U.S. patent application number 15/363908 was filed with the patent office on 2017-06-22 for devices and methods for sample characterization.
This patent application is currently assigned to Intabio. The applicant listed for this patent is Intabio, Inc.. Invention is credited to Erik GENTALEN.
Application Number | 20170176386 15/363908 |
Document ID | / |
Family ID | 58798061 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170176386 |
Kind Code |
A1 |
GENTALEN; Erik |
June 22, 2017 |
DEVICES AND METHODS FOR SAMPLE CHARACTERIZATION
Abstract
Devices and methods for characterization of analyte mixtures are
provided. Some methods described herein include performing
enrichment steps on a device before expelling enriched analyte
fractions from the device for subsequent analysis. Also included
are devices for performing these enrichment steps.
Inventors: |
GENTALEN; Erik; (Fremont,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Intabio, Inc. |
Fremont |
CA |
US |
|
|
Assignee: |
Intabio
Fremont
CA
|
Family ID: |
58798061 |
Appl. No.: |
15/363908 |
Filed: |
November 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62260944 |
Nov 30, 2015 |
|
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|
62338074 |
May 18, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/0268 20130101;
G01N 27/44791 20130101; G01N 27/44795 20130101; G01N 27/44721
20130101; G01N 2223/50 20130101; B01L 3/502715 20130101; B01L
2400/0421 20130101; B01L 2300/0816 20130101; B01L 2300/0654
20130101; B01L 2300/0861 20130101; G01N 27/44773 20130101; H01J
49/04 20130101; G01N 2223/40 20130101; H01J 49/167 20130101; G01N
2550/00 20130101; B01L 2200/143 20130101 |
International
Class: |
G01N 27/447 20060101
G01N027/447; B01L 3/00 20060101 B01L003/00 |
Claims
1. An apparatus, comprising: a substrate constructed of an opaque
material, the substrate having a thickness and a top surface in
which a microfluidic separation channel having a depth equal to the
thickness of the substrate is defined such that the microfluidic
separation channel defines an optical slit through the substrate;
and a layer disposed on a top surface of the substrate, a portion
of the layer disposed over the microfluidic separation channel
being transparent.
2.-4. (canceled)
5. The apparatus of claim 1, wherein the substrate, the
microfluidic separation channel, and the layer are collectively
configured such that when the apparatus is imaged, light is only
transmitted through the optical slit.
6. The apparatus of claim 1, wherein a portion of the layer is
opaque.
7. The apparatus of claim 1, wherein the portion of the layer that
is transparent extends over an entire length of the microfluidic
separation channel.
8. The apparatus of claim 1, wherein: a side surface of the
substrate defines an orifice in fluid communication with an end
portion of the microfluidic separation channel; the substrate
defines a reservoir fluidically coupled to the end portion of the
microfluidic separation channel such that fluid from the reservoir
forms a sheath solution around fluid from the microfluidic
separation channel when fluid is expelled from the orifice.
9. The apparatus of claim 1, wherein: the substrate further defines
a gas channel; a side surface of the substrate defines an orifice
in fluid communication with the substrate, the gas channel
configured to convey nebulizing gas that flanks fluid expelled from
the orifice.
10. The apparatus of claim 1, wherein the substrate further
defines: a first reservoir fluidically coupled to a first end
portion of the microfluidic separation channel; a second reservoir
fluidically coupled to a second end portion of the microfluidic
separation channel opposite the first end portion, the apparatus
further comprising: a first electrical contact electrically coupled
to the first reservoir; and a second electrical contact
electrically coupled to the second reservoir such that an electric
field can be applied to the microfluidic separation channel via the
first reservoir and the second reservoir to induce electrophoresis
within the microfluidic separation channel.
11. The apparatus of claim 1, wherein the microfluidic separation
channel is a chromatographic separation channel and the substrate
further defines an electrophoretic separation channel fluidically
coupled to the chromatographic separation channel such that at
least two phases of separation can be performed within the
apparatus.
12. The apparatus of claim 1, wherein the substrate further defines
an elution channel fluidically coupled to the microfluidic
separation channel, the elution channel configured to convey an
eluent to the microfluidic separation channel to elute an analyte
bound to a media disposed in the microfluidic separation
channel.
13. The apparatus of claim 1, wherein a side surface of the
substrate defines a countersunk surface having an orifice in fluid
communication with the microfluidic separation channel, the orifice
and the countersunk surface collectively configured such that a
Taylor cone emanating from the orifice is disposed entirely within
a volume defined by the countersunk surface.
14.-21. (canceled)
22. A method, comprising: injecting an analyte mixture into a
microfluidic device containing a first separation channel, the
first separation channel containing a media configured to bind to
an analyte from the analyte mixture; injecting an eluent into the
microfluidic device such that at least a fraction of the analyte is
mobilized from the media; imaging the first separation channel
while the analyte is mobilized; applying an electric field to a
second separation channel when the imaging detects that the
fraction is disposed at an intersection of the first separation
channel and the second separation channel such that the fraction is
mobilized into the second separation channel; and expelling at
least a portion of the fraction.
23. The method of claim 22, wherein the at least the portion of the
fraction is expelled via electrospray ionization.
24. (canceled)
25. The method of claim 22, further comprising: separating the
fraction of the analyte via capillary zone electrophoresis in the
second separation channel.
26. The method of claim 22, further comprising: separating the
fraction of the analyte via electrophoresis in the second
separation channel; and imaging the second separation channel while
the fraction of the analyte is separated.
27. The method of claim 22, wherein a first end portion of the
second separation channel intersects the first separation channel,
the method further comprising: injecting a sheath solution into a
second end portion of the separation channel opposite the first end
portion of the separation channel.
28. The method of claim 22, wherein the first separation channel
and the second separation channel arc orthogonal.
29. An apparatus, comprising: a substrate defining: a first
enrichment zone containing a media configured to bind to an
analyte; a second enrichment zone intersecting the first enrichment
zone; a surface; and an orifice defined by the recessed surface,
the orifice being an opening to a first end portion of the second
enrichment zone; a first electrode electrically coupled to the
first end portion of the second enrichment zone; and a second
electrode electrically coupled to a second end portion of the
second enrichment zone opposite the first end portion.
30. The apparatus of claim 29, wherein the first enrichment zone is
orthogonal to the second enrichment zone.
31. The apparatus of claim 29, wherein the first enrichment zone is
disposed before the second enrichment zone such that at least a
portion of a sample flows from the first enrichment zone to the
second enrichment zone.
32.-33. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional of and claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent
Application No. 62/260,944, filed Nov. 30, 2015, and U.S.
Provisional Patent Application No. 62/338,074, filed May 18, 2016,
each entitled "Devices, Methods, and Kits for Sample
Characterization," the disclosure of each of which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0002] Some embodiments described herein relate to devices and
methods for sample characterization and various uses thereof.
[0003] Separation of analyte components from a more complex analyte
mixture on the basis of an inherent quality of the analytes, and
providing sets of fractions that are enriched for states of that
quality is a key part of analytical chemistry. Simplifying complex
mixtures in this manner reduces the complexity of downstream
analysis. It can be advantageous to perform two or more enrichment
steps that are orthogonal, (e.g., based on different and/or
unrelated qualities). In many cases, however, the process of
performing orthogonal enrichment steps using known methods and/or
devices is cumbersome, and can dilute the analyte beyond the
sensitivity of the downstream analytical equipment. In addition,
complications can arise when attempting to interface known
enrichment methods and/or devices with analytical equipment and/or
techniques.
[0004] Methods have been used to interface protein sample
preparation techniques with downstream detection systems such as
mass spectrometers. A common method is to prepare samples using
liquid chromatography and collect fractions for mass spectrometry
(LC-MS). This has the disadvantage of requiring protein samples to
be digested into peptide fragments, leading to large number of
sample fractions which must be analyzed and complex data
reconstruction post-run. While certain forms of liquid
chromatography can be coupled to a mass spectrometer, for example
peptide map reversed-phase chromatography, these known techniques
are restricted to using peptide fragments, rather than intact
proteins, which limit their utility.
[0005] Another method to introduce samples into a mass spectrometer
is electrospray ionization (ESI). In ESI, small droplets of sample
and solution at a distal end of a capillary or microfluidic device
are ionized to induce an attraction to the charged plate of a mass
spectrometer. The droplet then stretches in this induced electric
field to a cone shape ("Taylor cone"), which then releases small
droplets into the mass spectrometer for analysis. Typically, this
is done in a capillary, which provides a convenient volume and size
for ESI. Capillaries however, provide a linear flow path that does
not allow for multi-step processing.
[0006] Other work has been pursued with microfluidic devices.
Microfluidic devices may be produced by various known techniques
and provide fluidic channels of defined width that can make up a
channel network designed to perform different fluid manipulations.
These devices offer an additional level of control and complexity
than capillaries. In connection with ESI, known devices include
outwardly tapered tips and conductive edges in an attempt to
enhance the ESI in these devices. The outward taper of known
microfluidic devices used for ESI, however, exposes the fragile
Taylor cone structure to potential disturbances by turbulent air
flow and results in a contact surface geometry that will support
only a limited range of cone radii, which limits control over the
volume introduced to the mass spectrometer through ESI.
Additionally, electrolysis of water at the conductive edge can lead
to gas bubble formation, which interferes with the cone
development.
[0007] One application for protein mass spectrometry is for
characterization during the development and manufacturing of
biologic and biosimilar pharmaceuticals. Biologics and biosimilars
are a class of drugs which include, for example, recombinant
proteins, antibodies, live virus vaccines, human plasma-derived
proteins, cell-based medicines, naturally-sourced proteins,
antibody-drug conjugates, protein-drug conjugates and other protein
drugs.
[0008] Regulatory compliance demands that biologics require
extensive testing during development and manufacture that is not
required for small molecule drugs. This is because the manufacture
of biologics has greater complexity due to, for example, using
living material to produce the biologic, greater complexity of
biologic molecule, greater complexity of the manufacturing process.
Characteristics required to be defined include, for example,
charge, efficacy, hydrophobic changes, mass, and glycosylation.
Currently these tests are done independent of each other leading to
a very time consuming and expensive process of characterizing
biologics.
SUMMARY
[0009] Some embodiments described herein relate to devices and
methods that can enable the analysis of analytes in an analyte
mixture. For example, many specific characterizations of biologic
proteins are required by regulatory agencies. Methods and devices
described herein can be suitable for characterizing proteins and/or
other analytes. In some embodiments, methods and devices described
herein can relate to characterizing an analyte mixture that
includes one or more enrichment steps performed to separate an
analyte mixture into enriched analyte fractions.
[0010] In some instances, these analytes can be, for example,
glycans, carbohydrates, DNA, RNA, intact proteins, digested
proteins, antibody-drug conjugates, protein-drug conjugates,
peptides, metabolites or other biologically relevant molecules. In
some instances, these analytes can be small molecule drugs. In some
instances, these analytes can be protein molecules in a protein
mixture, such as a biologic protein pharmaceutical and/or a lysate
collected from cells isolated from culture or in vivo.
[0011] Some embodiments described herein can include a first
enrichment step, in which fractions containing a subset of the
analyte molecules from the original analyte mixture are eluted one
fraction at a time; these enriched analyte fractions are then
subjected to another enrichment step. At the final enrichment step,
the enriched analyte fractions are expelled for further
analysis.
[0012] In some embodiments, one or more of the enrichment steps
will be solid-phase separations. In some embodiments, one or more
of the enrichment steps will be solution-phase separations.
[0013] In some embodiments, a final step concentrates the enriched
analyte fractions before expulsion.
[0014] In some embodiments, substantially all of the enriched
analyte fractions from the final enrichment step are expelled in a
continuous stream. In some embodiments, a portion of the analyte
mixture (e.g., a fraction of interest) will be expelled from a
microfluidic device via an outlet configured to interface with an
analytical instrument, such as a mass spectrometer or another
device configured to fractionate and/or enrich at least a portion
of the sample. Another portion of the analyte mixture (e.g.,
containing fractions other than the fraction of interest) can be
expelled via a waste channel.
[0015] In some embodiments, the expulsion is performed using
pressure, electric force, or ionization, or a combination of
these.
[0016] In some embodiments, the expulsion is performed using
electrospray ionization (ESI) into, for example, a mass
spectrometer. In some embodiments a sheath liquid is used as an
electrolyte for an electrophoretic separation. In some embodiments,
a nebulizing gas is provided to reduce the analyte fraction to a
fine spray. In some embodiments, other ionization methods are used,
such as inductive coupled laser ionization, fast atom bombardment,
soft laser desorption, atmospheric pressure chemical ionization,
secondary ion mass spectrometry, spark ionization, thermal
ionization, and the like.
[0017] In some embodiments, the enriched fractions will be
deposited on a surface for further analysis by matrix-assisted
laser desorption/ionization, surface enhanced laser
desorption/ionization, immunoblot, and the like.
[0018] Some embodiments described herein relate to devices and
methods for visualizing an analyte in an electrophoretic separation
before and during the expulsion of enriched fractions.
[0019] Some embodiments described herein relate to devices and
methods for visualizing an analyte during an enrichment step.
[0020] Some embodiments described herein relate to devices and
methods for visualizing an analyte in a channel between enrichment
zones.
[0021] In some embodiments, the visualization of an analyte can be
performed via optical detection, such as ultraviolet light
absorbance, visible light absorbance, fluorescence, Fourier
transform infrared spectroscopy, Fourier transform near infrared
spectroscopy, Raman spectroscopy, optical spectroscopy, and the
like.
[0022] Some embodiments described herein relate to devices that can
enable the analysis of analyte mixtures, in that they contain one
or more enrichment zones and an orifice to expel enriched analyte
fractions. In some embodiments, these devices include at least one
layer which is not transmissive to light of a specific wavelength,
and at least one layer which is transmissive to that specific
wavelength. One or more portions of the layer which is not
transmissive to light can define the one or more enrichment zones,
such that the enrichment zones serve as optical slits.
[0023] In some embodiments, an analyte mixture can be loaded into a
device through a tube or capillary connecting the device to an
autosampler. In some embodiments, an analyte mixture can be loaded
directly into a reservoir on the device.
[0024] In some embodiments, an orifice through which at least a
portion of a sample can be expelled from a device is countersunk
and/or shielded from air flow. In some embodiments, this orifice is
not electrically conductive. As used herein, countersunk should be
understood to mean that a portion of a substrate defines a recess
containing the orifice, irrespective of the geometry of the sides
or chamfers of the recess. Similarly stated, countersunk should be
understood to include counterbores, conical and/or frustoconical
countersinks, hemispherical bores, and the like.
[0025] Some embodiments described herein relate to an apparatus,
such as a microfluidic device that includes a substrate constructed
of an opaque material (e.g., soda lime glass, which is opaque to
ultraviolet light). The substrate can define a microfluidic
separation channel. Similarly stated, the microfluidic separation
channel can be etched or otherwise formed within the substrate. The
microfluidic separation channel can have a depth equal to the
thickness of the substrate. Similarly stated, the microfluidic
separation channel can be etched the full depth of the substrate
(e.g., from the top all the way through to the bottom). In this
way, the microfluidic separation channel can define an optical slit
through the substrate. A transparent layer (e.g., a top layer) can
be disposed on a top surface of the substrate, for example, sealing
the top surface of the substrate. A transparent layer (e.g., a
bottom layer) can also be disposed on a bottom surface of the
substrate, such that both the top and the bottom of the
microfluidic separation channel are sealed. In some embodiments,
only a portion of the top layer and/or the bottom layer may be
transparent. For example, the top layer and/or the bottom layer can
define a transparent window in an otherwise opaque material; the
window can provide optical access to, for example, the microfluidic
separation channel.
[0026] Some embodiments described herein relate to an apparatus,
such as a microfluidic device that includes a substrate. The
substrate can define one or more enrichment zones or channels. For
example, the substrate can define a first enrichment zone
containing a media configured to bind to an analyte. Such a first
enrichment zone can be suitable to separate an analyte mixture
chromatographically. The apparatus can further include two
electrodes electrically coupled to opposite end portions of a
second enrichment zone. Such a second enrichment zone can be
suitable to separate an analyte mixture electrophoretically. The
second enrichment zone can intersect the first enrichment zone such
that after a fraction of an analyte is separated, concentrated,
and/or enriched in the first enrichment zone, it can be further
separated, concentrated, and/or enriched in the second enrichment
zone. The device can also include a recessed orifice. The orifice
can be an outlet of the second enrichment channel and can be
disposed on a countersunk or otherwise recessed surface of the
substrate. The apparatus can be configured to expel a portion of an
analyte mixture from the orifice via ESI. The recess can provide a
stable environment for formation of a Taylor cone associated with
ESI and/or can be configured to accept an inlet port of a mass
spectrometer.
[0027] Some embodiments described herein relate to a method that
includes introducing an analyte mixture into a microfluidic device
that contains a separation channel. An electric field can be
applied across the separation channel to effect a separation of the
analyte mixture. The analyte mixture can be imaged during
separation via a transparent portion of the microfluidic device.
Similarly stated, a window and/or optical slit can provide optical
access to the separation channel such that the whole separation
channel or a portion thereof can be imaged while the separation is
occurring. A fraction of the analyte mixture can be expelled from
an orifice that is in fluid communication with the separation
channel. For example, the fraction can be expelled via ESI. In some
embodiments, the orifice can be disposed on a countersunk surface
of the microfluidic device such that a Taylor cone forms within a
recess defined by the countersunk surface.
[0028] Some embodiments described herein relate to a method that
includes injecting an analyte into a microfluidic device containing
a first separation channel and a second separation channel. The
first separation channel can contain a medium configured to bind an
analyte from the analyte mixture. Accordingly, when the analyte
mixture is injected into the microfluidic device at least a
fraction of the analyte mixture can be bound to the matrix and/or
impeded from flowing through the first separation channel. For
example, injecting the analyte into the microfluidic device can
effect a chromatographic separation in the first separation
channel. An eluent can be injected into the microfluidic device
such that at least a fraction of the analyte is mobilized from the
media. The first separation channel can be imaged while the analyte
is mobilized. Imaging the first separation can include whole column
(e.g., whole channel) imaging and/or imaging a portion of the
channel. An electric field can be applied to the second separation
channel when the imaging detects that the fraction is disposed at
an intersection of the first separation channel and the second
separation channel such that the fraction is mobilized into the
second separation channel. For example, in some embodiments, the
first separation channel can be orthogonal to the second separation
channel. Similarly stated the first separation channel and the
first separation channel can form a T-junction. The imaging can
detect when a portion of the fraction (e.g., a portion of interest)
is at the junction. Applying the electric field can mobilize the
portion of the fraction (and, optionally, not other portions of the
fraction that are not located at the junction) into the second
separation channel for a second stage of separation. At least a
portion of the fraction can be expelled from the microfluidic
device.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a schematic illustration of a device for two
dimensional separation and ESI of an automatically loaded sample,
according to an embodiment.
[0030] FIG. 2 is a schematic exploded view of a device having three
layers, according to an embodiment.
[0031] FIG. 3 is a schematic of a light path through a microfluidic
device, according to an embodiment.
[0032] FIG. 4 is a schematic illustration of a device for
isoelectric focusing (IEF) and ESI of an automatically loaded
sample, according to an embodiment.
[0033] FIG. 5 is a schematic illustration of a microfluidic device,
according to an embodiment.
[0034] FIG. 6 is a flowchart of an exemplary method for analyte
characterization.
[0035] FIG. 7 is a schematic of a microfluidic device, according to
an embodiment.
[0036] FIG. 8 is a schematic of a microfluidic device, according to
an embodiment.
DETAILED DESCRIPTION OF INVENTION
[0037] It is to be understood that both the foregoing general
description and the following description are exemplary and
explanatory only and are not restrictive of the methods and devices
described herein. In this application, the use of the singular
includes the plural unless specifically stated otherwise. Also, the
use of "or" means "and/or" unless stated otherwise. Similarly,
"comprise," "comprises," "comprising," "include," "includes" and
"including" are not intended to be limiting.
[0038] Devices
[0039] FIG. 1 is a schematic illustration of a device for two
dimensional separation and ESI of an automatically loaded sample,
according to an embodiment. A microfluidic network, 100, is defined
by a substrate 102. The substrate is manufactured out of material
which is compatible with the enrichment steps being performed. For
example, chemical compatibility, pH stability, temperature,
transparency at various wavelengths of light, mechanical strength,
and the like are considered in connection with selection of
material.
[0040] Substrate 102 may be manufactured out of glass, quartz,
fused silica, plastic, polycarbonate, polyfluorotetraethylene
(PTFE), polydimethylsiloxane (PDMS), silicon, polyfluorinated
polyethylene, polymethacrylate, cyclic olefin copolymer, cyclic
olefin polymer, polyether ether ketone and/or any other suitable
material. Mixtures of materials can be utilized if different
properties are desired in different layers of a planar substrate
and/or any other suitable material. Mixtures of materials can be
utilized if different properties are desired in different layers of
a planar substrate.
[0041] Channels 106, 110, 114, 116, 118, 124 122, 126,132, 136 and
140 form the microfluidic network 100 and are fabricated into
substrate 102. Similarly stated, the substrate 102 defines channels
106, 110, 114, 116, 118, 124 122, 126,132, 136 and/or 140.
[0042] Channels may be fabricated in the substrate through any
channel fabrication method such as, for example, photolithographic
etching, molding, machining, additive (3D) printing, and the
like.
[0043] Analyte mixtures and external reagents can be loaded through
tube/conduit 112, and excess reagent/waste can be removed through
tube/conduit 130.
[0044] Tubes 112 and 130 can be manufactured out of any material
compatible with the assay being performed, including, for example,
fused silica, fused silica capillary tubes, silicone tubing, and/or
PTFE tubing.
[0045] Channels 116 and 124 can be used to separate and/or enrich
an analyte and/or a portion (e.g., a fraction) of an analyte.
Channels 116 and/or 124 can be used to perform chromatographic
separations (e.g., reversed-phase, immunoprecipitation, ion
exchange, size exclusion, ligand affinity, dye affinity,
hydrophobic interaction chromatography, hydrophilic interaction
chromatography, pH gradient ion exchange, affinity, capillary
electrokinetic chromatography, micellar electrokinetic
chromatography, high performance liquid chromatography (HPLC),
amino acid analysis-HPLC, ultra performance liquid chromatography,
peptide mapping HPLC, field flow fractionation--multi angle light
scattering) or electrophoretic separations (e.g., isoelectric
focusing, capillary gel electrophoresis, capillary zone
electrophoresis, isotachophoresis, capillary electrokinetic
chromatography, micellar electrokinetic chromatography, flow
counterbalanced capillary electrophoresis, electric field gradient
focusing, dynamic field gradient focusing). For example, channel
116 can be derivatized or packed with material to perform a first
enrichment step.
[0046] The material disposed into channel 116 and/or 124 can be
selected to capture analytes based on, for example, hydrophobicity
(reversed-phase), immunoaffinity (immunoprecipitation), affinity
(efficacy), size (size exclusion chromatography), charge (ion
exchange) or by other forms of liquid chromatography.
[0047] Many different methods can be used to dispose the enrichment
material within channels 116 and/or 124. The walls can be directly
derivatized with, for example, covalently bound or adsorbed
molecules, or beads, glass particles, sol-gel or the like can be
derivatized and loaded into these channels.
[0048] After sample is loaded into channel 116 wash solution and
then elution reagent can be introduced through tube 112 and channel
114.
[0049] The elution process will depend on the enrichment method
performed in channel 116. A suitable eluent can be selected to
elute a fraction of the bound analyte. Some enrichment options may
not require an elution step (e.g., size exclusion chromatography,
electrophoretic separations, etc.).
[0050] The eluent or flow-through would then flow through channel
118 into channel 124. Channel 124 could be used to perform either a
chromatographic or electrophoretic enrichment step.
[0051] Electrophoretic separations can be performed in channel 124
by using a power supply to apply an electric field between
reservoir 108 and reservoir 120. Similarly stated, the device 100
can include electrodes in electrical contact with reservoir 108
and/or reservoir 120. The electrical ground of the power supply can
be connected to the electrical ground of a mass spectrometer to
provide continuity in the electric field from channel 124 to the
mass spectrometer.
[0052] Any capillary electrophoresis (CE) electrophoretic method
can be performed in channel 124--IEF, isotachophoresis (ITP),
capillary gel electrophoresis (CGE), capillary zone electrophoresis
(CZE), and the like. Alternately, non-electrophoretic enrichment
methods can be performed in the channel 124.
[0053] In the case of IEF or ITP, concentrated purified sample
bands would be mobilized, for example, by pressure or electrical
means towards confluence 126. Sheath solution from reservoirs 108
and 134 could serve as sheath and catholyte.
[0054] The sheath/catholyte can be any basic solution compatible
with the electrophoretic separation and mass spectrometry
(MeOH/N.sub.4OH/H.sub.2O for example). Anolyte can be any acidic
solution (e.g., phosphoric acid 10 mM).
[0055] Alternately, the electric field could be reversed and
catholyte (NaOH) could be loaded in reservoir 120, and anolyte
could be used as the sheath solution in reservoirs 108 and 134.
[0056] The confluence 126 is where the enriched analyte fraction
mixes with the sheath solution. As the analyte fractions in channel
124 are mobilized, solution will be pushed through confluence 126
out to orifice 128.
[0057] The orifice 128 can be disposed within a recess defined by
surface 127 of substrate 102. For example, surface 127 can be a
countersunk ESI surface. For example, as shown in FIG. 1, the
enriched analyte solution, being electrically grounded through well
108, can form a Taylor cone emanating from orifice 128, which is
disposed entirely within a recess defined by surface 127. The
orifice 128 and/or surface 127 can be oriented toward a mass
spectrometer inlet, which can have a voltage potential difference
relative to well 108. As spray breaks off from the cone structure
toward the mass spectrometer, it can be flanked by nebulizing gas
provided through channels 106 and 140 before it leaves the
substrate 102. The nebulizing gas can be any inert or non-reactive
gas (e.g., Argon, Nitrogen, and the like).
[0058] Additionally, using a sheath liquid and/or nebulizing gas
can allow for the use of an ion depleting step as the last
"on-device" step. The sheath liquid allows for replenishment of ion
potential lost during an IEF charge assay concentrating step prior
to ESI, and nebulization provides the sample in a fine mist for the
off line analysis.
[0059] By generating the Taylor cone on surface 127, the cone is
created in a stable pocket or recess and is protected from
disturbing air currents. Additionally, the conical geometry
surrounding the countersunk orifice has a naturally expanding
contact surface that will accommodate a wider range of Taylor cone
radial cross sections, allowing for a wider range of flow rates
into the mass spectrometer.
[0060] Orifice 128 can be positioned in proximity to an inlet port
of a mass spectrometer. In some instances, the surface 127 can be
configured such that an inlet port of a mass spectrometer can be
disposed within a recess defined by the surface 127.
[0061] FIG. 2 a schematic exploded view of a device 212 having
three layers, according to an embodiment. FIG. 2A shows a top layer
202 of device 212, according to an embodiment. FIG. 2B shows a
middle layer 206 of device 212, according to an embodiment. FIG. 2C
shows a bottom layer 210 of device 212, according to an embodiment.
FIG. 2D shows the device 212 as assembled, according to an
embodiment. Each of the three layers 202, 206, 210 may be made of
any material compatible with the assays the device 212 is intended
to perform.
[0062] In some embodiments, layer 202 will be fabricated from a
material which is transparent to a specific wavelength, or
wavelength range, of light. As used herein, "transparent" should be
understood to mean that the material has sufficient transmittance
to allow the amount of light having a specific wavelength or range
of wavelengths on one side of the material to be quantified by a
detector on the other side. In some instances, material with a
transmissivity of 30%, 50%, 80%, 95%, or 100% is transparent. In
some embodiments, a wavelength range of interest will include the
middle ultraviolet range (e.g., 200 nm-300 nm), and materials such
as, for example, glass, quartz, fused silica and UV-transparent
plastics such as polycarbonates, polyfluorinated polyethylene,
polymethacrylate, cyclic olefin polymer, cyclic olefin copolymer,
and other UV-transparent materials can be used as transparent
materials. In some embodiments, the light spectrum of interest will
be expanded beyond the visible spectrum (e.g., 200-900 nm).
[0063] Through-holes, 204, are fabricated in layer 202 to allow
pressure and electrical interface to a channel network in a lower
layer (e.g., layer 208) from outside the device.
[0064] FIG. 2B shows the internal middle layer 206 of device 212
containing the channel network 208. The channel network is designed
to interface with the through-holes fabricated in the top layer
202. The channel network 208 contains inlet and outlet
tubes/conduits 209, and orifice 205 for expelling enriched analyte
fractions, and a viewable enrichment zone 207. Enrichment zone 207
is fabricated so its depth is the full thickness of the layer 206.
In other embodiments, zone 207 can be less than the full thickness
of layer 206.
[0065] In some embodiments, layer 206 will be fabricated from a
material which is opaque and/or not transparent to a specific
wavelength, or wavelength range, of light. As used herein, "opaque"
should be understood to mean the material has insufficient
transmittance to allow the amount of light on one side of the
material to be quantified by a detector on the other side, and will
effectively block this light except in the regions where the zone
in the channel network is as deep as the full thickness of layer
206.
[0066] FIG. 2C shows a bottom layer 210 of device 212. Bottom layer
210 can be, for example, a solid substrate. In some embodiments,
bottom layer 210 can be fabricated from a material with the same
transmittance as layer 202.
[0067] FIG. 2D shows the device 212 including top layer 202, the
middle layer 206, and the bottom layer 210, as assembled, according
to an embodiment. Inlet and outlet tubes 209, reservoirs 204 and
orifice 205 can still be accessed after the device 210 is
assembled. In some embodiments, the entire top layer 202 and/or the
entire bottom layer 210 can be transparent. In other embodiments, a
portion of the top layer 202 and/or a portion of the bottom layer
210 can be opaque with another portion of the top layer 202 and/or
the bottom layer 210 being transparent. For example, the top layer
210 and/or the bottom layer 210 can define an optical window that
aligns with at least a portion of the enrichment zone 207 when the
device 212 is assembled.
[0068] FIG. 3 is a schematic of a light path through a microfluidic
device 302, according to an embodiment. FIG. 3A shows a top view of
the microfluidic device 302. FIG. 3B shows the microfluidic device
302 positioned between a light source 306 and a detector 308. The
detector 308 is positioned to measure light passing through the
device 302. While not illustrated in FIG. 3, the microfluidic
device 302 can have a similar channel structure as described in
FIGS. 1 and 2, but the channel structure is not shown for ease of
reference. In some embodiments, a portion of top surface of the
microfluidic device 302 is opaque and completely or substantially
obscures light projected from the light source 306 from reaching
the detector 308. The portion of the opaque top surface
substantially prevents the transmission of light through the device
at those portions where detection of sample properties is not
desired. For example, the microfluidic device 302 in some
embodiments is not opaque (e.g., allows some light to pass through)
over one or more channel region(s) 304, as the channel 304
transverses the entire thickness of a non-transparent layer.
[0069] In some embodiments, this transparent channel region(s) 304,
can be an enrichment zone, where optical detection can be used to
detect analyte, monitor the progress of the enrichment and/or
monitor enriched analyte fraction(s) as they are expelled from the
device. In some embodiments, changes in the amount of light passing
through transparent channel 304 will be used to measure the
absorbance of the analyte fractions while they are in this channel.
Thus, in some embodiments, channel region(s) 304 define an optical
slit, such that the light source 306 positioned on one side of the
microfluidic device 302 effectively illuminates the detector 308
only through the transparent channel region(s) 304. In this way,
stray light (e.g., light that does not pass thorough the
transparent channel regions(s) and/or a sample) can be effectively
blocked from the detector 308, which can reduce noise and improve
the ability of the detector 308 to observe sample within the
transparent channel region(s) 304. In some embodiments, the
transparent channel regions(s) 304 will be between two enrichment
zones, and can be used to detect analyte fractions as they are
eluted from the upstream enrichment zone.
[0070] Methods
[0071] FIG. 6 illustrates a method of analyte mixture enrichment
according to an embodiment. The method includes loading and/or
introducing an analyte mixture onto a microfluidic device, at 20.
The microfluidic device can be similar to the microfluidic devices
described above with reference to FIGS. 1-3. In some embodiments,
the analyte mixture can be, for example, glycans, carbohydrates,
DNA, RNA, intact proteins, digested proteins, peptides,
metabolites, vaccines, viruses and small molecules. In some
embodiments, the analyte mixture can be a mixture of proteins, such
as a lysate of cultured cells, cell-based therapeutics, or tumor or
other tissue derived cells, recombinant proteins, including
biologic pharmaceuticals, blood derived cells, perfusion or a
protein mixture from any other source. The analyte mixture may be
loaded directly onto the device, or may be loaded onto an
autosampler for serial analysis of multiple mixtures.
[0072] The microfluidic device can include a first separation
channel and/or enrichment zone. In some embodiments, the first
separation channel and/or enrichment zone can be configured for
chromatographic separation. For example, the first separation
channel and/or enrichment zone can contain a media configured to
bind an analyte from the analyte mixture and/or otherwise effect a
chromatographic separation. At 21, a first enrichment can be
performed; for example, a chromatographic separation can be
performed in the first separation channel and/or enrichment zone.
In some embodiments, such as embodiments in which the analyte
mixture is a protein mixture, the first enrichment, at 21, can
simplify the protein mixture. The first enrichment, at 21, can be
based on any discernable quality of the analyte.
[0073] This enriched analyte fraction is then eluted, at 22. For
example, an eluent can be injected into the microfluidic device to
mobilize the enriched analyte fraction from media disposed within
the first separation channel and/or enrichment zone. In some
embodiments, the enrichment and/or mobilization of the enriched
analyte fraction can be imaged. For example, as discussed above,
the first separation channel and/or enrichment zone can define an
optical slit. Light can be projected onto the microfluidic device
and a detector can detect light passing through the first
separation channel and/or enrichment zone. The sample, or a portion
thereof can be detected via absorbance and/or fluorescence imaging
techniques.
[0074] The microfluidic device can include a second separation
channel and/or enrichment zone. In some embodiments, the second
separation channel and/or enrichment zone can be configured for
electrophoretic separation. At 23, a second enrichment can be
performed, for example, on the eluate. For example, an electric
field and/or electric potential can be applied across the second
separation channel and/or enrichment zone.
[0075] In some embodiments, the second enrichment can be initiated,
at 23, when a fraction of the analyte mixture is disposed at an
intersection of the first separation channel and/or enrichment zone
and the second separation channel and/or enrichment zone. For
example, the first separation channel and/or enrichment zone can be
monitored (e.g., imaged) and an electric potential, and/or electric
field can be applied when a fraction of interest reaches the
intersection.
[0076] In some embodiments, the second enrichment, at 23, can
provide fractions enriched based on charge characteristics (charge
isoforms). Such enrichments can include, for example, gel
isoelectric focusing, isoelectric focusing with mobilization,
isoelectric focusing with whole column imaging, ion exchange
chromatography, pH gradient exchange chromatography,
isotachophoresis, capillary zone electrophoresis, capillary gel
electrophoresis or other enrichment techniques that are, for
example, charge-based.
[0077] Although the first enrichment, at 21, has been described as
a chromatographic enrichment and the second enrichment, at 23, has
been described as electrophoretic, it should be understood the any
suitable enrichment can be performed in any suitable sequence. For
example, the first enrichment, at 21, and the second enrichment, at
23, can both be chromatographic or both be electrophoretic. As
another example, the first enrichment, at 21, can be
electrophoretic, and the second enrichment, at 23, can be
chromatographic.
[0078] In some embodiments, one or more enrichments can provide
fractions enriched based on hydrophobic changes, such as oxidation.
Such enrichments can include, for example, reversed-phase
chromatography, hydrophobic interaction chromatography, hydrophilic
interaction chromatography, or other enrichment techniques that
are, for example, hydrophobicity-based.
[0079] In some embodiments, one or more enrichments can will
provide fractions enriched based on post-translational
modifications, glycoforms including galactosylation, fucosylation,
sialylation, mannose derivatives and other glycosylations, as well
as glycation, oxidation, reduction, phosphorylation, sulphanation,
disulfide bond formation, deamidiation, acylation, pegylation,
cleavage, antibody-drug conjugation (ADC), protein-drug
conjugation, C-terminal lysine processing, other naturally and
non-naturally occurring post-translational modifications and other
chemical and structural modifications introduced after translation
of the protein, and the like. Such enrichments can include, for
example, binding assays and the like.
[0080] In some embodiments, one or more enrichments can provide
fractions enriched based on hydrophobic changes, such as oxidation.
Such enrichments can include, for example, reversed-phase
chromatography, hydrophobic interaction chromatography, hydrophilic
interaction chromatography, or other enrichment techniques that are
hydrophobicity-based.
[0081] In some embodiments, one or more enrichments can provide
fractions enriched based on primary amino acid sequence, such as
caused by mutation, amino acid substitution during manufacture and
the like. Such enrichments can include, for example, separating by
charge isoforms, hydrophobic changes, or other enrichment
techniques that can distinguish between primary amino acid sequence
differences.
[0082] In some embodiments, one or more enrichments can provide
fractions enriched based on efficacy. Such enrichments can include,
for example, bioassays, enzyme inhibition assays, enzyme activation
assays, competition assays, fluorescence polarization assays,
scintillation proximity assays, or other enrichment techniques that
are efficacy-based and the like.
[0083] In some embodiments, one or more enrichments can provide
fractions enriched based on affinity. Such enrichments can include,
for example, solution phase binding to target, binding to bead
based targets, surface bound target, immunoprecipitation, protein A
binding, protein G binding and the like.
[0084] In some embodiments, one or more enrichments can provide
fractions enriched based on mass or size. Such enrichments can
include, for example, poly acrylamide gel electrophoresis,
capillary gel electrophoresis, size exclusion chromatography, gel
permeation chromatography, or other enrichment techniques that are
mass-based.
[0085] In some embodiments, the analyte mixture will go through
more than two enrichments and/or enrichment channels before being
expelled from the device.
[0086] At 24, an enriched analyte fraction can be expelled from the
device. In some embodiments, the enriched analyte fraction can be
expelled via IEF. Expelling the enriched analyte fraction, at 24,
can concentrate the analyte fractions before they are expelled
from.
[0087] In some embodiments the analyte fractions are expelled, at
24, using an ionization technique, such as electrospray ionization,
atmospheric pressure chemical ionization, and the like.
[0088] In some embodiments, the analyte fractions are expelled, at
24, using electrokinetic or hydrodynamic forces.
[0089] In some embodiments, the enriched protein fractions are
expelled, at 24, from the device in a manner coupled to a mass
spectrometer.
[0090] Mass of an analyte expelled from the microfluidic device
(e.g., a biologic or biosimilar) can be measured, for example,
through time-of-flight mass spectrometry, quadrupole mass
spectrometry, Ion trap or orbitrap mass spectrometry,
distance-of-flight mass spectrometry, Fourier transform ion
cyclotron resonance, resonance mass measurement, and nanomechanical
mass spectrometry.
[0091] In some embodiments pI markers are used to map pI ranges in
the visualized IEF channel (e.g., the first separation channel
and/or enrichment zone and/or the second separation channel and/or
enrichment zone). In some embodiments, pI markers or ampholytes can
be used to determine the pI of the analyte by their presence in
downstream mass spectrometry data.
[0092] In some embodiments, IEF can be monitored during the
mobilization and ESI. In this way, mass spectrometry data can be
correlated to peaks in the IEF, which can maintain and/or improve
peak resolution.
[0093] In some embodiments, the analyte mixture and/or a portion
thereof can be mobilized within the microfluidic device using
pressure source. In some embodiments, mobilization is done with
hydrostatic pressure. In some embodiments, mobilization is chemical
immobilization. In some embodiments, mobilization is electrokinetic
mobilization
[0094] FIG. 7 is a schematic of a microfluidic device, according to
an embodiment. A microfluidic network, 800, is disposed in and/or
defined by a substrate, 802. The substrate is manufactured out of
material which is compatible with the enrichment steps being
performed. For example, chemical compatibility, pH stability,
temperature, transparency at various wavelengths of light,
mechanical strength, and the like may be of concern when selecting
the material
[0095] Substrate 802 may be manufactured out of glass, quartz,
fused silica, plastic, polycarbonate, PTFE, PDMS, silicon,
polyfluorinated polyethylene, polymethacrylate, cyclic olefin
copolymer, cyclic olefin polymer, polyether ether ketone and/or any
other suitable material. Mixtures of materials can be utilized if
different properties are desired in different layers of a planar
substrate.
[0096] Channels 806, 808, 810, 811, 817, 814, 812 form a channel
network and are fabricated into (e.g., defined by) substrate
802.
[0097] Channels may be fabricated in the substrate through any
channel fabrication method such as photolithographic etching,
molding, machining, additive (3D) printing, and the like.
[0098] Analyte mixtures and external reagents can be loaded through
tube 804, and excess reagent/waste can be removed through tube 810
and 818.
[0099] Tubes 804 810, and/or 818 can be manufactured out of any
material compatible with the assay being performed, including fused
silica, fused silica capillary tubes, silicone tubing, PTFE tubing,
and the like.
[0100] Channels 806 and 814 can be designated as
separation/enrichment zones. Either of channel 806 and/or 814 can
be used to perform chromatographic separations (reversed phase,
immunoprecipitation, ion exchange, size exclusion, ligand affinity,
dye affinity, hydrophobic interaction, affinity, capillary
electrokinetic chromatography, micellar electrokinetic
chromatography and/or the like) or electrophoretic separations
(isoelectric focusing, capillary gel electrophoresis, capillary
zone electrophoresis, isotachophoresis, capillary electrokinetic
chromatography, micellar electrokinetic chromatography, flow
counterbalanced capillary electrophoresis, electric field gradient
focusing, dynamic field gradient focusing, and/or the like). For
example, channel 806 can be derivatized or packed with material to
perform a first enrichment step, represented by darker circles in
channel 806.
[0101] The material disposed into channel 806 can be selected to
capture analytes based on hydrophobicity (reversed phase), affinity
(efficacy), size (size exclusion chromatography), charge (ion
exchange), immunoaffinity (immunoprecipitation), protein-protein
interaction, DNA-protein interaction, aptamer-base capture, small
molecule-base capture or by other forms of liquid chromatography
and the like.
[0102] Many different methods can be used to dispose the enrichment
material within channel 806 and/or 814. The walls can be directly
derivatized with covalently bound or adsorbed molecules, or beads,
glass particles, sol-gel or the like can be derivatized and loaded
into these channels, or channels can be packed with a sieving
material such as--linear polymer solutions such as linear
polyacrylamide (LPA), polyvinylpyrrolidone (PVP), polyethylene
oxide (PEO), dextran, and the like, cross-linked polymer solutions
such as polyacrylamide and the like, matrices for liquid
chromatography, or other materials.
[0103] Chemically reactive solutions may be added depending on the
particular assay performed. In some cases, derivatization of
material may occur after it is loaded into channel 806 (or channel
814), by adding molecules which will adsorb or covalently bond to
the loaded material, or can chemically cross link reactive elements
to the material. For example, material coated with an
antibody-binding molecule such as protein A, protein G, epoxy or
the like, could be disposed into channel 806. Subsequent rinsing
with an antibody solution would leave the material coated with
antibody and able to participate in immunoaffinity capture. In some
cases, the antibody may be mixed with a target analyte or lysate so
that the antibody can bind its target in free solution before being
coated onto the material.
[0104] After enrichment materials are loaded onto device, sample is
loaded via tube 804 into channel 806. Subsequently, wash solutions
and elution reagents can be introduced through tube 804 to channel
806.
[0105] In some cases, detection reagents will be added to bind to
captured material. Numerous labeling reagents are available that
can covalently attach detection moieties such as fluorophores,
chromophores or other detection molecules to the target proteins at
terminal ends of the polypeptide, and by attachment to amino acid
side chains such as lysine, cysteine and other amino acid moieties.
Covalently bound detection moieties allow for the protein to be
detected through fluorescence excitation, chromophoric assay, or
other indirect means. In some cases, the target protein can remain
unlabeled and detected through native absorbance at 220 nm, 280 nm
or any other wavelength at which the protein will absorb light, or
native fluorescence. In some cases, the protein will be detected
using non-covalently bound fluorogenic, chromogenic, fluorescent or
chromophoric labels, such as SYPRO.RTM. ruby, Coomassie blue and
the like.
[0106] In some cases, detection reagents will be added directly to
channel 814 to aid detection.
[0107] The elution process will depend on the enrichment method
performed in channel 806. It will be selected to elute at least a
fraction of the bound analyte. In some cases, this can be
accomplished with a combination of heat and sodium dodecyl sulfate
(SDS), or other detergents, glycine, urea, or any other method
which will induce the release of the captured analyte. Some
enrichment options may not require a direct elution step (e.g. size
exclusion chromatography). In some cases, elution will be followed
by denaturation.
[0108] The eluent would then flow through channel 808 into the next
separation/enrichment zone, channel 814. Channel 814 could be used
to perform either a chromatographic or electrophoretic enrichment
step.
[0109] Electrophoretic separations can be performed in channel 814
by using a power supply to apply an electric field between
reservoir 812 and reservoir 816. When eluate from channel 806
passes through the intersection of channels 808 and 814, the
electric field can be enabled, loading analyte into channel 814. In
some case, the analyte will be negatively charged, such as in the
standard gel electrophoresis mode where protein analyte is
saturated with a negatively charged detergent like SDS. However,
the polarity of channel 814 can easily be reversed to accommodate
systems where for example, a protein analyte is saturated with a
positively charged detergent such as cetyl trimethylammonium
bromide (CTAB) or the like. In other cases, a protein analyte may
be coated with a neutral detergent, or no detergent--such as in
native gel electrophoresis. In this case, polarity will be selected
based on the anticipated charge of the protein target in the buffer
system selected, so that the protein analyte will migrate into
channel 814.
[0110] Any CE electrophoretic method can be performed in channel
814--IEF, ITP, CGE, CZE, and the like. Alternately,
non-electrophoretic enrichment methods can be performed in the
channel.
[0111] Analyte in channel 814 can be viewed by whole column
imaging, partial column imaging, and/or by single point
detection.
[0112] In some cases, the enrichment material in channels 806, 814
or both may be removed and replenished with fresh material so that
the device can be used on another analyte sample.
[0113] In some cases, a channel design such as FIG. 7 may be
repeated multiple times on a device, so that more than one analyte
sample may be analyzed in parallel.
EXAMPLES
[0114] Aspects of embodiments may be further understood in light of
the following examples, which should not be construed as limiting
in any way.
Example 1
Characterize Protein Charge on Chip Before Mass Spectrometry
(MS)
[0115] For this example, the channel network shown in FIG. 4 is
fabricated from a plate of soda lime glass, which has very low
transmission of 280 nm light using a standard photolithographic
etching technique. The depth of the enrichment channel 418 is the
same as the thickness of the glass layer 402, i.e., the enrichment
channel 418 passes all the way from the top to bottom of this glass
plate 402. The device 400 can be illuminated by a light source
disposed on one side of device 400 and imaged by a detector on
disposed on an opposite side of device 400. Because substrate 402
is opaque, but enrichment channel 418 defines an optical slit, the
substrate 402 can block light that does not pass through the
enrichment channel 418, blocking stray light and improving
resolution of the imaging process.
[0116] The glass layer 402 is sandwiched between two fused silica
plates, which are transmissive (e.g., transparent) to 280 nm light.
As in FIG. 2, the top plate contains through holes for the
instrument and user to interface with the channel network, while
the bottom plate is solid. The 3 plates are bonded together at
520.degree. C. for 30 minutes. The inlet and outlet tubing is
manufactured from cleaved capillary (100 .mu.m ID, polymicro),
bonded to the channel network.
[0117] The device is mounted on an instrument containing a nitrogen
gas source, heater, positive pressure pump (e.g., Parker,
T5-1IC-03-1EEP), electrophoresis power supply (Gamm High Voltage,
MC30) terminating in two platinum-iridium electrodes (e.g.,
Sigma-Aldrich, 357383), UV light source (e.g., LED, qphotonics,
UVTOP280), CCD camera (e.g., ThorLabs, 340UV-GE) and an autosampler
for loading samples onto the device. The power supply shares a
common earth ground with the mass spectrometer. The instrument is
controlled through software (e.g., labView).
[0118] Protein samples are pre-mixed with ampholyte pH gradient and
pI markers before placing into vials and loading onto the
autosampler. They are serially loaded from an autosampler via the
inlet 412 onto the microfluidic device 400 through the enrichment
channel 418 and out of the device to waste 430 through the outlet
434.
[0119] The sheath/catholyte fluid (50% MeOH, N.sub.4OH/H.sub.2O) is
loaded onto the two catholyte wells 404, 436, anolyte (10 mM
H.sub.3PO.sub.4) onto the anolyte well 426, and the source of
heated nitrogen gas is attached to the two gas wells 408, 440.
[0120] After all reagents are loaded, an electric field of +600
V/cm is applied from anolyte well 426 to catholyte wells 404, 436
by connecting the electrodes to the anolyte well 426 and catholyte
wells 404, 436 to initiate isoelectric focusing. The UV light
source is aligned under the enrichment channel 418, and the camera
is placed above the enrichment channel 418 to measure the light
that passes through the enrichment channel 418, thereby detecting
the focusing proteins by means of their absorbance. The glass plate
402, being constructed of soda-lime glass, acts to block any stray
light from the camera, so light not passing through the enrichment
channel 418 is inhibited from reaching the camera, increasing
sensitivity of the measurement.
[0121] Images of the focusing proteins can be captured continuously
and/or periodically during IEF. When focusing is complete, low
pressure will be applied from the inlet 412, mobilizing the pH
gradient toward the orifice 424. The electric field can be
maintained at this time to maintain the high resolution IEF
separation. Continuing to image the enrichment channel 418 during
the ESI process can be used to determine the pI of each protein as
it is expelled from the orifice 424.
[0122] As the enriched protein fraction moves from the enrichment
channel 418 into the confluence 420, it will mix with the sheath
fluid, which can flow from the catholyte wells 404, 436 to the
confluence 420 via sheath/catholyte fluid channels 406, 438. Mixing
enriched protein fractions with the sheath fluid can put the
protein fraction in a mass spectrometry compatible solution, and
restore charge to the focused protein (IEF drives proteins to an
uncharged state), improving the ionization.
[0123] The enriched protein fraction then continues on to the
orifice 424, which can be defined by a countersunk surface 422 of
the glass plate 402. The enriched protein fraction can create a
Taylor cone once caught in the electric field between the sheath
fluid well ground and mass spectrometer negative pole.
[0124] As solution continues to push at the Taylor cone from the
enrichment channel 418, small droplets of fluid will be expelled
from the Taylor cone and fly towards the mass spectrometer inlet.
Nitrogen gas (e.g., at 150.degree. C.) can flow from the gas wells
408, 440, down gas channels 410, 432 and form nitrogen gas jets
which flank the Taylor cone which can convert droplets emanating
from the Taylor cone to a fine mist before leaving the microfluidic
device, which can aid detection in the mass spectrometer. Adjusting
pressure from the inlet 412 can adapt Taylor cone size as needed to
improve detection in mass spectrometer.
Example 2
Reversed-Phase.fwdarw.IEF.fwdarw.MS
[0125] Example 2 can be similar to example 1, but is described with
reference to FIG. 1. The channel 116 can be a first enrichment zone
loaded with sol-gel derivatized with C18. After loading protein, a
volume of eluent (MeCN/H.sub.2O with IEF ampholytes and standards)
can be loaded into channel 116 to elute the least hydrophobic
proteins trapped on the sol gel. The eluate is directed to channel
124, which can be a second enrichment zone where IEF, UV absorbance
monitoring and finally ESI take place as described in example 1.
Once the ESI of the first eluate is complete, a volume of higher
MeCN concentration is used to elute the next lowest hydrophobic
protein fraction.
Example 3
Efficacy.fwdarw.IEF.fwdarw.MS
[0126] Example 3 can be similar to example 2, but biologic drug
target derivatized beads can be loaded into channel 116 and used to
capture protein. Affinity of reaction is characterized through
elution by solution phase target (competitive), salt, pH, or the
like.
Example 4
Reversed-Phase.fwdarw.Capillary Zone Electrophoresis.fwdarw.MS
[0127] Example 4 can be similar to example 2, but is described with
reference to FIG. 5. A protein mixture can be loaded through inlet
521 and pass through to enrichment zone 510, which can contain
beads derivatized with C18 for reversed-phase chromatography.
During loading, fluid passes through the zone 510, through viewing
region 511 and out outlet 522 to waste. Viewing region 510
transverses an internal layer made of soda-lime glass, which is
opaque to 280 nm UV light, while the top and bottom layers are made
of fused silica, which are transparent to 280 nm light.
[0128] A 280 nm light source is positioned below viewing region 511
and a CCD detector is placed above viewing region 511.
[0129] A solution of 20% MeCN/H.sub.2O is loaded through inlet 521
through enrichment zone 510. This solution will elute a fraction
enriched for the least hydrophobic proteins in the mixture. Viewing
region 511 is monitored for the absorbance of the enriched protein
fraction at 280 nm as it moves from enrichment zone 510 to the
outlet 522. When the fraction is positioned at the intersection of
enrichment zone 510 and enrichment zone 515, a power supply is
turned on creating an electric field between a positive electrode
in reservoir 514 and ground at reservoir 504. This polarity can
easily be reversed by switching the polarity of the power supply.
Once the electric field is present, the enriched protein fraction
will migrate down enrichment zone 515 separating proteins by
capillary zone electrophoresis. The separated proteins will mix
with the sheath, electrolyte solution at confluence 516, and form a
Taylor cone on surface 518. Nebulizing Nitrogen gas line is
connected to the device at ports 508 and 528, and moves through
channels 512 and 530 to flank material from the electrospray as it
exits the device via orifice 520.
[0130] Alternatively, hydrodynamic pressure could be used to load
the enriched protein fraction into enrichment zone 515.
Example 5
Immunoprecipitation.fwdarw.Capillary Gel Electrophoresis of Protein
Lysates
[0131] In this example, a microfluidic channel layer represented by
the layout in FIG. 7 is fabricated from a cyclic olefin copolymer.
Similarly stated, substrate 802 of microfluidic device 800 defines
a channel network. For many applications, for example, if
fluorescent detection is employed, microfluidic device 800 could be
manufactured using a single material, provided that this material
will transmit the wavelength range of light needed to detect the
analyte.
[0132] Protein A coated beads are loaded into channel 806. These
beads are rinsed with a solution of antibody raised against a
target of interest, which will bind to the protein A beads. To
reduce antibody shedding interfering with analyte detection, the
antibody is then covalently cross-linked to the antibody to the
bead using commercially available cross linking reagents, such as
Dimethyl pimelimidate (DMP), Bis(sulfosuccinimidyl)suberate (BS3)
and the like. After immunoprecipitation beads are prepared and
loaded in channel 806, lysate analyte sample can be loaded via tube
804. After analyte is given sufficient time to be captured by
immobilized antibody, unbound proteins are washed and cleared to
waste via tube 822.
[0133] Next, the protein is eluted from the antibody beads so it
can be analyzed. Elution is accomplished by loading solution of
sodium dodecyl sulfate (SDS) and heating to 50 C for 10 minutes.
Once released, the eluted analyte is flowed through channel 808
toward the intersection of channel 808 and 814. When the analyte
plug reaches the intersection of channel 808 and 814, an electric
field is turned on between a negative pole at reservoir 812 and a
positive pole at reservoir 816, causing the negatively charged
protein to migrate through a dextran linear polymer solution in
channel 814, which has been loaded with the fluorogenic protein dye
SYPRO.RTM. ruby.
[0134] Fluorescently labeled target protein can be visualized
during CGE in channel 814 using whole column imaging. Similarly
stated, the entirety of channel 814 can be imaged while the
SYPRO.RTM. ruby dye is excited with 280 nm light and emitted light,
at 618 nm, is measured by a detector.
Example 6
Variations of Microfluidic Design Without Mass Spectrometer
Interface
[0135] In some cases, it will be advantageous to have two designs
of a microfluidic layer, that differ by presence or absence of the
mass spectrometer interface. Once an analyte is characterized,
confirmatory characterization may be done in the absence of the
mass spectrometry data. By doing the confirmatory characterization
in nearly the same microfluidic design, when an anomaly is
identified, it will be simple to transfer the assay back to the
chip with the mass spec interface for mass identification. This can
eliminate the work otherwise needed to show that the anomaly in the
confirmatory data is being analyzed in the mass spectrometry
data.
[0136] As an example, FIG. 8 shows a microfluidic design similar to
microfluidic device 400 shown in FIG. 4, without orifice 424 and
countersunk surface 422. Analyte is still introduced to the chip
through an inlet 904 and channel 906 to an enrichment channel 908,
but after analysis the sample will be flushed out through an outlet
channel 910, rather than conducting electrospray ionization at an
orifice. This design could be run for general operation, and then
at times when mass identification is required, the same enrichment
can be performed on the microfluidic device 400, shown in FIG. 4,
ensuring identification of the analyte variants see on microfluidic
device 900 of FIG. 8.
[0137] The foregoing descriptions of specific embodiments of the
invention have been presented for purposes of illustration and
description. They are not intended to be exhaustive or to limit the
invention to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. Although various embodiments have been described as
having particular features and/or combinations of components, other
embodiments are possible having a combination of any features
and/or components from any of embodiments where appropriate. The
embodiments were chosen and described in order to best explain the
principles of the invention and its practical application, to
thereby enable others skilled in the art to best utilize the
invention and various embodiments with various modifications as are
suited to the particular use contemplated. It is intended that the
scope of the invention be defined by the claims appended hereto and
their equivalents.
[0138] Where methods and/or schematics described above indicate
certain events and/or flow patterns occurring in certain order, the
ordering of certain events and/or flow patterns may be modified.
Additionally certain events may be performed concurrently in
parallel processes when possible, as well as performed
sequentially. While the embodiments have been particularly shown
and described, it will be understood that various changes in form
and details may be made.
[0139] All patents, patent applications, publications, and
references cited herein are expressly incorporated by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
* * * * *