plasma on the spots.
[0057] With reference to FIG. 3 an embodiment of the present
invention is illustrated. This is an exploded side-view of a
portion of an exemplary microfluidic device as described in FIG. 1
with at least some embodiments of the present invention. Each
circle represents an individual single lipid coated particle within
a single circular spot as depicted in FIGS. 1 and 2.
[0058] The device of the present invention comprises at least one
microfluidic channel. The microfluidic device may include a
plurality of fluid-filled receiving passages, which are capable of
receiving fluid at a receiving end and allowing the fluid to flow
through a microfluidic channel to a collection point or terminal
end. One or more microfluidic channels may be present and may spilt
into multiple channels, thereby resulting in a number of terminal
ends. The number of receiving ends may equal the number of terminal
ends. The configuration and design of the microfluidic channels can
vary without departing from the scope of the present invention.
[0059] In addition to comprising at least one microfluidic channel,
the microfluidic device may also comprise at least one substrate
surface which intersects one or more of the microfluidic channels.
In addition, the substrate surface can be functionalized. In this
step, the substrate surface may be treated with
3-aminopropyl-trimethoxysilane (APTMS), thereby creating a
monolayer of APTMS on the upper surface of the substrate. Methods
of rendering substrates, such as glass substrates, hydrophilic are
well known in the art. Method of functionalizing the substrate
include, without limitation, rendering the substrate and/or the
substrate surface hydrophilic, hydrophobic, reactive (via amine or
carboxylic acid groups) or some other chemistry. In one embodiment,
silane chemistries may be used on the substrates. The substrate may
be any composed of any material including but not limited to glass,
plastic, gold, quartz, silicon, silicon nitride, silicon dioxide,
polydimethylsiloxane, polystyrene, polymethyl methacrylate and
combinations thereof, or any other type of known substrate material
used in surface chemistry. Additionally, the substrate is a size
that allows for complete immersion of the substrate and/or
substrate surface into the microfluidic channel.
[0060] The substrate surface comprises a plurality of lipid coated
particles that are immobilized on the substrate surface. The lipid
coated particles may be comprised of silica such as silica glass or
ceramics, including but not limited to silica beads that are
synthesized by methods known to those of skill in the art,
including but not limited to the Stober process. The resulting
silica beads may be silica micro beads and may range in diameter
from about 0.1 micrometer to about 100 micrometers. Preferably, the
resulting silica beads are 1 to 10 micrometers in diameter.
[0061] In the case of lipid coated particles formed using silica
beads, once the silica beads are synthesized they may be made
hydrophilic by using known methods including but not limited to
treatment with hydrogen peroxide and dilute organic acid. Once the
beads are synthesized and made hydrophilic, their surfaces may be
coated with lipid bilayers comprised of one or more phospholipid
structures. These phospholipid structures include but are not
limited to phosphatidylserine, phosphatidylcholine, phosphatidic
acid, phosphatidylethanolamine, phophoinositides,
phosphosphingolipids, and combinations thereof. The composition of
the lipid bilayer may be altered to mimic the surface of various
cell types, such as platelets, leukocytes, erythrocytes,
endothelial cells or smooth muscle cells. This alteration may be
accomplished by mixing different ratios of the phospholipid
structures, such as phosphatidylserine and phosphatidylcholine and
phosphatidylethanolamine. Other combinations of two or more of the
phospholipid structures may also be mixed.
[0062] The lipid coated particles in addition to being coated with
lipid bilayers, also are comprised of one or more functional
molecules which are contained within the lipid bilayers. The
functional molecule may be any transmembrane protein. Such
transmembrane protein may include transmembrane proteins that are
known to regulate blood coagulation including but not limited to
tissue factor, thromobomodulin, endothelial cell protein C receptor
and combinations thereof. Thrombin is a known serine protease that
creates a biopolymer of fibrin by cleaving fibrinopeptide from the
plasma protein of fibrinogen. Fibrin forms a highly entangled
hydrogel that provides the scaffold onto which a blood clot grows.
Generally, high concentrations of thrombin are created during the
extrinsic or tissue factor pathway of the coagulation cascade,
hence why tissue factor is known as a coagulation cascade inducing
agent. Other transmembrane proteins may include proteins that are
known to be receptors for cell to cell adhesion including but not
limited to glycoprotein IIb/IIIa, glycoprotein VI, glycoprotein
Ib/IX/V, P-selectin, glycoprotein IV, CD9, platelet endothelial
cell adhesion molecule (PECAM-1), Ras-related protein 1b (rap1b),
c-type lectin-like receptor 2 (CLEC-2), intracellular adhesion
molecule 1 (ICAM-1), intracellular adhesion molecule 2 (ICAM-2) and
combinations thereof. The lipid coated particles may contain
various concentrations of one or more of the functional
molecules.
[0063] Once the lipid coated particles are synthesized and coated
as discussed above, they are immobilized to the substrate surface.
Methods to immobilize silica beads, such as the lipid coated
particles of the present invention, are known to those of skill in
the art and include but are not limited to covalent bonding,
electrostatic interactions and hydrogen bonding. The immobilization
method provides an adequate attractive force between the lipid
coated particles and the substrate surface to withstand shear
stresses during the assay.
[0064] In a further aspect, the lipid coated particles are
immobilized and patterned to the substrate surface. The patterning
may be achieved by a subtractive technique such as microblotting or
a lift-off process such as microstenciling. In regards to
microblotting, a blotting device may be loaded onto a mechanical
press and lowered onto the substrate surface comprising a plurality
of the lipid coated particles until the blotting device is in full
contact with the substrate surface comprising the immobilized lipid
coated particles. Once the blotting device is removed, defined
sections and/or areas (i.e. the resulting pattern) of the
immobilized lipid coated particles remain on the substrate surface
(see FIG. 4). The defined sections and/or areas may be of any
geometric shape including but not limited to circular shape, square
shape, oval shape, rectangular shape or unshaped and combinations
thereof. A circular shape may be referred to as a "spot". Each
defined section and/or area is comprised of a plurality of the
lipid coated particles. In each defined section and/or area, there
is at least more than one lipid coated particle. Preferably, there
are hundreds to thousands of lipid coated particles present in each
defined section and/or area. The number of individual lipid coated
particles within each defined section and/or area can vary and can
be in a range from 1 to about 1,000,000 individual lipid coated
particles. It is possible for the defined section and/or area to be
comprised of a single lipid coated particle. As used herein a
plurality of lipid coated particles refers to at least more than
one lipid coated particle. The diameter of each defined section
and/or area can vary as determined by the patterning method. One or
more defined sections and/or areas may result depending on the
patterning method. At least one defined section and/or area
comprising a plurality of the lipid coated particles results
depending on the pattern. The number of defined sections and/or
areas found on the substrate surface can vary as determined by the
patterning method, the composition of the lipid bilayer and the
measurement being taken. The number of defined sections and/or
areas can be from about 1 to 1000 defined sections and/or areas.
The spacing between two or more defined sections and/or areas of
the plurality of the lipid coated particles on the substrate
surface may vary depending on the pattern. As an example, the space
between two of the defined sections and/or areas may be about 1
.mu.m apart to about 1 mm apart. Each lipid coated particle within
the defined section and/or area is smaller than the diameter of the
microfluidic channel itself.
[0065] The width of the substrate surface comprising the
immobilized lipid coated particles may vary depending upon the size
of the microfluidic channel. In some embodiments the width may be
about 10 micrometers (.mu.m), about 20 .mu.m, about 30 .mu.m, about
40 .mu.m, about 50 .mu.m, about 100 .mu.m, about 150 .mu.m, about
200 .mu.m, about 250 .mu.m, about 300 .mu.m, about 350 .mu.m, about
400 .mu.m, about 450 .mu.m, about 500 .mu.m, about 600 .mu.m, about
700 .mu.m, about 800 .mu.m, about 900 .mu.m or about 1000 .mu.m.
The actual width of the substrate surface can have a greater or
lesser size without departing from the scope of the present
invention.
[0066] After the plurality of the lipid coated particles are
immobilized and patterned, the particles are integrated into at
least one microfluidic channel by aligning either manually, such as
by using alignment marks made on the substrate surface, or having
posts on the substrate that align with holes that can be found on
the microfluidic device or by other methods known to those of skill
in the art. Once aligned, the substrate and the device are bonded.
Such bonding method includes but is not limited to vacuum assisted
bonding.
[0067] In a further embodiment, the microfluidic device has the
capability of hydrodynamic focusing. Additional buffer, such as
HEPES buffered saline (HBS) may be infused through additional side
channels of the device to provide focusing of the sample, such as a
sample of a blood product, which is perfused through a middle
channel. As the buffer solution is perfused in from the side it
forces the sample to flow in the middle part of the channel. This
design prevents edge effects, most notably, the accumulation of the
deposition in the corners of the channel of the sample and/or
sample product (see FIG. 5B).
[0068] Once the device is produced, in accordance with at least
some embodiments of the present invention, the microfluidic device
may be hygienically sealed in a sterile environment (e.g. hermetic
plastic package) such that the microfluidic device can be
distributed as a clot testing kit to medical personnel and other
interested parties. In addition, the substrate surface comprising
the lipid coated particles can be kept in an aqueous environment.
Accordingly, prior to hermetically sealing the microfluidic device
in a sterile environment, an aqueous solution may be injected into
the hermetic packaging prior to the final sealing. Alternatively,
the substrate surface may be kept in a dry environment.
[0069] Another embodiment of the invention is a microfluidic
channel through which a blood product is capable of flowing. The
channel may comprise at least one substrate provided as part of at
least a portion of one surface of the channel. The substrate
surface comprises a plurality of the lipid coated particles of the
invention. These particles comprise at least one functional
molecule that is embedded in the lipid coating the particle. The
functional molecule is as defined herein. As used herein the term
blood product refers to whole blood, plasma, platelet rich plasma
(defined as having no red or white blood cells, while containing
plasma and platelets), and platelet poor plasma (defined as having
no platelets). The blood product of the present invention may be
from an individual, such as whole blood or plasma taken from an
individual or the blood product may be synthetically produced by
methods known in the art.
[0070] In accordance with at least some embodiments of the present
invention, once the microfluidic device has been prepared, one or
more blood component samples can be passed or perfused through the
microfluidic channels of the device under flow conditions to
evaluate the blood product for coagulation products associated with
at least one of the functional molecules of the coated lipid
particles. As noted previously, the functional molecules may be one
or more transmembrane proteins including but not limited to tissue
factor, thromobomodulin, endothelial cell protein C receptor,
glycoprotein IIb/IIIa, glycoprotein VI, glycoprotein Ib/IX/V,
P-selectin, glycoprotein IV, CD9, platelet endothelial cell
adhesion molecule (PECAM-1), Ras-related protein 1b (rap1b), c-type
lectin-like receptor 2 (CLEC-2), intracellular adhesion molecule 1
(ICAM-1), intracellular adhesion molecule 2 (ICAM-2) and
combinations thereof. In a preferred embodiment, the functional
molecule initiates coagulation, such a tissue factor. In another
embodiment, the functional molecule inhibits coagulation, such as
thrombomodulin.
[0071] The flow conditions and rate can be defined by the user of
the device. Preferably, the flow conditions simulate hemodynamic
conditions of an individual for which the blood product is obtained
from. The flow conditions may include a wall shear rate of in the
range of about 50 sec.sup.-1 to about 2600 sec.sup.-1, which
corresponds to the normal range shear rates in the human
vasculature. The flow conditions may also include wall shear rates
in a range from zero up to about 500,000 sec.sup.-1 for testing
conditions in which the pathological flow conditions exist.
Pathological flow conditions may occur if the flow has been
retarded, as in the case of individuals diagnosed deep vein
thrombosis, or if blood is forced to pass through a partially
occluded or stenosed vessel, as in the case of individuals
diagnosed with atherosclerosis.
[0072] As the one or more blood component samples are perfused
through the microfluidic channel and over the substrate surface
comprising a plurality of the lipid coated particles comprising one
or more functional molecules of the present invention, and the
sample is perfused at a user defined flow rate, coagulation
products can be detected as one or more coagulation products
associates with the coated lipid particles comprising the
functional molecule. Coagulation products include but are not
limited to thrombin, fibrin, thrombin-antithrombin complex,
fibrinopeptide A, fibrinopeptide B, D-dimer, prothrombin fragment
1+2, activated factor X, activated factor V, activated factor
VIIIa, activated factor IXa, activated factor XIa, activated factor
XIIa, activated protein C, activated protein S, and mixtures
thereof. The coagulation products may be detected and/or measured
by various methods including but not limited to brightfield
microscopy, darkfield microscopy, fluorescence microscopy,
multi-photon excitation, second harmonic generation, third harmonic
generation, atomic force microscopy, scanning electron microscopy,
and absorbance. For example, thrombin and/or fibrin amounts can be
detected and/or measured by using a fluorescent substrate such as
boc-VPR-AMC for thrombin and for fibrin by adding exogenous
fibrinogen with a fluorescent label. Thrombin generation could also
be indirectly detected and/or measured by measuring collecting the
effluent at the outlet of the device and measuring the
concentration of thrombin-antithrombin complex (TAT) or the release
of fibrinopeptides (peptides that are released off of fibrinogen
following cleavage by thrombin). Fibrin could also be detected
and/or measured using a fibrin specific antibody or by other
microscopy techniques such as differential contrast, phase contrast
and Hoffman modulation. Any of the other transmembrane protein
could be detected and/or measured in similar ways to those
described above. An individual's result may be compared to results
that have been obtained under identical conditions using plasma
pooled from normal donors (i.e. normal pooled plasma (NPP) which is
plasma that has been pooled from a number of normal donors (donors
without known blood coagulation conditions)). The NPP can be plasma
standards that are available commercially. Additionally, an
individual's results can be compared to results that have been
obtain under identical conditions using factor deficient plasmas
such as factor II (prothrombin), factor VIII, factor IX, factor and
factor XI deficient plasmas.
[0073] Another embodiment of the present invention relates to a
method for determining an individual's coagulation potential,
comprising perfusing an individual's blood product (such as plasma)
over the substrate surface comprising the coated lipid particles of
the present invention and one or more functional molecules, wherein
one or more coagulation products associate with the lipid
particles; and detecting one or more coagulation products
associated with the lipid particles. In one aspect, the lipid
particles comprise functional molecules. The functional molecules
can initiate coagulation or can inhibit coagulation. In a preferred
aspect, the functional molecules are tissue factor and
thrombomodulin. The flow rate of the blood product can be a rate
which mimics hemodynamic conditions of the individual. The normal
range of shear rates in the human vasculature is about 50 to 2600
sec.sup.-1.
[0074] Another embodiment of the invention relates to a method for
determining an individual's response to an agent comprising
perfusing the individual's blood product (such as plasma) over the
substrate surface comprising the coated lipid particles of the
present invention and one or more functional molecules wherein one
or more coagulation products associate with the lipid particles;
and detecting one or more coagulation products associated with the
lipid particles. In one aspect, the agent is an anticoagulant agent
or coagulating agent.
[0075] In still another embodiment, the invention relates to a
method to diagnose and/or monitor an individual for bleeding
comprising perfusing the individual's blood product (such as
plasma), over the substrate surface comprising the coated lipid
particles of the present invention and one or more functional
molecules wherein one or more coagulation products associate with
the lipid particles; and detecting one or more coagulation products
associated with the lipid particles.
[0076] In yet another embodiment, the invention relates to a method
to determine the dose of one or more anticoagulation agents or
coagulation agents to be administered to an individual comprising
perfusing the individual's blood product (such as plasma), over the
substrate surface comprising the coated lipid particles of the
present invention and one or more functional molecules, wherein one
or more coagulation products associate with the lipid particles;
and detecting one or more coagulation products associated with the
lipid particles.
[0077] Yet another embodiment relates to a method to screen for
anticoagulation agents or coagulation agents comprising perfusing
the individual's blood product (such as plasma), over the substrate
surface comprising the coated lipid particles of the present
invention and one or more functional molecules, wherein one or more
coagulation products associate with the lipid particles; and
detecting one or more coagulation products associated with the
lipid particles.
[0078] Still another embodiment relates to a method to screen for
coagulation agents comprising perfusing the individual's blood
product (such as plasma), over the substrate surface comprising the
coated lipid particles of the present invention and one or more
functional molecules, wherein one or more coagulation products
associate with the lipid particles; and detecting one or more
coagulation products associated with the lipid particles.
[0079] The individual in the invention can include any mammal,
including human and non-human mammals.
[0080] The following experimental results are provided for purposes
of illustration and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
[0081] This example demonstrates the fabrication of lipid coated
particles of the present invention to detect fibrin formation.
[0082] In order to initiate fibrin formation in the plasma-based
model for coagulation using the microfluidic device of the present
invention, silica microbeads are used to simulate the catalytic
activity of platelets. Once the silica beads are made hydrophilic,
their surfaces are coated with lipid bilayers, which contain varied
concentrations of functional molecules such as tissue factor (TF)
and thrombomodulin (TM) that initiate and inhibit coagulation. The
composition of the lipid bilayer can be manipulated to mimic the
surface of various cell types. This is accomplished by mixing
different ratios of phospholipids such as phosphatidylserine (PS),
phosphatidylcholine (PS), and phosphatidylethanolamine (PE). A
description of the fabrication of silica beads and coating them
with phospholipids is found below:
[0083] 1. Weigh or measure out 2.1 mL of deionized water, 15.4 mL
of anhydrous ethanol, and 6.5 mL of ammonium hydroxide in a plastic
container.
[0084] 2. Stir for 10 minutes with a magnetic stirrer, and then add
1.5 mL tetraethyl orthosilicate (TEOS) dropwise.
[0085] 3. Stir the solution for 2 hours at the room
temperature.
[0086] 4. After 2 hours, centrifuge the precipitated silica beads,
and wash in ethanol 4 times.
[0087] 5. Resuspend the bead solution of 400 mg in 1 ml of
deionized water.
[0088] 6. Dilute the silica particles to a concentration of 5 mg/ml
in DI water
[0089] 7. Add hydrogen peroxide and HCl solution to the mixture to
final concentrations of 4% vol and 0.4 M, respectively.
[0090] 8. Stir the solution at 85.degree. C. for 10 minutes, then
cool the mixture to room temperature.
[0091] 9. Wash the solution of beads by centrifuging the beads at
2000 RPM for 5 minutes and re-suspending in buffered saline to wash
the beads. Repeat 5 times
[0092] 10. Pipette 200-500 uL of the silica bead in a
microcentrifuge tube and centrifuge the silica beads at 2000 RPM
for 3 minutes.
[0093] 11. Pipette out the supernatant buffer without drying out
the silica beads and add 100 uL of liposome .Vortex the mixture
until the silica beads are re-suspended in the lipid solution
[0094] 12. Incubate the lipid-bead mixture for 24 hours at
4.degree. C. to allow the lipid bilayers to rupture on the silica
bead surfaces
[0095] 13. Centrifuge the silica beads at 2000 RPM for 5 minutes,
then pipette off the supernatant solution. Do not dry out the
beads.
[0096] 14. Rinse the beads thoroughly with a 1.times. HBS
buffer
[0097] 15. Re-suspend the lipid coated silica beads to
concentration of 1 mg/mL and store at 4.degree. C. until ready to
use.
Example 2
[0098] This example demonstrates an example of the patterning of
lipid coated particles of the present invention.
[0099] Once the lipid coated particles are made as described in
Example 1, they need to be immobilized and patterned onto a
substrate. The immobilization of the particles can rely on methods
such as covalent bonds (e.g. streptavidin-biotin), electrostatic
interactions, or hydrogen bonding. The patterning may be achieved
either by a subtractive technique such as microblotting or a
lift-off process such as a microstencil. A microblotting technique
is described below:
[0100] 1. Obtain a 100 .mu.g/mL solution of silica beads
functionalized with lipid bilayer as explained in Example 1.
[0101] 2. Incubate a 100 .mu.g/mL solution of the lipid coated
particles in HBS buffer onto and APTES functionalized glass slide
for 1 hour at room temperature. Wash substrates three times in HBS
buffer to remove unbound beads.
[0102] 3. Place glass slide into a 6-inch petri dish with 100 mL of
deionized 18 M.OMEGA. water.
[0103] 4. Load blotting devices onto a customized mechanical press
and lower onto the slide until the device is in full contact with
the slide. The device is contacted on the slide for 3 minutes.
[0104] 5. Thoroughly rinse the patterned slide in PBS or HBS for 5
min. Store the patterned slide in buffer until use in the flow
assay.
Example 3
[0105] This example demonstrates an example of the integration of
the lipid coated particles into microfluidic channels of the
present invention.
[0106] After immobilizing and/or patterning the lipid coated
particles, the microfluidic device of the present invention is
aligned and bonded to the substrate. Because the lipid bilayers on
the lipid coated particles should remain hydrated, this step should
be done while the substrate is immersed or covered in buffer
solution. The device may be aligned either manually or using
alignment marks on the substrate. Once in contact, the device may
be bonded to the substrate by vacuum assisted bonding.
Example 4
[0107] The example demonstrates how to measure coagulation under
flow conditions.
[0108] The procedure for the assay itself using the microfluidic
device of the present invention comprises perfusing plasma through
the microfluidic channels and over the plurality of the lipid
coated particles at a user defined flow rate. Coagulation can be
monitored by a variety of optical methods: (1) fibrin generation by
brightfield microscopy (phase contrast, Hoffman modulation
contrast, differential interference contrast), (2) fibrin
generation by fluorescence microscopy (epifluorescence or
confocal), which requires that either some fibrinogen is labeled
with a fluorophore or the inclusion of a fibrinogen or fibrin
fluorescence labeled antibody into the plasma, (3) fibrin
generation by nonlinear optical methods (two photon excitation,
second harmonic generation, third harmonic generation), (4)
thrombin generation by monitoring the fluorescence or absorbance of
a thrombin substrate (e.g. boc-VPR-AMC), (5) thrombin generation by
measuring thrombin-antithrombin (TAT) complex, (6) fibrin
deposition by measurement of the D-dimer following digestion by
plasmin, (7) fibrin generation by measurement of fibrinopeptides A
and B.
Example 5
[0109] This example demonstrates the use of the microfluidic device
of the present invention to measure transient fibrin deposition and
thrombin generation. The Inventors demonstrate that for a given
Tissue Factor (TF) concentration, flow profoundly influenced fibrin
deposition, fiber diameter, fiber orientation and local thrombin
concentration. The microfluidic device can also be used to
investigate the effects of different factor deficiencies on the
dynamics of fibrin production and thrombin generation.
[0110] These findings suggest that for significant fibrin formation
to occur, coagulation reactants and products must be protected from
transport away from a clot either by a reduction in shear rate
(i.e. occlusion or within secondary flows downstream of stenosis)
or within the interstitial spaces of a platelet aggregate.
Preparation of TF Lipid Coated Particles and Assay Conditions Using
the Microfluidic Device
[0111] TF bearing lipid coated particles were synthesized by
coating 1 .mu.m silica beads with 0.5, 5, or 50 molecules
TF/.mu.m.sup.2 in a lipid bilayer (PS:PC 30:70). The lipid coated
particles were patterned as 100 .mu.m spots on a glass substrate
using a microblotting technique. (FIG. 5A). Normal pooled plasma
(NPP), factor deficient plasmas FII, FVIII, FX and FXI were
perfused over the TF spots at wall shear rates of 50, 100, 250, 500
and 1000 sec.sup.-1 for 10 min. (FIG. 5B). Fibrin formation and
thrombin generation were measured in real-time by epifluorescence
using labeled fibrinogen and the thrombin substrate boc-VPR-AMC,
respectively. (FIGS. 5C-5H). Following the assay, fibrin gels were
either (i) fixed and further imaged by confocal or scanning
electron microscopy or (ii) digested by plasmin to measure the rate
of lysis and to quantify the amount of fibrin deposited using a
D-dimer ELISA.
Shear Rate Dependent Fibrin Deposition and Thrombin Generation
[0112] NPP was perfused over surface TF concentration of 0.5, 5 and
50 molecules/.mu.m.sup.2 at wall shear rates of 50, 100, 250, 500
and 1000 sec.sup.-1. For each experiment, fibrin deposition and
thrombin generation were monitored in real-time. Fibrin deposition
and thrombin generation decreased with increasing wall shear rate
and decreasing TF concentration. (FIGS. 6A-6F). Three metrics were
used to quantify the dynamics of fibrin formation and thrombin
generation; (i) the lag time to fibrin fiber and thrombin
generation, (ii) the maximum fibrin density and thrombin
fluorescence, and (iii) the rate of fibrin and thrombin
generation.
[0113] At a given TF concentration either of 50, 5 and 0.5
molecules/.mu.m.sup.2 there was a decrease in the lag time and
fibrin generation rate with increasing wall shear rate. As a result
of the decrease in the rate of fibrin production, the maximum
fibrin deposited also decreased with an increase in shear rate. The
thrombin generation followed the same trend as the fibrin
deposition.
[0114] At the highest shear rates and lowest tissue factor
concentration, no fibrin fibers were observed in the time period of
the experiments therefore there was a subthreshold amount thrombin
produced to induce fibrin formation.
[0115] The cumulative fibrin deposited on all spots over the 10
minute flow assay was measured by D-dimer concentration following
plasmin digestion. The threshold nature of fibrin formation is
evident at all three TF concentrations. For 5 and 50
molecules/.mu.m.sup.2, fibrin formation was supported at wall shear
rates less than or equal to 250 s.sup.-1. For 0.5
molecules/.mu.m.sup.2, fibrin formation was supported at wall shear
rates of less than or equal to 100 s.sup.-1.
[0116] After plasma perfusion, the fibrin fibers produced on the TF
spots were digested with plasmin over time and monitored by the
decrease in fluorescence intensity over time. The analysis of the
rate of digestion shows that the fibers produced at the lowest
shear rate digested the fastest (46.1 RFU/s) (i.e. relative
fluorescence units/second), while the fibers produced at the
highest shear rate digested the slowest (17.3 RFU/s). Overall, the
D-dimer analysis shows a decrease in the quantity of D-dimer
fragments as shear rate is increased and tissue factor
concentration is decreased (FIG. 7). The D-Dimer results also
confirm that at the lowest TF concentration and the highest shear
rates there was little or no fibrin observed to be produced because
the signals were the same as that of the ELISA background.
Cross-Talk Between Spots
[0117] In each assay there were 142.+-.11 fibrin(ogen) lipid coated
particle spots with a spot-to-spot distance of 200 .mu.m. There was
an increase in accumulation of fibrin from upstream to downstream
spots at shear rates of 50 s.sup.-1 and 100 s.sup.-1. There were
also vertical spot to spot interactions at all shear rates where
fibrin was produced, depending on the distance from the leading
spot upstream. Fibrin monomers were being transported downstream
with flow. This trend was evident at all shear rates where fibrin
was produced.
Shear Rate Effects on Fibrin Morphology
[0118] The final gel-height of the fibers accumulated on the
individual spots also showed the same trend as the fibrin
deposition (FIG. 8A). The lowest shear rate had a height of 15.3
.mu.m, while the highest shear rate had a height of 2.1 .mu.m.
Fibrin fibers align in the direction of flow in a shear rate
dependent manner (FIGS. 8B-8F). At 50 s.sup.-1 and 100 s.sup.-1,
the fibers were isotropically oriented in a starburst pattern. With
increasing wall shear rates of 250 s.sup.-1 to 1000 s.sup.-1, the
fibers become more orientated with flow. Fibrin fiber diameter also
decreases with increasing wall shear rate (FIGS. 8G-8K). The lowest
shear rate had the largest diameters, with individual fibers
appearing to be composed of smaller fibers joint together to form
bigger ones. At the highest shear rate, the spots were scanned and
no discernable fibers could be found under the scanning conditions
used.
The Effect of FVIII, FIX, and FXI Deficiencies on Fibrin
Deposition
[0119] NPP and FII, FVIII, FIX, FX and FXI deficient plasma were
perfused in the microfluidic device at the conditions of low shear
rates and high TF concentrations. As controls, FII and FX deficient
plasmas showed no visible fibrin production at 50 s.sup.-1 or at
any other shear rates tested. At 50 molecules/.mu.m.sup.2, there
was a slightly prolonged lag time for FVIII, FIX and FXI deficient
plasma compared to NPP, however the final fibrin deposition was
similar (FIG. 9A). The difference between NPP and these deficient
plasmas at high TF concentrations was more evident in the thrombin
generation data (FIG. 9C). FVIII deficient plasma was
indistinguishable from NPP. FIX deficient plasma had a reduced rate
of thrombin generation. FXI deficient plasma had a prolonged lag
time compared to NPP. At low TF concentration (5
molecules/.mu.m.sup.2), there was reduced fibrin deposition and
thrombin generation for FVIII, FIX, and FXI deficient plasmas
compared to NPP. There was almost a complete absence of fibrin for
FVIII and FIX deficient plasma, while the fibrin deposition for the
FXI deficient plasmas was significantly reduced. The trends for
thrombin generation were similar to fibrin deposition at low TF
(FIG. 9B and 9D).
Materials and Methods and Data Analysis for Example 5
[0120] Materials: L-.alpha.-phosphatidylcholine (PC) and
L-.alpha.-phosphatidylserine (PS) were purchased from Avanti Polar
Lipids (Alabaster, Ala., USA). Texas red
1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) was
purchased from Invitrogen (Carlsbad, Calif. USA). Bio-Beads SM-2
were purchased from BioRad Laboratories (Hercules, Calif., USA).
Sodium deoxycholate was purchased from CalBiochem (Gibbstown, N.J.,
USA). Boc-Val-Pro-Arg-MCA
[t-Butyloxyl-carbonyl-L-Valyl-L-Prolyl-L-Arginine-4-Methyl-Coumaryl-7-Ami-
de], was purchased from Peptide Institute Inc, Osaka, Japan and a
10 mM stock solution was prepared according to manufacturer
instruction. Recombinant human tissue factor (TF), IMUBIND tissue
factor ELISA and D-Dimer ELISA were from America Diagnostica
(Stamford, Conn.), and were used according to the manufacturer's
instructions. Plasmin was purchased from Enzyme Research
Laboratories (South Bend, Ind.). Normal pooled plasma (NPP) and
factor XI, X, IX, VIII, VII and II deficient plasmas were purchased
from George King Biomedical (Overland Park, Kans.). Alexa Fluor 488
protein labeling kit (Invitrogen, Carlsbad, Calif., USA) was used
to label fibrinogen according to the manufacturers instruction.
Polydimethylsiloxane (PDMS) used for microfluidic devices (Dow
Corning, Sylgard 184) was purchased from Ellsworth Adhesives
(Germantown, Wis.). HEPES buffered saline (HBS, 20 mM HEPES, 150 mM
CaCl.sub.2, pH 7.4) was made in house. 3-[(2-Aminoethylamino)
propyl] trimethoxysilane (APTMS), tetraethyl orthosilicate (TEOS)
and all other chemical were purchased from Sigma Aldrich (St.
Louis, Mo.).
Preparation of Lapidated Tissue Factor
[0121] Recombinant human tissue factor was incorporated into
liposomes according to previously developed protocols (Smith and
Morrissey, Journal of Thrombosis and Haemostasis, 2:1155-1162).
Briefly, PC, PS and DHPE lipids were mixed at a 80:19.5:0.5 molar
ratio in chloroform and dried under vacuum for 1 h. The dried film
was resuspended in 1 mL of 20 mM sodium deooxycholate in HBS, and
allowed to hydrate for 1 h at room temperature. TF was then added
to the lipid mixture and incubated for 10 minutes (8700:1
lipid:TF). Detergent was removed from the lipid solution with 50 mg
of Biobeads under agitation for 90 min. Next, an additional 350 mg
of Biobeads were added to the same solution, and agitated for
another 90 minutes. Finally, the beads were allowed to settle and
the supernatant TF was collected. The concentration of the
lipidated TF was determined by ELISA to be 460 nM.
Preparation of Silica Beads
[0122] The Stober process was used to synthesize silica beads used
in this study. Tetraethylorthosilicate (TEOS) was added drop wise
to a mixture of water, ethanol and ammonium hydroxide, and the
solution was stirred for 2 h at room temperature. The resulting
solution was centrifuged at 2000 rpm for 5 minutes, washed in
ethanol and suspended in HBS buffer. Finally, transmission electron
microscopy (TEM) was used to characterize the size distribution of
these silica particles as ranging from 800 to 1000 nm.
Preparation of TF Lipid Coated Particles
[0123] To promote the formation of lipidated TF on the surface of
the 1 .mu.m silica beads, the beads were first made hydrophilic by
suspending them at a concentration of 5 mg/ml in 4% peroxide and
0.4 M HCl solution. Then, the suspension was heated to
80-90.degree. C. for 10 min, cooled to 25.degree. C., centrifuged
at 2000 rpm for 5 min, washed three times with deionized water and
finally re-suspended in FIBS buffer. To confirm that the surface
chemistry was successful, Fourier transform infrared spectroscopy
(FTIR) was used to confirm the presence of deposited surface
silanol groups (SiOH groups) on the silica particles. Next, the
desired concentration of beads was pipetted from the stirred stock
suspension, centrifuged, and the supernatant was replaced by the
desired lipidated TF concentration. The suspension was gently
vortexed for 30 min. and then allowed to sit undisturbed for 5 min.
Finally, the beads were finally centrifuged and washed three times
with HBS buffer to remove unbound lipids from the solution.
Microblotting TF Lipid Coated Particles
[0124] Clean glass slides were incubated in a 40 wt % APTMS in
ethanol solution for 45 minutes. The amine group on the APTMS
renders the surface positively charged. The negatively charged
TF-coated silica beads were incubated for 4 h on the positively
charged glass slides and then rinsed with HBS to remove excess
silica beads. Next, a PDMS microblot with 100 .mu.m holes spaced
200 .mu.m center-to-center was used selectively remove beads from
the surface. Electrostatic interactions between the beads and the
surface provides an adequate attractive force to withstand the
shear stresses during the flow assays.
Plasma Flow Assay
[0125] A polydimethylsiloxane (PDMS) microfluidic hydrodynamic
focusing device (w=1000 .mu.m, g=100 .mu.m) was vacuum-sealed to
the glass slide containing the patterned lipid coated particles.
Within each channel there was 5.times.22 array of 100 .mu.m bead
spots. HBS was infused through the two side channels to provide the
focusing of plasma, which was perfused through the middle channel
(FIG. 5B). The total flow rate (HBS and plasma) through the main
channel was set to achieve the desired wall shear rate using the
expression: .gamma.=6.times.Q/H.sup.2 W where .gamma. is the shear
rate, Q is the volumetric flow rate, W is the width and H is the
height. As the buffer solution was perfused in from the side
(mid-shaded regions adjacent to the lightest shaded regions
indicated as the "Buffer Area") it forced the plasma (lightest
shaded region indicated as the "Blood Product Area") to flow in the
middle part of the channel. This design prevents edge effects,
notably the preferential accumulation of fibrin deposition in the
corners of the channel.
[0126] Citrated normal pooled plasma (NPP), FII, FX, FVIII, FIX and
FXI deficient plasmas were defrosted at 37.degree. C. immediately
before perfusion through the microfluidic flow device. The citrated
plasma (400 .mu.L) was re-calcified by adding 20 .mu.L of a
solution of CaCl.sub.2 (500 mM) and withdrawn with a syringe pump
at wall shear rates of 50, 100, 250, 500 and 1000 s.sup.-1. To
monitor fibrin formation, Alexa 488 labeled fibrinogen was added to
the plasma at 17.5 .mu.g/mL. Thrombin generation was monitored
through the cleavage of a fluorogenic substrate, Boc VPR-AMC.
Data Acquisition and Image Analysis
[0127] Fibrin deposition and thrombin generation were measured for
10 minutes, and images were recorded every 50 s by epifluorescence
microscopy using a 40.times. objective. The data was taken starting
from the leading spot upstream where the plasma first encountered
the lipidated-TF lipid coated particle bead spot pattern. Image J
software was used to determine the integrated fluorescence of the
fibrin or thrombin generated on single bead spots.
Plasmin Digestion and D-Dimer Level Measurements of Fibrin
Deposits
[0128] After the plasma perfusion, the heparin wash buffer was used
to rinse the channel for 5 min. at the same shear rate as the
experiment. Next, a 250 .mu.L plasmin solution (0.48 mg/ml diluted
in HBS containing 1 mM Tris/HCl, pH 7.4) was perfused through the
microfluidic channel at a flow rate of 5 .mu.L/min for 10 min, and
then flow was stopped for 30 min to allow for sufficient time for
fibrin digestion by the plasmin. Finally, the remaining plasmin
solution was perfused through the channel at the same shear rate.
The digested fibrin samples were collected and snap frozen at
-70.degree. C. until assayed for D-dimer.
Scanning Electron Microscopy
[0129] After plasma perfusion and the heparin wash buffer wash, the
glass slide with the fibrin deposit was immersed in a glass slide
holder containing 2.5% glutaraldehyde solution for 5 minutes, then
immersed in another glass slide holder containing de-ionized water
for an additional 5 minutes. The slide was then rinsed in graded
ethanol solutions (50%, 70%, 80%, 100% and 100%) for 5 min, and
dehydrated once in 50%, and twice in 100% hexamethylsilazane for 5
min. Next, a 10-20 nm layer of gold was sputtered on the dehydrated
fibrin deposits. Images were taken with JOEL 7000 field emission
SEM (Hitachi, Tokyo, Japan) at an accelerating voltage of 1.5kV and
a working distance of 6 mm. The diameters of 20-30 fibrin fibers
were measured with Image J software, averaged and reported with
standard deviations.
[0130] All of the documents cited herein are incorporated herein by
reference.
[0131] The foregoing description of the present invention has been
presented for purposes of illustration and description.
Furthermore, the description is not intended to limit the invention
to the form disclosed herein. Consequently, variations and
modifications commensurate with the above teachings, and the skill
or knowledge of the relevant art, are within the scope of the
present invention. The embodiments described hereinabove are
further intended to explain the best mode known for practicing the
invention and to enable others skilled in the art to utilize the
invention in such, or other, embodiments and with various
modifications required by the particular applications or uses of
the present invention. It is intended that the appended claims be
construed to include alternative embodiments to the extent
permitted by the prior art.
* * * * *