U.S. patent application number 15/053092 was filed with the patent office on 2016-09-01 for microfluidic system having monolithic nanoplasmonic structures.
This patent application is currently assigned to National Research Council of Canada. The applicant listed for this patent is National Research Council of Canada. Invention is credited to Lidija MALIC, Keith MORTON, Teodor VERES.
Application Number | 20160250634 15/053092 |
Document ID | / |
Family ID | 46829979 |
Filed Date | 2016-09-01 |
United States Patent
Application |
20160250634 |
Kind Code |
A1 |
MALIC; Lidija ; et
al. |
September 1, 2016 |
Microfluidic System Having Monolithic Nanoplasmonic Structures
Abstract
A microfluidic system, particularly suited as a cell culture
system, is provided having a single monolithic biocompatible
substrate with both a surface having an ordered array of nano-scale
elements required for plasmonic response monitoring and a network
of microchannels for precisely controlling cellular environment.
The system has the additional advantages of low-volume consumption,
rapid low-cost fabrication of molds with easily interchangeable
microfluidic channel layouts, amenability to mass production, and
in situ label-free real-time detection of cellular response,
viability, behavior and biomolecular binding using plasmonic
techniques. A ratio of greater than 0.2 between the cross-sectional
dimension and the spacing distance of the nano-scale elements is
useful for plasmonic response monitoring. A process for producing
such a system involves fabrication of a master mold containing the
nano-scale elements etched into a hard substrate, and the
micro-scale and meso-scale features, such as channels and chambers,
provided in a soft membrane bonded to the hard substrate. A stamp
may be created by setting a settable liquid polymer or metal placed
in the master mold and then the features of the intended device
transferred to a polymeric substrate using the stamp.
Inventors: |
MALIC; Lidija; (Montreal,
CA) ; MORTON; Keith; (Saint Bruno, CA) ;
VERES; Teodor; (Montreal, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Research Council of Canada |
Ottawa |
|
CA |
|
|
Assignee: |
National Research Council of
Canada
Ottawa
CA
|
Family ID: |
46829979 |
Appl. No.: |
15/053092 |
Filed: |
February 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14005347 |
Sep 16, 2013 |
9291567 |
|
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PCT/CA2012/000203 |
Mar 12, 2012 |
|
|
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15053092 |
|
|
|
|
61452868 |
Mar 15, 2011 |
|
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Current U.S.
Class: |
264/129 |
Current CPC
Class: |
G01N 21/65 20130101;
B01L 3/502707 20130101; B01L 2300/0887 20130101; B01L 2300/044
20130101; B01L 2300/12 20130101; Y10T 137/8593 20150401; B82Y 30/00
20130101; C12M 23/16 20130101; B01L 2300/0861 20130101; B01L
3/502715 20130101; B01L 2300/0896 20130101; B01L 2300/0822
20130101; G01N 21/554 20130101; B01L 2300/0654 20130101; B01L
2400/086 20130101; B01L 2200/12 20130101; G01N 21/658 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 21/552 20060101 G01N021/552; C12M 3/06 20060101
C12M003/06 |
Claims
1. A stamp for patterning a polymeric substrate, the stamp
comprising: a stamp body formed of a polymer or metal of sufficient
hardness to be able to pattern the polymeric substrate, the stamp
body having a surface bearing a pattern of micro-scale and/or
meso-scale reliefs for forming one or more channels and/or
microfluidic chambers, wherein: one or more of the micro-scale
and/or meso-scale reliefs has a surface with one or more nano-scale
relief patterns for forming one or more ordered arrays of
nano-scale elements in the polymeric substrate; the nano-scale
elements have cross-sectional dimensions in a range of from 10 nm
to 1000 nm; and the arrays have a spacing distance between the
elements where a cross-sectional dimension to spacing distance
ratio is greater than 0.2.
2. The stamp according to claim 1, wherein the polymer or metal of
sufficient hardness comprises a photocured thermoset polymer.
3. The stamp according to claim 1, wherein size, spacing, geometry
or any combination thereof of the nano-scale relief patterns have
standard deviations from their respective averages of no more than
3%.
4. The stamp according to claim 1, wherein the cross-sectional
dimension to spacing distance ratio is in a range of from 0.2 to
1.5.
5. The stamp according to claim 1, wherein the cross-sectional
dimension to spacing distance ratio is in a range of from 0.5 to
1.
6. The stamp according to claim 1, wherein the nano-scale relief
patterns are for forming one or more of nanopillars, nanoposts,
nanodots, nanorods, nanopyramids, nanocrescents, nanodisks,
nanodomes, nanoholes, nanogratings and nanogrooves in the polymeric
substrate.
7. The stamp according to claim 1 wherein: the nano-scale relief
patterns are patterned on microstructural reliefs to provide two
levels of topographical cues in the polymeric substrate; an aspect
ratio of individual nano-scale reliefs is in a range of from 10:1
to 1:10; or at least one nano-scale relief pattern is on a
micro-scale and/or meso-scale relief for forming a microfluidic
chamber.
8. A method for forming a stamp for producing a patterned polymeric
substrate, the method comprising: micro-patterning a polymeric film
to form a membrane comprising a first surface bearing a pattern of
micro-scale and/or meso-scale features for defining one or more
channels and/or chambers, one or more of the micro-scale and/or
meso-scale features comprising at least one through-hole;
patterning a hard substrate to provide one or more ordered
patterns; placing the membrane on the hard substrate with a surface
opposite the first surface against the hard substrate, the at least
one through-hole aligned to expose the one or more ordered
patterns, and applying pressure sufficient to seal lips of the
membrane surrounding the through-holes against the hard substrate;
and placing a settable liquid polymer or metal in the through-holes
and over the first surface and setting the settable liquid polymer
or metal to form a stamp, the stamp comprising micro- scale and/or
meso-scale reliefs for defining one or more channels and/or
microfluidic chambers and further comprising one or more nano-scale
relief patterns on the micro-scale and/or meso- scale reliefs that
complement the one or more ordered patterns.
9. The process according to claim 8, wherein size, spacing,
geometry or any combination thereof of the nano-scale elements in
the ordered patterns have standard deviations from their respective
averages of no more than 3%.
10. The process according to claim 8, wherein the cross-sectional
dimension to spacing distance ratio is in a range of from 0.2 to
1.5.
11. The process according to claim 8, wherein the cross-sectional
dimension to spacing distance ratio is in a range of from 0.5 to
1.
12. The process according to claim 8, wherein: the hard substrate
comprises a cyclo-olefin polymer; the nano-scale elements comprise
one or more of nanopillars, nanoposts, nanodots, nanorods,
nanopyramids, nanocrescents, nanodisks, nanodomes, nanoholes,
nanogratings and nanogrooves; the polymeric film comprises a
thermoplastic elastomer; the micro-scale and/or meso-scale features
further define one or more valves, conduits, inlets or outlets; the
nano-scale elements are patterned on microstructures to provide two
levels of topographical cues; or an aspect ratio of individual
nano-scale elements is in a range of from 10:1 to 1:10.
13. The process according to claim 8, wherein at least one
additional membrane comprising a pattern of micro-scale and/or
meso-scale features for defining one or more channels and/or
chambers is stacked on the membrane placed on the hard
substrate.
14. The process according to claim 8, wherein the stamp is formed
from a settable polymer, the settable polymer comprises a
photocurable polymer and the photocurable polymer is cured by
exposing the photocurable polymer to ultraviolet light.
15. The process according to claim 8, wherein at least one ordered
array of nano-scale elements is in a microfluidic chamber.
16. A utilization of the stamp of claim 1, for forming a
nanoplasmonic microfluidic device, the utilization comprising:
stamping a polymeric substrate with the stamp to form in the
polymeric substrate, the one or more channels and/or microfluidic
chambers in the polymeric substrate, and one or more surfaces
having one or more nano-scale elements determined by the one or
more ordered patterns etched onto the hard substrate.
17. The utilization of claim 16 further comprising treating the
nano-scale elements of the ordered array to enhance or alter its
properties.
18. The utilization of claim 17 wherein treating the nanoscale
elements comprises metallization of the nano-scale elements.
19. The utilization of claim 18 wherein metallization of the
nano-scale elements are metallized with one or more of: silver,
gold, copper, platinum and palladium by evaporation, sputtering or
plating.
20. The utilization of claim 17 wherein treating the nanoscale
elements comprises chemical surface modification of the nano-scale
elements.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/005,347 filed Sep. 16, 2013, which is a
national stage entry of International Patent Application
PCT/CA2012/000203 filed Mar. 12, 2012 and claims the benefit of
United States Provisional Patent Application No. 61/452,868 filed
Mar. 15, 2011, the entire contents of which are hereby incorporated
by reference.
FIELD OF THE INVENTION
[0002] The present invention is related to microfluidic systems and
devices having nanoplasmonic features, particularly for use in cell
culture applications, and to processes of producing such systems
and devices.
BACKGROUND OF THE INVENTION
[0003] Analysis of molecular binding and cell behavior are
important for disease diagnostics, biomedical research, and drug
discovery. The vast majority of array-based studies of bioaffinity
interactions employ fluorescently labeled biomolecules or
enzyme-linked colorimetric assays. However, there is a need for
methods that detect bioaffinity interactions without molecular
labels, especially for biomolecular and cellular interactions,
where labeling is problematic and can interfere with their
biological properties. The development of simple and specific
biosensors to detect biomarkers and measure cellular response has
far-reaching implications in their timely detection which is of
great concern to human health and safety.
[0004] The advances in genomics and proteomics have unveiled an
exhaustive catalogue of biomarkers that can potentially be used as
diagnostic and prognostic indicators of genetic and infectious
diseases. The antibody and nucleic acid fluorescence-based
detection approaches currently consist of complex, multi-step, time
consuming, and labor intensive assay formats and target analyte
analysis to ensure the specificity of detection. Additionally,
these methods are not suitable for the rapid pathogen or cancer
detection as they require extensive blood culture of the pathogen
or diseased tissue in the central laboratory prior to the detection
of antibodies.
[0005] The analysis of bio-molecular interactions is also a key
part of the drug discovery process which involves determining the
binding affinity of the drug to the target protein of interest.
Even though developments in the field of high-throughput screening
(HTS) and computational chemistry greatly accelerated and
facilitated the drug finding process, there are significant
limitations to overcome. An example is the fluorescence-based HTS
assay, which may generate false positive (e.g. binding to the
reporter enzyme or direct hydrophobic interaction of the label with
the target) or false negative results (e.g. occluding of the
binding site). The application of novel and efficient label-free
technologies is of high importance to the drug discovery process,
since they will lower development costs and decrease the time to
market.
[0006] For drug discovery as well as in the biomedical research,
the study of the effect of the specific cues (e.g. chemical,
topographical, flow, etc.) on cell attachment and motility, cell
viability, cell proliferation and cell cycle is of paramount
importance. Inducing and subsequent measurement of a specific
cellular response requires providing the cells with the appropriate
cues, to control the conditions in the cell microenvironment, and
to monitor cellular responses on multiple hierarchical levels
within a large number of parallel experiments. Currently employed
assays that rely on cell culture in Petri dishes and subsequent
fluorescence-based live-cell imaging and biomolecule detection are
slow, cumbersome and cannot meet these requirements. Multi-well
plate assays can increase the throughput through automatic imaging
afforded by high content cell screening (HCCS). However, an
important consideration for the multi-well assays is ensuring
uniform patterning or treatment of each well which is often
precluded by variations in the volume of liquid dispensed into each
well. The resulting variability in the concentration of applied
reagents hinders fair and quantitative comparisons and limits the
ability of HCCS to resolve small differences in cell signaling
responses. This issue is exacerbated in more complex protocols,
such as sequential exposure of cells to different media, because of
errors that accumulate when changing media. Moreover repeated media
aspirations might unintentionally remove cells from the wells.
Because these assays are also difficult to miniaturize, HCCS
experiments may consume large quantities of expensive or valuable
cells and reagents. Finally HCCS still relies on fluorescent tags
which may trigger unwanted steric hindrance effects.
[0007] Consequently, the research into the effect of cues (single
or multiple) on cellular response to date has been limited by the
lack of robust and reproducible methods for homogeneous material
production, precise control of the cell culture conditions and in
situ real-time label-free monitoring of cellular response, cell
behavior, cell viability or biomolecular binding interactions.
Specifically, the material production methods have lacked the
control required to reproducibly fabricate homogeneous surfaces
that will allow investigations into specific interactions between
cells and isolated variables i.e. a precisely defined nanoscale
patterns in a defined space with control over the induced change in
topography and associated changes to surface energy. The commonly
employed well-based cell culture methods are costly and suffer
errors in liquid dispensing, both manually and robotically, thus
precluding uniform handling of each well which in turn limits how
finely signaling responses may be resolved. Finally, while the use
of fluorescent imaging techniques for cell analysis can provide
information not easily attainable by other methods, they are
usually confounded by the need to over-express the signaling
protein of interest and by possible effects of the fluorescent
marker on the protein's function. Therefore due to the
phenomenological nature of current studies, the responses achieved
have been heterogeneous at both single cell and cell population
levels (Balasundaram 2007; Barbucci 2003; Blummel 2007; Curtis
2006; Dalby 2007a; Ernsting 2007; Kimura 2007; Salber 2007).
[0008] Fluorescence and chemiluminescent detection are the most
common methods employed for biomolecule recognition. Both schemes
require the use of a labeled recognition element which binds to a
molecule of interest thus producing a selective signal upon binding
(Marquette 2006). Currently, the detection and quantification of
genomic and proteomic biomarkers from serum or other physiological
samples rely on solid-phase detection, where strong amplification
chemistry is often needed to produce a readout. In the case of DNA
markers, the state of the art relies on polymerase chain reaction
(PCR), while for the protein markers enzyme-linked immunosorbent
assay (ELISA) prevails. Many attempts at miniaturizing bench-top
systems using microfluidics in order to increase the detection
limits and reduce incubation times, reagent consumption and sample
size have been reported with impressive results (Zhang 2009a; Zhang
2009b; Lim 2007; Lee 2006; Malic 2007). However, despite a growing
focus from the microfluidic research community, both PCR and ELISA
rely on fluorescence labels, which increase the complexity and cost
of the assay. In addition to the requirement for a labeled
recognition element, these techniques typically require complex
optical systems which typically consist of a large microscope or a
microplate reader. As a result, the field of microfluidics has yet
to produce many commercial devices for disease diagnostics (Myers
2008). There is a need for coupling and integrating microfluidics
with direct label-free detection methods that base themselves on
physical characteristics of biologic phenomena and have the
potential to reduce reagent costs and test complexity (Weigl
2008).
[0009] The development of minimally invasive techniques to induce a
specific cellular response is focused on controlling the direct
contact and interaction between a given cell type and a well
defined material. One way of controlling cell adhesion and
subsequent morphology is by nanotopography. Research has shown that
cells can detect and respond to an array of topographies and can be
affected by the level of order of an induced topography, with clear
effects on cell functionality (Dalby 2009; Dalby 2008; Dalby 2007b;
Dalby 2007c; Dalby 2007d; Hochbaum 2010). Similarly, bacteria also
respond to topographical (spatial and mechanical) cues and
spontaneous bacteria patterning on a periodic nanostructure array
has recently been shown (Hochbaum 2010).
[0010] Another method employs chemically modified surface to induce
cellular response (Cavalcanti-Adam 2008). For both strategies to
prove successful the material must be homogenous, robust and
fabricated or functionalized in a reproducible manner. To date,
several methods have been used for this purpose, including
electron-beam lithography, nanoimprint lithography and dip-pen
nanolithography (Cavalcanti-Adam 2007; Curran 2010). However, the
assays in these studies were performed using traditional
cell-culture methods and analyzed using live fluorescence
microscopy with inherent drawbacks of these techniques which may
have resulted in misleading interpretation of results due to error
in liquid handling, perturbation caused by fluorescent markers and
low throughput in which only a few cells were imaged for each
experimental run.
[0011] Flow cytometry (FC) and laser scanning cytometry (LSC) are
the most widely used techniques for cell analysis with well
characterized distributions of cellular behaviour. Both techniques
use fluorescent dyes to label biomolecules of interest within the
cell in order to reveal the information about the quantity of
biomolecules within the cell. Flow cytometry involves the
hydrodynamic isolation of individual cells thus affording high
throughput serial analysis. However, FC is limited to
characterizing fluorescent signals (GFP-fusion proteins,
immunofluorescence, and fluorogenic substrates to intracellular
enzymes) (Fayet 1991; Nolan 1998; Krutzik 2006), which can lead to
steric hindrance and is incapable of important time-dependent
measurements of the cell population. Conversely, laser scanning
cytometry (LSC) relies on the use of a scanning laser to excite the
dyes on surface immobilized cells (Griffin 2003; Bedner 1993)
thereby allowing kinetic measurement of time-dependent information
in individual cells. However, only a limited region of a plate can
be scanned thus limiting the throughput of the technique.
Additionally, the introduction of reagents is performed using a
pipette and only slow time dependent changes after solution
exchange are meaningful. This is particularly due to uneven
introduction of solution over the whole slide or plate, and the
serial process of laser scanning. Furthermore, cells analyzed using
both methods are usually grown in traditional flasks, slides or
Petri-dishes before analysis, and so uniformity of environment is
limited to that of the flask or dish. Notably, cell-cell contact is
not controllable, and diffusible secretions are maintained in the
culture environment.
[0012] To overcome some of these limitations, research has recently
shifted towards the exploitation of the precise chemical delivery
capabilities of microfluidic devices. The single most popular
approach for the fabrication of microfluidic devices for cell-based
assays is based on the soft-lithography of polydimethylsiloxane
(PDMS). PDMS is an elastomer which is casted over a mold typically
fabricated using photolithography and cured for several hours
resulting in a transfer of features from the mold to the PDMS. Its
wide use as a material of choice is due to its mechanical property,
which is amenable to integration of fluidic valves, essential
elements for major microfluidic applications. PDMS platforms for
cell culture have been reported in the past especially for
two-dimensional morphological cells, such as epithelial cells, and
several designs have been the subject of patent applications (Jin
2010; Lee 2010). However, most of these studies coupled
microfluidic device to traditional macroscale equipment (i.e.
fluorescence microscopes) and relied on the use of fluorescence
imaging for cellular response analysis.
[0013] Moreover, there is surprisingly little work reported on the
combination of nanopatterned surfaces and microfluidics, especially
in a way advantageous for studying topographically induced cellular
response. This is in part due to the difficulty in reproducibly
fabricating nanostructured surfaces within microfluidic cell
culture devices. Several production processes have been reported
for nanostructure fabrication inside microchannels including vapor
deposition of nanoparticles (Song 2009), in situ formation of
nanoparticles inside the channels from catalytic reaction (Fonverne
2009), and polymerization of a polymer around an anodic aluminum
oxide template (Soper 2008). However, controlling the regularity,
geometry and/or spacing of the nanoparticle arrays using these
techniques is difficult to achieve limiting the reproducibility of
the experimental measurements.
[0014] In order to obtain spatially controlled geometry and spacing
of the nanostructures within a PDMS microfluidic channel,
multilayer mold comprising nano- and micro- structures are required
with fabrication procedures involving sequential electron-beam
lithography, interference lithography or nanoimprint lithography in
concert with photolithography of SU-8 resist. The compatibility of
materials and reagents involved in these processes is difficult to
achieve. Additionally, once the mold is fabricated and the
microchannels have been defined, the slightest change of the
microfluidic layout would require the repetition of complete
fabrication process, starting with the nanostructured substrate.
This can result in topographical surface variations induced by
sample-to-sample fabrication differences. Furthermore, PDMS
soft-lithography fabrication technique itself is not well suited
for mass production of microfluidic devices which hinders their
application in industry, including medical diagnosis and
pharmaceutics. Finally, the use of PDMS as a material for in vitro
models for cell culture needs to be considered in a biological
context due to the leaching of uncured oligomers from the polymer
network into microsystem media (Regehr 2009).
[0015] Further, microfluidic devices with nanostructured
hydrophobic surfaces have been developed to control surface tension
and liquid pressure in fluid flow channels (Extrand 2005), but the
standard techniques used to nano-pattern the channel surfaces are
insufficiently flexible to permit simple and fast patterning of
nanostructures, especially different nanostructured patterns, at
specific locations in the channels or chambers of the device but
not at others. Thus, different design features within the same
device are difficult to accomplish and the designs are difficult to
adapt to the requirements of plasmonic detection techniques.
[0016] In general, prior art systems have one or more deficiencies.
There is a lack of an integrated microfluidic system that relies on
non-invasive, label-free detection technologies including plasmonic
techniques such as surface plasmon resonance (SPR) (e.g.
reflection-mode SPR, transmission-mode SPR, localized surface
plasmon resonance (LSPR)) and surface-enhanced Raman spectroscopy
(SERS) for monitoring cell behavior, cell-substrate interactions,
cell response to stimuli and biomolecule detection. There is a lack
of fabrication techniques allowing monolithically integrated
nanostructured cell culture system in long-term biocompatible
materials with simultaneous cell guiding functionality and
plasmonic detection capability using topographical cues and
nanostructure plasmonic response, respectively. There is poor
control of cellular microenvironment in Petri-dish or microwell
plates. There is lack of reproducible and robust surface topography
(nanopatterning) for precise control of cellular response and
cell-substrate interaction studies. There is a lack of integrated
nanostructured surface within microfluidic channels. And, there is
a lack of low-cost and rapid mold fabrication techniques that allow
interchangeable nano- and micro-structure design.
[0017] There remains a need for an integrated system that can meet
one or more of the following requirements: (i) efficient control of
initial cell adhesion; (ii) efficient control of the cellular
response to the specific stimulus over a prolonged period; (iii) in
situ, label-free and real-time monitoring of cellular response,
cell mobility, cell behavior, cell-viability or biomolecule
detection in order to avoid false response due to cellular
secretion of the molecules to which they respond and steric
hindrance induced by the fluorescent tags. Additionally, the system
is ideally low-cost, portable and amenable to mass-production.
SUMMARY OF THE INVENTION
[0018] In one aspect of the present invention, there is provided a
process of producing a patterned polymeric substrate comprising:
etching one or more ordered patterns onto a hard substrate, the
ordered patterns comprising ordered arrays of nano-scale elements
having cross-sectional dimensions in a range of from 10 nm to 1000
nm, the arrays having a spacing distance between their respective
elements where cross-sectional dimension to spacing distance ratio
is greater than 0.2; micro-patterning a polymeric film to form a
membrane comprising a first surface bearing a pattern of
micro-scale and/or meso-scale features for defining one or more
channels and/or chambers, one or more of the micro-scale and/or
meso-scale features comprising through-holes; placing the membrane
on the hard substrate with a surface opposite the first surface
against the hard substrate, the through-holes aligned to expose the
one or more ordered patterns, and applying pressure sufficient to
seal lips of the membrane surrounding the through-holes against the
hard substrate; placing a settable liquid polymer or metal in the
through-holes and over the first surface and setting the settable
liquid polymer or metal to form a stamp from the settable liquid
polymer or metal, the stamp comprising micro-scale and/or
meso-scale reliefs for defining one or more channels and/or
microfluidic chambers and further comprising one or more nano-scale
relief patterns on the micro-scale and/or meso-scale reliefs that
complement the one or more ordered patterns; and, patterning a
polymeric substrate by stamping the polymeric substrate with the
stamp to form the one or more channels and/or microfluidic chambers
in the polymeric substrate, the polymeric substrate comprising one
or more surfaces having one or more ordered patterns that are
substantially identical to the one or more ordered patterns etched
onto the hard substrate, the one or more ordered patterns in the
polymeric substrate suitable for plasmonic resonance reading of a
fluid within the one or more channels and/or chambers.
[0019] In another aspect of the present invention, there is
provided a stamp for patterning a polymeric substrate, the stamp
comprising: a polymer or metal of sufficient hardness to be able to
pattern the polymeric substrate; and, a pattern of micro-scale
and/or meso-scale reliefs for forming one or more channels and/or
microfluidic chambers, one or more of the micro-scale and/or
meso-scale reliefs having top surfaces comprising one or more
nano-scale relief patterns for forming one or more ordered arrays
of nano-scale elements in the polymeric substrate, the nano-scale
elements having cross-sectional dimensions in a range of from 10 nm
to 1000 nm, the arrays having a spacing distance between the
elements where cross-sectional dimension to spacing distance ratio
is greater than 0.2.
[0020] In yet another aspect of the present invention, there is
provided a microfluidic device comprising a monolithic polymeric
substrate patterned with one or more micro-scale channels in fluid
communication with one or more microfluidic chambers, a surface in
the polymeric substrate comprising an ordered array of nano-scale
elements suitable for plasmonic resonance reading of a fluid on the
surface, the nano-scale elements having cross-sectional dimensions
in a range of from 10 nm to 1000 nm, the array having a spacing
distance between the elements where cross-sectional dimension to
spacing distance ratio is greater than 0.2.
[0021] Microfluidic devices of the present invention are monolithic
microfluidic structures in a polymeric substrate having
nanostructures monolithically integrated in the substrate. The
devices have at least one micro-scale channel in fluid
communication with at least one microfluidic chamber. Channels
include, for example, sample loading channels, cell loading
channels, medium perfusion channels, mixing channels, particle
separation or fractionation channels, gradient generating channels
and high resistance perfusion conduits, which may have different
channel dimensions dictated by the specific application.
Microfluidic chambers include, for example, cell culture chambers,
bacteria or cell capture chambers, biomolecular interaction
chambers or mixing chambers. Other microfluidic structures may also
be present, for example valves and pumps for controlling fluid
flow, conduits, inlets, outlets, and the like.
[0022] At least one surface in a channel or microfluidic chamber of
the device is patterned with an ordered array of nano-scale
elements suitable for plasmonic resonance reading of a fluid on the
surface. More than one surface may be patterned and the pattern may
be the same or different. The patterned surface or surfaces may be
anywhere in the device, and could even be everywhere in the device.
The location of the patterned surface or surfaces is dictated by
the ultimate use of the device. Such patterned surfaces may be
called "nanoplasmonic surfaces", and the elements may be called
"nanoplasmonic elements". The nano-scale elements may serve a dual
purpose as both nanoplasmonic elements and nano-topographical cues.
To function as a nanoplasmonic surface, the ordered array of
nano-scale elements has a highly regular or periodic pattern
designed to have a specific plasmonic resonance to permit
label-free, real-time optical reading using plasmonic techniques.
The regularity of the pattern is reflected in very consistent size,
spacing and/or geometry of the nano-scale elements, and arises from
the highly reproducible process employed in the present invention
to produce the patterned polymeric substrate of the device.
Preferably, for each of the size, spacing and/or geometry of the
elements in the array, the standard deviation from the respective
average is no more than about 3%, preferably no more than about
2.5%, and may be no more than about 1%.
[0023] The nano-scale elements have cross-sectional dimensions in a
range of from about 10 nm to about 1000 nm, which is in the
nano-scale range. Preferably the cross-sectional dimensions are in
a range of from about 10 nm to about 750 nm. Individual nano-scale
elements preferably have an aspect ratio (height to width) of about
100 or less, more preferably 50 or less, yet more preferably 10 or
less. Aspect ratios may be in a range of from 100:1 to 1:100, 50:1
to 1:50 or 10:1 to 1:10.
[0024] Spacing of nano-scale elements in the ordered array is an
important factor in maintaining suitable regularity for plasmonic
resonance reading techniques. Ideal spacing distance is dependent
on the size of the nano-scale elements. For plasmonic techniques,
cross-sectional dimension to spacing distance ratio is generally
greater than about 0.2. Preferably, the ratio of cross-sectional
dimension to spacing distance is in a range of from about 0.2 to
about 1.5, or about 0.5 to about 1. The ratio of cross-sectional
dimension to spacing distance is commonly about 0.5 or about 1.
[0025] The nano-scale elements may have any nanostructure geometry
suitable for the reading technique to be used. Suitable geometries
include, for example, nanopillars, nanoposts, nanodots, nanorods,
nanopyramids, nanocrescents, nanodisks, nanodomes, nanoholes,
nanogratings or nanogrooves. Multiple arrays having different
nanostructure geometries for different functionalities (e.g.
gratings for light-coupling or nanopillars for SPR electromagnetic
field enhancement) can be integrated within the same device.
Multiple arrays having different nanostructure geometries can be
co-mingled to occupy the same surface, or different arrays can be
on different surfaces in the device. Different arrays can resonate
at different wavelengths permitting implementation of a multiple
frequency interrogation scheme for parallel multichannel detection
of different targets.
[0026] Ordered arrays of nano-scale elements may be integrated onto
a surface of micro-scale features (e.g. micropillars) in the
device. In cell culture applications, this can provide two levels
of topographical cues (spatial and mechanical cues) on the micro-
and nano-scales for control of cell attachment and motion while
retaining plasmonic detection capability for studying cell behavior
and interactions. Further, micro-optic features (e.g. microlenses,
blazed gratings, etc.) may be formed into the microfluidic device
for various purposes, including enhancing light coupling or
improving light collection efficiency, depending on the particular
reading techniques used.
[0027] Nano-scale elements may be further treated to enhance or
alter the properties of the ordered array. Such treatments may
include, for example, metallization of the nano-scale elements to
increase reflectivity of the array or chemical surface modification
to permit attachment of biolmolecules. For plasmonic techniques,
metallization of one side of the nano-scale elements to form
single-sided metallic nano-elements is particularly preferred.
Silver, gold, copper, platinum and palladium are preferred metals
for metallization, particularly for SERS applications.
Metallization may be accomplished by any suitable method, for
example, evaporation, sputtering or electroplating. Chemical
surface modification includes modification with specific reactive
end-groups, for example --COOH, --OH, --NH.sub.3, -biotin, -silane,
etc., to enable subsequent attachment of antibodies,
oligonucleotides, aptamers or proteins for cell, bacteria or
biomolecule capture.
[0028] The polymeric substrate may comprise any polymeric material
that is soft enough to be stamped by the stamp. Preferably, the
polymeric material is suitable for fabrication of microfluidic
devices. Preferably, the polymeric material comprises a
cyclo-olefin polymer (e.g. Zeonor.TM.), a thermoplastic polymer
(e.g. polyolefins), a biodegradable polymer (e.g. starch,
poly-lactic acid), an elastomer (e.g. thermoplastic elastomer (TPE)
or any blend thereof. More preferably, the polymeric material
comprises a cyclo-olefin polymer (COP) (e.g. Zeonor.TM.) or a
thermoplastic elastomer (TPE).
[0029] The stamp may comprise any polymer or metal that is settable
from liquid form to produce a polymer or metal that is harder than
the polymeric substrate and hard enough to impress the ordered
pattern on the polymeric substrate. If a metal is used, the metal
in its liquid state must not be hot enough to melt the hard
substrate and polymeric film. Preferably, a settable polymer is
used and the polymer is a curable polymer, preferably a curable
thermoset polymer. The polymer for the stamp may be settable or
curable thermally, chemically or with light. Photo-curable
polymers, especially ones cured by UV light, are particularly
preferred. Some examples of photcurable polymers include MD-700 and
Darcour.TM. blend. The stamp is used to transfer all of the
features of the microfluidic device into the polymeric substrate in
one processing step. The processing step may involve any suitable
method for patterning polymeric substrates using stamp or dies, for
example, hot embossing, nanoimprint lithography or injection
moulding. The stamp may be treated to facilitate patterning of the
substrate, for example, treating the stamps with a release agent
can facilitate separation of the patterned polymeric substrate from
the stamp. Since the stamp comprises reliefs for all of the
features of the microfluidic device, the microfluidic device can be
formed completely in one step resulting in a monolithic device
having all of the features of the device integrated into the
polymeric substrate. Further, the stamp can be re-used to make more
devices and the use of the stamp provides pattern and dimensional
consistency between devices produced in different production runs.
These are distinct advantages over prior art processes for
producing microfluidic devices.
[0030] The stamp may be fabricated from a master mold by
transferring features from the master mold to the settable polymer.
The master mold comprises a hard substrate and one or more
membranes placed on the hard substrate. The hard substrate may
comprise a metal, a silicon wafer, a glass substrate or a hard
polymer. The term "hard substrate" refers to a substrate that is
harder than the membranes placed on the substrate. Preferably, the
hard substrate comprises a hard polymer, for example, a
cyclo-olefin polymer (e.g. Zeonor.TM.), a polymethylmethacrylate
(PMMA), a polycarbonate (PC) or a polyetheretherketone (PEEK). The
one or more membranes comprise polymeric films of a soft polymer,
for example, polydimethylsiloxane (PDMS), a soft thermoplastic
polymer (e.g. a soft polyolefin) or a soft thermoplastic elastomer
(e.g. Kraton.TM. Mediprene.TM., CL-30 or
styrene-ethylene-butadiene-styrene (SEBS)). The polymeric films are
preferably films of soft thermoplastic elastomer (TPE). The term
"soft polymer" refers to a polymer that is softer than the hard
substrate.
[0031] One or more ordered patterns comprising nano-scale elements
are etched onto the hard substrate. Etching can be accomplished by
any suitable means appropriate for the hard substrate. For example,
etching may be accomplished with lasers, with ion bombardment or
with chemical etching, for example reactive ion etching, deep
reactive ion etching, wet chemical etching, electron beam
lithography, nanoimprint lithography, ion beam milling, laser
ablation or interference lithography. The membranes comprise
polymeric films having one surface onto which micro-scale and/or
meso-scale features have been micro-patterned. Patterning of the
polymeric films may be accomplished by any suitable means, for
example, hot embossing, injection moulding, nanoimprint lithography
or roll-replication. The micro-scale and/or meso-scale features in
the membranes define one or more microchannels and/or microfluidic
chambers that will eventually be created in the final microfluidic
device. One or more of the features patterned in the polymeric film
may be through-holes that are aligned to expose the one or more
ordered patterns on the hard substrate. The through-holes have the
shape of the microfluidic features (e.g. microchannels,
microfluidic chambers, micropillars, etc.) that are intended bear
the ordered patterns in the final device.
[0032] The membranes are placed on the hard substrate so that the
surfaces bearing the micro-scale features are also exposed. More
than one membrane may be stacked on the hard substrate and the
through-holes aligned with the ordered patterns to obtain the
desired microstructural features in the device. Use of stacked
membranes is particularly useful for forming microstructural
features, such as micropillars, having a top surface covered with
the nano-scale elements. When placing the membranes on the hard
substrate, a seal around the through-holes may be achieved by
applying sufficient pressure to seal lips of the membrane
surrounding the through-holes against the hard substrate.
[0033] The master mold thus formed may be used to fabricate the
stamp by placing the settable polymer in the through-holes and on
the membrane surface onto which the micro-scale and/or meso-scale
features have been micro-patterned. The settable polymer is
typically poured or injected in liquid form onto and into the
master mold and then set as described above. In this manner, the
settable polymer is in contact with the features on the membrane
and the ordered pattern on the hard substrate, so when the settable
polymer hardens, the micro-scale and/or meso-scale features and the
ordered pattern of nano-scale elements are transferred to the set
polymer. Once the settable polymer has hardened, the stamp so
fabricated can be demolded and then used to pattern the polymeric
substrate to form the final device. Thus, the master mold is an
exact replica of the final device, and the stamp is the toll used
to transfer the pattern in the master mold to the polymeric
substrate to produce the final device. It is an advantage of the
present invention that when a different channel layout needs to be
used with the same ordered pattern of nano-scale elements, the new
master mold can be easily fabricated by delaminating the soft
membranes from the hard substrate an replacing the membranes with
membranes having the new layout.
[0034] The present invention advantageously provides low-cost
monolithic integrated microfluidic systems with multiplexing
capability (e.g. valving, pumping) for precise control of cell
culture conditions that can simultaneously integrate label-free
enhanced plasmonic techniques such as surface plasmon resonance
(SPR) (e.g. reflection-mode SPR, transmission-mode SPR, localized
surface plasmon resonance (LSPR)) or surface-enhanced Raman
scattering (SERS). The present monolithic integrated polymer-based
microfluidic system has micro- and nano-structures that provide
topographical cues for cell attachment and culture for controlling
cell behavior while permitting monitoring of cellular behavior,
motility, attachment, viability, biomolecule interactions or any
combination thereof using plasmonic detection. The system is
fabricated using a simple, robust and cost-effective process in a
single step.
[0035] The present system is advantageous over both conventional
and recently reported processes employed for cell culture and
monitoring of cell behavior as the present system integrates in a
single monolithic biocompatible substrate both a nanostructured
surface required for plasmonic response monitoring and a network of
microchannels for precisely controlling cellular environment, with
additional advantages of low-volume consumption, rapid low-cost
fabrication of molds with easily interchangeable microfluidic
channel layouts, amenability to mass production, and in situ
label-free real-time detection of cellular response, viability,
behavior and biomolecular binding using enhanced SPR
(reflection-mode SPR, transmission-mode SPR, LSPR) or SERS.
[0036] The present invention has application to such problems as
screening molecular or cellular targets, cellular identification,
screening single cells for RNA or protein expression, monitoring
cell response to different stimuli (chemical, topographical, flow,
etc.), genetic diagnostic screening at the single cell level, or
performing single cell signal transduction studies.
[0037] Further features of the invention will be described or will
become apparent in the course of the following detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] In order that the invention may be more clearly understood,
embodiments thereof will now be described in detail by way of
example, with reference to the accompanying drawings, in which:
[0039] FIG. 1 depicts a schematic diagram of a process of the
present invention for producing a monolithic integrated
nanoplasmonic microfluidic cell culture system.
[0040] FIG. 2A illustrates a schematic cross-section of a chamber
through a perfusion channel and perfusion conduit of a
nanostructured nanoplasmonic microfluidic cell culture system.
[0041] FIG. 2B illustrates a schematic cross-section of a chamber
through a cell-loading channel of a nanostructured nanoplasmonic
microfluidic cell culture system.
[0042] FIG. 2C illustrates a schematic cross-section through a
chamber showing integrated micropillars with nanostructured top
surface of a nanostructured nanoplasmonic microfluidic cell culture
system.
[0043] FIG. 2D illustrates a schematic cross-section through a
chamber of a nanostructured nanoplasmonic microfluidic cell culture
system showing an integrated micro-optic element on the bottom side
of the flow layer substrate.
[0044] FIGS. 2E,F depict 3D views of the chamber bottom depicted in
FIG. 2A containing micropillars with nanostructured top surface
area used to (E) control cell attachment/motility and (F) study
cell-substrate interactions.
[0045] FIG. 3A-D depicts SEM micrographs of fabricated structures
showing nanoplasmonic nanostructures including nanoholes (A),
nanopillars (B), nanoposts (C) and nanogratings (D).
[0046] FIG. 3E is a SEM micrograph with two enlargements and an
inset showing three-dimensional prism-shaped microstructures with
nano-structures defined in a single substrate using a one step
fabrication process of the present invention.
[0047] FIG. 3F is a SEM micrograph with two enlargements and an
inset showing three-dimensional cylindrical microstructures bearing
nano-structures defined in a single substrate using a one step
fabrication process of the present invention.
[0048] FIG. 4 depicts a schematic of a nanoplasmonic microfluidic
cell culture system with plasmonic detection capability.
DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
Process for Fabricating a Monolithic Integrated Nanoplasmonic
Microfluidic Cell Culture System
[0049] A monolithic integrated nanoplasmonic microfluidic cell
culture system of the present invention may be produced generally
as shown in FIG. 1, which illustrates the process showing a single
cell chamber and none of the channels, conduits, valves or other
microfluidic features for clarity. A soft thermoplastic elastomer
film is hot-embossed to form micro-scale and meso-scale features
(microchannels, conduits, chambers, etc.) in TPE membrane 10
including through-hole 11. This is performed at an applied pressure
ranging from 5 kN to 15 kN, for 5-30 min, at a temperature in a
range of from 100.degree. C. to 160.degree. C., depending on the
desired features. Hard Zeonor.TM. substrate 12 is patterned by
hot-embossing at an applied pressure of 10 kN to 20 kN for 10-30
min at a temperature ranging from 140.degree. C. to 170.degree. C.
depending on the specific Zeonor.TM. grade to form a regular array
of nano-scale grating elements 13. With the micro-scale and
meso-scale features facing up, the TPE membrane is placed on the
Zeonor.TM. substrate such that the through-hole is aligned with the
grating elements. The membrane is then reversibly bonded to the
Zeonor.TM. substrate to seal the membrane around the through-hole
against the Zeonor.TM. substrate to form master mold 14 at room
temperature. Photocurable polymer 15 is poured into the
through-hole and onto the membrane to cover the membrane and the
micro-scale and meso-scale features thereon. Glass or metal backing
plate 16 is placed over top of the photocurable polymer and the
photocurable polymer is then exposed to UV radiation 17 to cure the
polymer. When a metal backing plate is used, the assembly is
flipped upside down to UV cure the polymer. After curing, master
mold 14 and glass (or metal) plate 16 are removed to provide
working stamp 18 having reliefs 19 comprising a reverse image of
the micro-scale and meso-scale features and the regular array of
nano-scale grating elements. The working stamp is then used to
hot-emboss "hard or soft" thermoplastic polymer substrate 20 (e.g.
Zeonor.TM., PMMA or a thermoplastic elastomer such as CL-30,
Mediprene.TM., etc.) to provide, in one step, a monolithic
microfluidic cell culture system having micro-scale and meso-scale
features 21 and regular array of nano-scale grating elements 22
therein.
[0050] Microfluidic cell culture systems produced in this manner
may comprise any number of cell culture chambers, microchannels,
conduits, valves, etc. More detailed schematic drawings of one cell
culture chamber in the monolithic integrated nanoplasmonic
microfluidic cell culture system produced by this process are shown
in FIG. 2. Referring to FIGS. 2A to 2D, flow layer 40 of the cell
culture system comprises cell loading channels 41, perfusion
channels 42, perfusion conduits 43 and culture chambers 44, which
have different dimensions dictated by the specific application. As
shown in FIGS. 2A, 2B and 2D, the bottom of the cell culture
chambers may be patterned with an ordered array of nanostructures
45, in this case a nanograting. Alternatively, as shown in FIG. 2C,
the bottom of the cell culture chamber may have integrated
micropillars 46 having nanostructures 47 patterned thereon. As
shown in FIGS. 2E to 2F, such nanostructured micropillars can
provide two-levels of topographical (spatial and mechanical) cues
on the micro- and nano- scale for controlling attachment/motion
(cell isolation or confinement) of cells 48, while retaining
plasmonic detection capability for the study of cell behavior and
interactions. Further, as shown in FIG. 2D, flow layer 40 can be
fabricated to include micro-optic elements, such as microlens 49 of
nanograting 45, for enhanced light coupling or improved light
collection efficiency, depending on the particular interrogation
scheme (e.g. transmission or reflection SPR, LSPR or SERS).
[0051] Control layer 50 and thin membrane 51 may be placed on top
of flow layer 40 to control fluid flow in the channels and conduits
of the microfluidic cell culture system. Control layer 50 contains
a network of channels used to supply pressure on thin membrane 51
sandwiched between the control layer and the flow layer in order to
close the valves and control fluid flow. While for certain
application, the use of valves for fluidic management might not be
necessary, for high-level microfluidic integration of the system it
is of great importance in order to allow two-dimensional addressing
of each individual chamber. The monolithic integration of
nanostructures with the flow layer allows the use of the control
layer for the integration of valves which would otherwise be
impossible by simply assembling a bottom nanostructured SPR layer
with a top microfluidic structure.
[0052] Referring to FIG. 3, sample scanning electron microscope
(SEM) micrographs of possible nanostructures and their monolithic
integration within microfluidic chambers of the microfluidic cell
culture system are shown. In FIGS. 3A-D, from left to right are
shown nanoholes, nanopillars, nanoposts and nanogratings. In each
of FIGS. 3E and 3F from left to right are shown successive
magnifications of SEM micrographs of monolithic three-dimensional
microstructures and nanostructures fabricated using the present
method, where the SEM on the left shows the microstructures, the
SEM on the right shows the nanostructures in a microstructure and
the SEM in the middle has a magnification in between the left and
right. In FIGS. 3E and 3F, the SEM in the middle has a
magnification 2.5.times. greater than the SEM on the left, and the
SEM on the right has a magnification 20.times. greater than the SEM
in the middle. The field of view for the SEM on the left is 500
.mu.m.
EXAMPLE 2
Use of a Monolithic Integrated Nanoplasmonic Microfluidic Cell
Culture System in Plasmonic Detection
[0053] In operation a monolithic integrated nanoplasmonic
microfluidic cell culture system of the present invention employs
pressure-driven flow to transport cells in suspension from a
plurality of reservoirs through a plurality of cell-loading
channels to a plurality of nanostructured cell culture chambers by
closing valves of the perfusion channels and opening valves on the
cell-loading channels. A plurality of cell-lines are loaded using a
plurality of different reservoirs. Following initial cell
attachment on the bottom of the nanostructured chambers, the valves
on the cell-loading channels are closed, and fresh media is
continuously injected in each of the perfusion channels. Multiple
high resistance perfusion conduits ensure equal distribution of the
media within the chamber while minimizing the shear-stress exerted
on the cells.
[0054] Once the cell culture chambers are loaded with cells,
plasmonic resonance readings are taken using optical detection
methods of Reflection or Transmission-mode Surface Plasmon
Resonance, Localized Surface Plasmon Resonance or Surface Enhanced
Raman Spectroscopy. FIG. 4 illustrates the configuration of
microfluidic device 60 in relation to light source 62 and detector
64 of the optical detection method. With these detection methods,
cell-substrate interaction can be monitored in situ, in real-time
and without any labels by analyzing the shift in the plasmonic
peaks of the nanostructured substrate response. Resulting shifts in
plasmonic peaks for surface plasmon resonance (SPR) and localized
surface plasmon resonance (LSPR) or surface enhanced Raman
spectroscopy (SERS) are illustrated at the left and right,
respectively, in FIG. 4.
[0055] Additionally, the present design allows monitoring of
cellular response due to different bio-chemical cues which can be
supplemented in the perfusion media. Furthermore, prior to cell
loading, using the same microchannels, the bottom of the chambers
can be functionalized by flowing different chemicals and/or
biological species for monitoring of cell-substrate interactions or
for the detection of biochemical targets excreted or extracted from
the cell.
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[0094] Other advantages that are inherent to the structure are
obvious to one skilled in the art. The embodiments are described
herein illustratively and are not meant to limit the scope of the
invention as claimed. Variations of the foregoing embodiments will
be evident to a person of ordinary skill and are intended by the
inventor to be encompassed by the following claims.
* * * * *