U.S. patent application number 11/524426 was filed with the patent office on 2007-01-25 for multicapillary device for sample preparation.
Invention is credited to Yuri P. Belov, Ksenia Lvova, Carlo G. Pantano.
Application Number | 20070017870 11/524426 |
Document ID | / |
Family ID | 46123976 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070017870 |
Kind Code |
A1 |
Belov; Yuri P. ; et
al. |
January 25, 2007 |
Multicapillary device for sample preparation
Abstract
A multicapillary sample preparation device, especially useful
for handling biological samples, comprising a plurality of uniform
capillary tubes coated with a stationary phase, and arranged in a
monolithic element. The multicapillary device is suitable for
attachment to a pipette, micropipette, syringe, or other analytical
or sample preparation instrument.
Inventors: |
Belov; Yuri P.; (State
College, PA) ; Pantano; Carlo G.; (Pennsylvania
Furnace, PA) ; Lvova; Ksenia; (State College,
PA) |
Correspondence
Address: |
MCQUAIDE BLASKO
811 UNIVERSITY DRIVE
STATE COLLEGE
PA
16801
US
|
Family ID: |
46123976 |
Appl. No.: |
11/524426 |
Filed: |
September 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10955377 |
Sep 30, 2004 |
|
|
|
11524426 |
Sep 20, 2006 |
|
|
|
60507474 |
Sep 30, 2003 |
|
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Current U.S.
Class: |
210/656 ;
210/198.2; 422/70; 436/161 |
Current CPC
Class: |
G01N 2030/567 20130101;
G01N 30/6043 20130101; G01N 30/56 20130101; G01N 30/6078
20130101 |
Class at
Publication: |
210/656 ;
210/198.2; 436/161; 422/070 |
International
Class: |
B01D 15/08 20060101
B01D015/08 |
Claims
1. A sample preparation device, especially useful for handling
biological samples, comprising: (a) an element for receiving a
sample at an upper end thereof and discharging a concentrated,
purified, or separated sample at a lower end of the element, said
element defining a cavity; (b) a plurality of capillary tubes
arranged within said cavity, each capillary tube defining an inner
bore having an inner wall and first and second openings; and (c) a
housing of substantially cylindrical or conical configuration for
retaining the multicapillary element, said housing being suitable
for attachment to a sample preparation or analytical
instrument.
2. The sample preparation device of claim 1 wherein a stationary
phase is deposited on the inner wall of said capillary tubes
without an intermediary constituent.
3. The sample preparation device of claim 2 wherein the inner bore
of said capillary tubes is unobstructed by said stationary phase;
and the stationary phase comprises a thickness that is correlated
with the radius of individual capillary tubes for high
efficiency.
4. The sample preparation device of claim 2 or 3 wherein the
stationary phase is insoluble.
5. The sample preparation device of claim 4 wherein the insoluble
stationary phase is chemically bonded to the inner wall of said
capillary tubes.
6. The sample preparation device of claim 4 wherein the insoluble
stationary phase is cross-linked to the inner wall of said
capillary tubes.
7. The sample preparation device of claim 4 wherein the insoluble
stationary phase is both cross-linked and chemically bonded to the
inner wall of said capillary tubes.
8. The sample preparation device of claim 2 or 3 wherein the
thickness of said stationary phase coating is proportional to the
radius of said capillary tubes in power n, where n is greater than
1.
9. The sample preparation device of claim 2 or 3 wherein the
following relationship holds: d.sub.f(r)=c.sub.fr.sup.n where
d.sub.f=insoluble stationary phase thickness; c.sub.f=constant;
r=capillary radius; and n>1.
10. A sample preparation device, especially useful for handling
biological samples, comprising: (a) an element for receiving a
sample at an upper end thereof and discharging a concentrated,
purified, or separated sample at a lower end of the element, said
element defining a cavity; (b) a plurality of capillary tubes
arranged within said cavity, each capillary tube defining an inner
bore having an inner wall and first and second openings, said
capillary inner wall having an imperforate structure; (c) a housing
of substantially cylindrical or conical configuration for retaining
the multicapillary element, said housing being suitable for
attachment to a sample preparation or analytical instrument; and
(d) wherein the imperforate structure of said capillary inner wall
prevents the sample from diffusing from one capillary tube to
another during separation.
11. The sample preparation device of claim 10 wherein a stationary
phase is deposited on the imperforate inner wall of said capillary
tubes without an intermediary constituent.
12. The sample preparation device of claim 11 wherein the inner
bore of said capillary tubes is unobstructed by said stationary
phase; and the stationary phase comprises a thickness that is
correlated with the radius of individual capillary tubes for high
efficiency.
13. The sample preparation device of claim 11 or 12 wherein the
stationary phase is insoluble so as to prevent discharge of said
stationary phase into a mobile phase during separation.
14. The sample preparation device of claim 2 or 3 wherein the
following relationship holds: d.sub.f(r)=c.sub.fr.sup.n where
d.sub.f=insoluble stationary phase thickness; c.sub.f=constant; r
=capillary radius; and n>1.
15. The sample preparation device of claim 1 or 10 wherein said
multicapillary element is formed of fused silica, glass, ceramic,
stainless steel, or polyetheretherketone.
16. The sample preparation device of claim 1 or 10 wherein an inner
diameter of one or more of said capillary tubes is in the range of
about 0.1 .mu.m to about 200 .mu.m.
17. The sample preparation device of claim 1 or 10 wherein an outer
diameter of said multicapillary element is in the range of about
0.1 mm to about 20 mm.
18. The sample preparation device of claim 1 or 10 wherein a length
of said multicapillary element is in the range of about 0.1 mm to
about 250 mm.
19. The sample preparation device of claim 1 or 10 wherein a volume
of said housing is in the range of about 0.1 .mu.L to about 100
mL.
20. The sample preparation device of claim 1 or 10 wherein said
housing is formed of polyolefin, polyetheretherketone, glass, fused
silica, ceramic, or stainless steel.
21. The sample preparation device of claim 1 or 10 wherein the
inner wall of said capillary tubes includes particles of inert
material for increasing surface area of the tubes.
22. The sample preparation device of claim 1 or 10 wherein the
inner wall of said capillary tubes includes a nodular surface for
increasing surface area of the tubes.
23. The sample preparation device of claim 1 or 10 wherein said
sample comprises a protein, peptide, polynucleotide, biopolymer,
virus, spore, cell, microorganism, nucleic acid, or other
biological specimen.
24. The sample preparation device of claim 1 or 10 wherein said
housing comprises a tip, pipette, micropipette, or syringe.
25. A method of preparing a sample preparation device, especially
useful for handling biological samples, comprising: (a) introducing
a stationary phase solution into an element containing a plurality
of capillary tubes for receiving a sample at a first end of the
element and discharging a separated sample at a second end of the
element; (b) simultaneously exposing the element to an environment
that facilitates evaporation of the solution; (c) cross-linking or
chemically bonding the stationary phase to the interior of said
capillary tubes; and (d) wherein the stationary phase comprises a
thickness that is correlated with the radius of individual
capillary tubes for high efficiency.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. patent application
No. 10/955,377 filed Sep. 30, 2004, and U.S. Provisional Patent
Application No. 60/507,474 filed Sep. 30, 2003.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a multicapillary sample
preparation device especially useful for handling biological
samples. In particular, the multicapillary device is suitable for
use with a pipette, micropipette, syringe, or other similar
analytical instrument.
[0004] 2. Background Art
[0005] Many biological samples are commonly separated by gel
electrophoresis and analyzed by matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS). One
disadvantage of these techniques, however, is that analysis is
strongly affected by the presence of salts, buffers and low
molecular weight organic compounds commonly used in the preparation
of biological samples. In order to improve the sensitivity and
selectivity of analyses, adsorptive and membranous devices are
frequently used to purify and concentrate the sample prior to
analysis. Such devices feature a bed of porous adsorbent or a
semipermeable membrane fixed in a housing of a suitable dimension
and shape that traps desired constituents, while allowing
contaminants to pass.
[0006] To handle samples in the 0.01 to 100 microgram (.mu.g)
range, pipettes, micropipettes, syringes or similar analytical
instruments (collectively referred to hereinafter as "pipettes")
are commonly employed. The tip of these pipettes is fitted with one
or more adsorptive or membranous plugs capable of purifying,
concentrating, or fractionating peptides and other
biomolecules.
[0007] A principal shortcoming of adsorptive and membranous plugs,
however, is that porous materials are generally not effective at
separating smaller biomolecules such as proteins and
polynucleotides. Porous plugs are also deficient with respect to
isolating and purifying larger biological materials and nucleic
acids such as DNA, RNA and cells. This shortcoming derives from the
fact that during sample processing, molecules must wend through a
labyrinth of sponge-like, expansive and porous adsorbent
silica.
[0008] There is little uniformity, consistency, and reproducibility
of porous materials used for sample preparation. Sample loss in
existing pipette tips is typically about 40-60%. Poor sample
recovery is largely due to the fact that a sample must travel
through irregular voids in the porous material, whereby a portion
of the sample lodges in small voids and is unrecoverable. Moreover,
in order to achieve adequate results, samples must be passed
through porous materials multiple times (e.g., ten). The sample
preparation devices are usually not reusable and fit poorly with
automatic instrumentation because poor sample recovery may give
rise to contamination due to sample carry-over.
[0009] Spin columns and other apparatus operated by a centrifuge
rotor are commonly used for the isolation and purification of
biological and nucleic acid samples. However, it is desirable in
certain applications to avoid the use of a centrifuge for rotating
a specimen to be isolated and purified. This is due, in part, to
the fact that horizontal separation may result in centrifugal
forces of up to, for example, 4,000 RPM, being exerted on or
transmitted along the vertical axis of the spin column and sample
in order to achieve satisfactory separation. Air resistance
negatively affects the spin column by generating drag and friction,
which heat the spin column and its contents. Considerable breakage
of sample fragments is unavoidable due to the heat transfer, acute
centrifugal force and accompanying air resistance. The impaired
quality of biological and nucleic acid samples extracted during
spin column and centrifugal processing is highly undesirable to the
user.
[0010] It can be seen, therefore, that the purification and
concentration of biological and nucleic acid samples using porous
materials prior to instrumental analysis is time consuming, is
poorly reproducible, has low throughput, and requires repeated
passing of a sample through the porous plug.
[0011] Accordingly, it is an object of the present invention to
provide an efficient sample preparation device for use in isolating
(immunoassay), purifying and concentrating samples of proteins,
peptides, nucleic acids (e.g., DNA and RNA), and other biological
materials (e.g., cells) prior to analysis.
[0012] It is also an object of the invention to provide a sample
preparation device with high sample capacity that increases
throughput and reduces sample loss.
[0013] It is a further object of the invention to provide a highly
reproducible sample preparation device that achieves uniformity,
consistency, and nearly identical pathways for sample passage.
[0014] It is a still further object of the invention to provide a
sample preparation device that is simple, cost-effective, and does
not require the use of a silica type porous substrate or special
equipment such as a centrifuge.
SUMMARY OF THE INVENTION
[0015] The invention is a high surface area multicapillary sample
preparation device especially useful for handling biological
samples. The multicapillary device does not require use of a silica
type porous substrate. Rather, the device incorporates a plurality
of parallel capillary tubes, wherein the cavity of each tube
remains open and unobstructed throughout sample processing. The
capillary tubes of the device function independently of one another
so that sample molecules are incapable of being physically
exchanged or diffusing from one capillary to another. The
multicapillary device is preferably disposed in a housing that is
suitable for attachment to a "pipette" or other sample preparation
or analytical instrument, enabling the isolation, purification,
concentration and/or fractionation of nucleic acids or biological
samples in the micro- and nanoliter range, as well as larger mass
loads and volumes. In an embodiment of the invention, the
multicapillary device features a monolithic element pierced with
multiple uniform capillaries. The monolithic element is typically
mounted in the lower end of a pipette tip, syringe needle or
tubing, and is operated using a pipette.
[0016] For protein separation and purification applications, an
insoluble stationary phase material is deposited onto the interior
surfaces (walls) of each capillary tube, without employing a
supporting or intermediary constituent.
[0017] The invention also includes a method of preparing a
multicapillary device for protein sample preparation. In such
method, inner walls of the capillary tubes are first coated with a
stationary phase material, and then the monolithic element is
mounted in an appropriate housing. Alternatively, the monolithic
element can first be fixed in a housing, after which the capillary
walls can be coated with the stationary phase. For operation, the
multicapillary sample preparation device is attached to a pipette,
micropipette, tube, syringe or similar analytical instrument.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a perspective view of a multicapillary device for
sample preparation in accordance with an embodiment of the present
invention. Individual capillaries of the device are shown in the
enlarged, cross-sectional views of FIGS. 2A, 2C and 2D (SEM). FIG.
2B is an exploded, perspective view of an individual capillary
tube.
[0019] FIG. 3 depicts SEM images showing cross-sectional views of a
conventional sample preparation device.
[0020] FIGS. 4A-4D are perspective views of pipette tips and a
pipette format multicapillary device, respectively, in accordance
with the present invention.
[0021] FIGS. 5A-5C show perspective views of a syringe format
multicapillary device for sample preparation according to the
present invention.
[0022] FIG. 6 is a chromatogram showing the separation of a three
component mixture in a multicapillary device for sample preparation
according to the present invention.
[0023] FIG. 7A is a chromatogram illustrating the performance of a
multicapillary sample preparation device in sample enrichment as
compared to a standard SPE cartridge, FIG. 7B.
[0024] FIGS. 8A and 8B are mass spectra demonstrating the
performance of a multicapillary sample preparation device in
desalting of complex peptide mixtures.
[0025] FIGS. 9A and 9B are mass spectra demonstrating the
performance of a multicapillary sample preparation device in
fractionating of complex peptide mixtures.
[0026] FIG. 10 shows gels demonstrating sample capacity and
recovery performance (10A), reproducibility (10B), and time
performance (10C) of a conventional device versus a multicapillary
sample preparation device according to the present invention.
Comparative data is shown in Table 2.
[0027] FIG. 11A is a perspective view of a conventional spin column
used for DNA purification. FIGS. 11B and 11C are SEM images of the
spin column.
[0028] FIGS. 12A and 12B, respectively, show pulsed field and
agarose gel electrophoresis analyses performed to determine DNA
quality and size of a conventional spin column versus a
multicapillary sample preparation device according to the present
invention.
[0029] FIGS. 13A and 13B, respectively, show sample purification
results of conventional spin columns versus a multicapillary sample
preparation device of the present invention in terms of DNA yield
and time required for sample preparation
DETAILED DESCRIPTION OF THE INVENTION
[0030] In accordance with the present invention, a parallel
capillary array or multicapillary sample preparation device 12 is
provided for use with commercially available pipettes to permit the
isolation, purification, concentration and/or fractionation of
biological samples in the micro- and nanoliter range, as well as
larger mass loads and volumes. The invention includes both
detachable and integrally embedded multicapillary devices 12
adapted for use with manual and automatic pipettes, micropipettes
20, syringes 22 and other sample handling or analytical
instruments. Notably, the multicapillary device 12 does not require
use of a silica type porous substrate.
[0031] Referring now to FIGS. 1 and 2, there is shown a
multicapillary device for sample preparation 12 comprising a
monolithic element (rod, tube, etc.) 14 that has an upper end and a
lower end, and defines a chamber. Capillary tubes 16 of uniform
internal diameter and length are arranged within the chamber. Each
capillary tube 16 includes a non-porous or imperforate wall having
an inner and an outer surface, which defines an inner bore. Each
tube 16 also includes a first and a second opening at opposing
ends, so that resistance and backpressure are low.
[0032] For protein or peptide separation and purification
applications, it is preferable to deposit an insoluble stationary
phase 18 on imperforate inner walls of the capillary tubes 16. For
the separation and purification of polar compounds such as nucleic
acids (e.g., DNA and RNA), it is preferable that the insoluble
stationary phase 18 comprise a polar material. In a preferred
embodiment of the invention, the thickness of the stationary phase
18 is correlated with the radius of individual capillary tubes 16
to optimize efficiency of the multicapillary device 12. As a
result, during application of the stationary phase 18, a greater
amount settles on the inner surface of wider capillaries; while a
smaller amount settles on the inner surface of narrower
capillaries. Through this process, the capillaries 16 achieve
quasi-uniformity, which substantially increases the efficiency of
the multicapillary device 12. The following relationship for high
peak efficiency has been derived by the inventors:
d.sub.f(r)=c.sub.rr.sup.n (Equation 1)
[0033] The stationary phase film thickness d.sub.f is proportional
to capillary radius r in power n, where n>1; c.sub.f is a
constant.
[0034] In a more preferred embodiment, the thickness of the
stationary phase coating 18 is proportional to the radius of the
capillary tubes 16 in power n, where n is greater than 1.
[0035] To achieve the highest peak efficiency, the stationary phase
thickness d.sub.f is proportional to capillary radius r in power
3.
[0036] Stationary phase media 18 is retained on the interior
surfaces of the imperforate, hollow capillary tubes 16 via stable
chemical bonding or cross-linking. There is, therefore, no
discharge of stationary phase media 18 into the mobile phase during
separation, reducing sample contamination. In the open tubular
system of the multicapillary device 12, supporting intermediary
constituents and adsorptive and membranous plugs (e.g., porous
adsorbent silica particles or fibers) are unnecessary. The lumen or
inner cavity of each capillary tube 16 remains unobstructed and
impediment free throughout the protein, peptide, nucleic acid or
biological sample separation process. In this stable,
surface-mediated mechanism of separation, sample molecules are
incapable of diffusing between and through the imperforate walls of
the capillary tubes 16 or from one capillary to another. Individual
capillary tubes 16 remain physically and functionally independent
of one another.
[0037] The multicapillary device for sample preparation 12 may be
detachably mounted (mechanically) or fixedly inserted (e.g., by
melting or adhesion) about the end portion of a pipette tip,
needle, tubing or other housing of suitable shape and dimension
that is attachable to a pipette 20. The multicapillary device 12
receives a sample in a mobile (liquid) phase at its first end, and
a concentrated and purified sample, devoid of contaminants such as
salts and buffers, is discharged at a second end of the device
12.
[0038] As shown in FIGS. 2A, 2B and 2C (Scanning Electron
Microscope image), the structure of the multicapillary device 12 is
distinctive and dissimilar to conventional spin columns and
silica-based adsorptive and membranous "plugs" (see FIGS. 3 and
11), which feature irregular voids and vastly different sample
pathways that entrap biological samples, such that more than half
of the sample is usually unrecoverable. The multicapillary device
12 comprises a plurality of uniform capillary tubes 16 having an
insoluble stationary phase media 18 on internal surfaces thereof,
thereby permitting a sample inserted into the flow passage of the
multicapillary device 12 to advance through open and virtually
identical pathways. The inner cavity or flow passage of each
capillary tube 16 thus remains unobstructed and impediment free
throughout the sample separation process.
[0039] As shown in FIGS. 12 and 13, purified (DNA) sample fragments
extracted from the open and unobstructed capillary channels of the
multicapillary device 12 comprise considerably larger fragments,
representing a significant decrease in fragment breakage or
"shearing." These larger fragments generally reflect a better
quality of purified sample as compared to conventional porous
silica plugs used for sample processing. In the conventional
adsorptive and membranous plugs, sample must travel through
tortuous and irregular voids in the porous material, whereby
portions of the sample lodge in small voids and are unrecoverable.
Fragment shearing is thus unavoidable. With its open and
unobstructed channel structure, the multicapillary device 12 of the
present invention achieves substantial uniformity and consistency
as compared to the sponge-like and expansive porous silica
materials currently used for sample preparation.
[0040] Due to the significant reduction in sample loss enabled by
the open and unobstructed channel structure of the multicapillary
device 12, it is unnecessary for sample to be passed through the
separation materials multiple times, as required in existing porous
silica plugs, particles (for proteins and peptides), and fibers
(for DNA and RNA). As a result, the multicapillary device 12 is
generally reusable and fits conveniently with automatic
instrumentation because there is no contamination due to sample
carry-over. In short, the present multicapillary device 12
demonstrates superior characteristics over conventional adsorptive
and membranous plugs with respect to binding capacity, recovery,
increased throughput, uniformity and reproducibility.
[0041] In one embodiment of the invention, the imperforate inner
walls of the capillary tubes 16 include particles of inert material
or a nodular or uneven surface for increasing the surface area of
the multicapillary device 12. In such case, the inner wall may be
altered using an etching process in combination with a solvent such
as, for example, a mineral acid or base, or an organic acid or
base.
[0042] The present invention encompasses the use of any stationary
phase 18 and surface chemistry adapted for liquid chromatography
and sample preparation applications. In some embodiments, the
stationary phase media 18 deposited on inner surfaces of the
capillary wall comprises a monolayer of organic molecules,
biopolymers or larger particles. Such molecules and particles
include, but are not limited to, hydrocarbons and their C-, N-, S-
, and P-derivatives; proteins, nucleic acids, and polysaccharides;
linear and cross-linked polysiloxanes and other polymers; and
viruses and cells. In other embodiments, a stationary phase coating
18 is formed by treating inner surfaces of the capillaries 16 with
organosilicone compounds and further modifying these groups with
appropriate reagents and particles.
[0043] An alternative technique for the deposition of a stationary
phase 18 involves polymerization of unsaturated compounds, such as
butadiene, styrene, divinylbenzene, and others, on inner walls of
the capillary tubes 16.
[0044] The techniques used for deposition of a stationary phase
material 18, described in the following Examples, render the
stationary phase material insoluble in organic and water-organic
solvents commonly used in sample preparation and liquid
chromatography, such as acetonitrile, methanol, isopropanol,
acetone, dimethylsulfoxide, dimethylformamide and urea; acetic,
iodoacetic, trifluoroacetic and formic acids; and phosphate, acetic
and carbonate buffers, etc.
[0045] The multicapillary sample preparation device 12 is
advantageously suited for use with a wide range of sample
preparation and analytical instruments. As shown in FIGS. 1, 4, and
5, these include, but are not limited to, manual and automatic
pipettes and micropipettes 20, syringes 22, disposable devices, and
automatic sample handling instruments. In some embodiments, the
multicapillary device 12 is inserted about the terminal portion of
a pipette tip 20 or other appropriate housing by, for example,
sliding or press fitting, and is detachably retained in place by
mechanical means such as elastic sealing rings and/or walls of the
housing.
[0046] In other embodiments, the multicapillary device 12 is
integrally and permanently embedded (e.g., cast-in-place) about the
terminal region of a pipette tip 20 or other housing by melting,
heat shrinking or adhesion in order to fuse the monolithic element
14 to the surface of a pipette tip 20 or other housing commonly
made from polypropylene or other thermoplastic material. As an
alternative, plasma and/or chemical means may be used to accomplish
adhesion of the monolithic element 14 to the surface of the pipette
tip 20 or other housing.
[0047] In yet another embodiment, the multicapillary device 12 is
adapted to be detachably or fixedly engaged or aligned with the
hollow flow passage(s) of a substantially cylindrical, conical or
other housing configuration. The multicapillary device 12 is
suitably sized and shaped to be integrated in housings of varying
sizes and configurations. However, it is preferable that the
housing have a volume in the range of about 0.1 .mu.L to about 100
mL, more preferably in the range of about 1 to about 1000 .mu.L,
and most preferably in the range of about 2 to about 200 .mu.L.
[0048] It will be understood that any technique used to install the
multicapillary device 12 into a pipette tip or other housing for
operation with a pipette 20 should accurately direct sample in a
liquid phase through the capillary tubes 16 of the monolithic
element 14 without bypassing the element 14. In such adaptation,
the multicapillary device 12 receives the sample at its first end,
and a concentrated and/or purified sample, devoid of contaminants
such as salts and buffers, is discharged at a second end. The
multicapillary device 12 of the present invention is freely
permeable not only to proteins, peptides, polynucleotides, and
other molecules and biopolymers, but also to viruses, spores, cells
(e.g., cancer and stem), and microorganisms. It will be understood,
however, that sample molecules are prevented from diffusing from
one capillary tube 16 to another
[0049] Preferred materials for fabricating the monolithic element
14, capillary tubes 16, tips, pipettes 20, syringes 22 and other
housings of the present invention include, but are not limited to,
glass, fused silica, ceramic, metal (e.g., stainless steel), and
plastic (e.g., polypropylene, polyethylene, polyolefin, or
polyetheretherketone). In sample preparation and chromatographic
applications, it is desirable to employ a large number (e.g.
hundreds or thousands) of capillary tubes to provide an abundant
surface area for higher sample loading capacity. It will be
understood, however, that the number and dimensions of the
multicapillary device 12, stationary phase media 18, solvent, tips
and other housings employed in the invention will vary according to
application.
[0050] As an example, the number of capillary tubes 16 provided in
a multicapillary device 12 may range from about 100 to about
1,000,000. The inner diameter of each capillary tube 16 may range
from about 0.1 .mu.m to about 200 .mu.m. The outer diameter of the
monolithic element 14 may range from about 0.1 mm to about 1 m, and
the length may range from about 0.1 mm to about 2 m. In a preferred
embodiment, the number of capillary tubes 16 ranges from about 1000
to about 10,000, the inner diameter of each capillary ranges from
about 5 .mu.m to about 100 .mu.m, the outer diameter of the
monolithic element 14 ranges from about 1 mm to about 20 mm, and
the length ranges from about 1 mm to about 250 mm.
EXAMPLES
Example 1
[0051] C-1 Stationary Phase
[0052] A 5% solution of trimethylchlorosilane in toluene is pumped
at 10 .mu.L/min for six hours through a 1 mm outer
diameter.times.25 mm long multicapillary glass rod pierced with
approximately 4400 capillaries of 10 .mu.m diameter at 105.degree.
C. The multicapillary rod is rinsed with toluene, acetone and
methanol, and dried with a nitrogen stream.
Example 2
[0053] C-4 Stationary Phase
[0054] A 10% solution of butyldimethylchlorosilane in toluene is
pumped at 40 .mu.L/min for six hours through a 2 mm outer
diameter.times.300 mm long multicapillary glass rod pierced with
approximately 4600 capillaries of 25 .mu.m diameter at 105.degree.
C. The multicapillary rod is rinsed with toluene, acetone and
methanol, and dried with a nitrogen stream.
Example 3
[0055] C-8 Stationary Phase
[0056] A 10% solution of octyltrichlorosilane in toluene is pumped
at 50 .mu.L/min for six hours through a 2.3 mm outer
diameter.times.25 mm long multicapillary glass rod pierced with
approximately 1400 capillaries of 40 .mu.m diameter at 105.degree.
C. The multicapillary rod is rinsed with toluene, acetone and
methanol, and dried with a nitrogen stream.
Example 4
[0057] C-12 Stationary Phase
[0058] A 5% solution of dodecyltrichlorosilane in toluene is pumped
at 75 .mu.L/min for six hours through a 6 mm outer
diameter.times.300 mm long multicapillary glass rod pierced with
approximately 3300 capillaries of 65 .mu.m diameter at 105.degree.
C. The multicapillary rod is rinsed with toluene, acetone and
methanol, and dried with a nitrogen stream.
Example 5
[0059] C-18 Stationary Phase 1
[0060] A 10% solution of octadecyltriethoxysilane in toluene is
pumped at 10 .mu.L/min for six hours through a clean and dry 2.3 mm
outer diameter.times.300 mm multicapillary glass rod pierced with
approximately 4,000 capillaries of 20 .mu.m diameter at 105.degree.
C. While pumping the solution, an opposite end of the
multicapillary device is moved at a linear speed of 0.5 mm/min
inside an oven heated to 150.degree. C. The device is rinsed with
toluene, acetone and methanol, and dried with a nitrogen
stream.
Example 6
[0061] C-18 Stationary Phase 2
[0062] Twenty 1 mm outer diameter.times.2.5 mm long multicapillary
glass rods pierced with approximately 4400 capillaries of 10 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
octadecyldimethylchlorosilane in toluene and equipped with a reflux
condenser and a calcium chloride tube. The mixture is slowly
refluxed for six hours. The liquid phase is separated and the
multicapillary rods are repeatedly washed with toluene,
tetrahydrofuran and methanol, and dried at room temperature.
Example 7
[0063] C-16, C-30, Phenyl, Naphthyl, and Cyano Stationary
Phases
[0064] In accordance with the conditions described in Example 6,
the stationary phases with C-16, C-30, phenyl, naphthyl and cyano
groups are prepared, correspondingly, from
hexadecyltrichlorosilane, triacontyltrichlorosilane,
phenethyltrichlorosilane, (1-naphthylmethyl)trichlorosilane and
3-cyanopropyltrichlorosilane.
Example 8
[0065] Epoxide Stationary Phase
[0066] Twenty 2.3 mm outer diameter.times.5 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
(3-glycidoxypropyl)trimethoxysilane in toluene and equipped with a
reflux condenser. The mixture is slowly refluxed for five hours
while the condenser is maintained at a temperature of 70.degree. C.
to remove the methanol formed from the reaction. The liquid phase
is separated and the multicapillary rods are repeatedly washed with
toluene, tetrahydrofuran and methanol, and dried at room
temperature.
Example 9
[0067] Diol Stationary Phase 1
[0068] Twenty 2.3 mm outer diameter.times.3 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
(3-glycidoxypropyl)trimethoxysilane in toluene and equipped with a
reflux condenser. The mixture is slowly refluxed for three hours
while the condenser is maintained at a temperature of 70.degree. C.
The liquid phase is separated and the multicapillary rods are
repeatedly washed with toluene, tetrahydrofuran, methanol and
water. 50 mL of water is added and the pH is adjusted to 2.0 with
nitric acid. The mixture is slowly agitated for two hours at room
temperature. The liquid phase is separated and the multicapillary
rods are washed with water until the wash is neutral, then washed
three times with methanol, and dried at room temperature.
Example 10
[0069] Diol Stationary Phase 2
[0070] 2.5 g of (3-glycidoxypropyl)trimethoxysilane is dropped into
a flask containing 50 mL of water, while maintaining the pH between
5 and 6 with 0.01 M potassium hydroxide. Twenty 2.3 mm outer
diameter.times.5 mm long multicapillary glass rods pierced with
approximately 1400 capillaries of 40 .mu.m diameter are placed in a
flask. The mixture is slowly refluxed for three hours with a reflux
condenser. The liquid phase is separated and the multicapillary
rods are repeatedly washed with water, methanol and
tetrahydrofuran. 50 mL of water is added and the pH is adjusted to
2.0 with nitric acid. The mixture is slowly agitated for two hours
at room temperature. The liquid phase is separated and the
multicapillary rods are washed with water until the wash is
neutral, then washed three times with methanol, and dried at room
temperature.
Example 11
[0071] Amino Stationary Phase
[0072] Twenty-five 1 mm outer diameter.times.2.5 mm long
multicapillary glass rods pierced with approximately 4400
capillaries of 10 .mu.m diameter are placed in a flask containing
50 mL of a 5% solution of 3-aminopropyltrimethoxysilane in toluene
and equipped with a reflux condenser. The mixture is slowly
refluxed for five hours while the condenser is maintained at a
temperature of 70.degree. C. The liquid phase is separated and the
multicapillary rods are repeatedly washed with toluene,
tetrahydrofuran and methanol, and dried at room temperature.
Example 12
[0073] Trimethylammonium Stationary Phase
[0074] Thirty 2.3 mm outer diameter.times.5 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
3-aminopropyltrimethoxysilane in toluene and equipped with a reflux
condenser. The mixture is slowly refluxed for six hours while the
condenser is maintained at a temperature of 70.degree. C. The
liquid phase is separated and the multicapillary rods are
repeatedly washed with toluene, tetrahydrofuran and methanol. 30 mL
of a 5% solution of trimethylamine in methanol is added to the
flask. The flask is equipped with a calcium chloride tube, and the
mixture is slowly agitated at 0-5.degree. C. for 48 hours. The
liquid phase is separated and the multicapillary rods are
repeatedly washed with methanol, water, 0.01 M HCl, water and
tetrahydrofuran, and dried at room temperature.
Example 13
[0075] Carboxylic Acid Stationary Phase
[0076] Twenty 2.3 mm outer diameter.times.5 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 50 mL of a 5% water
solution of carboxyethylsilane triol (sodium salt). The pH is
adjusted to 2.0 by adding hydrochloric acid. The mixture is slowly
refluxed for three hours with a reflux condenser. The liquid phase
is separated and the multicapillary rods are washed with water
until the wash is neutral, then washed three times with methanol,
and dried at room temperature.
Example 14
[0077] Sulfonic Stationary Phase
[0078] Twenty-five 2.3 mm outer diameter.times.3 mm long
multicapillary glass rods pierced with approximately 1400
capillaries of 40 .mu.m diameter are placed in a flask containing
50 mL of a 5% water solution of
3-(trihydroxysilyl)-1-propanesulfonic acid. The mixture is slowly
refluxed for three hours with a reflux condenser. The liquid phase
is separated and the multicapillary rods are washed with water
until the wash is neutral, then washed three times with methanol,
and dried at room temperature.
Example 15
[0079] Phosphonic Stationary Phase
[0080] Thirty 2.3 mm outer diameter.times.3 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 50 mL of a 5% water
solution of (3-trihydroxysilylpropyl)methylphosphonate sodium salt.
The mixture is acidified to pH 2.0 with HCL and slowly refluxed for
three hours with a reflux condenser. The liquid phase is separated
and the multicapillary rods are washed with water until the wash is
neutral, then washed three times with methanol, and dried at room
temperature.
Example 16
[0081] Iminodiacetic Acid Stationary Phase
[0082] Twenty 1 mm outer diameter.times.2.5 mm long multicapillary
glass rods pierced with approximately 4400 capillaries of 10 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
(3-glycidoxypropyl)trimethoxysilane in toluene and equipped with a
reflux condenser. The mixture is slowly refluxed for five hours
while the condenser is maintained at a temperature of 70.degree. C.
The liquid phase is separated and the multicapillary rods are
repeatedly washed with toluene, tetrahydrofuran, methanol and
water. 20 mL of a 2 M iminodiacetic acid solution in 0.1 M sodium
borate buffer, pH 8.5, is added and the mixture is slowly agitated
for 24 hours at room temperature. The liquid phase is separated and
the multicapillary rods are washed with water until the wash is
neutral, then washed three times with methanol, and dried at room
temperature.
Example 17
[0083] Cystein Stationary Phase
[0084] Twenty-five 1 mm outer diameter.times.2.5 mm long
multicapillary glass rods pierced with approximately 4400
capillaries of 10 .mu.m diameter are placed in a flask containing
50 mL of a 5% solution of 3-bromopropyltrimethoxysilane in toluene
and equipped with a reflux condenser. The mixture is slowly
refluxed for five hours while the condenser is maintained at a
temperature of 70.degree. C. The liquid phase is separated and the
multicapillary rods are repeatedly washed with toluene,
tetrahydrofuran and methanol. 50 mL of a 1% solution of cystein in
methanol and 1 mL of triethylamine are added, and the mixture is
slowly refluxed with a reflux condenser for five hours. The liquid
phase is separated and the multicapillary rods are repeatedly
washed with methanol, water, methanol and methylene chloride, and
dried at room temperature.
Example 18
[0085] Glutathione Stationary Phase
[0086] Twenty 1 mm outer diameter.times.2.5 mm long multicapillary
glass rods pierced with approximately 4400 capillaries of 10 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
11-bromoundecyltrimethoxysilane in toluene and equipped with a
reflux condenser. The mixture is slowly refluxed for five hours
while the condenser is maintained at a temperature of 70.degree. C.
The liquid phase is separated and the multicapillary rods are
repeatedly washed with toluene, tetrahydrofuran and methanol. 50 mL
of a 0.5% solution of glutathione in methanol and 1 mL of
triethylamine are added, and the mixture is slowly refluxed with a
reflux condenser for five hours. The liquid phase is separated and
the multicapillary rods are repeatedly washed with methanol, water,
methanol and methylene chloride, and dried at room temperature.
Example 19
[0087] Chiral Stationary Phase
[0088] A 5% solution of
(R)-N-1-phenylethyl-N-triethoxysilylpropylurea in toluene is pumped
at 10 .mu.L/min for six hours through a 1 mm outer
diameter.times.300 mm long multicapillary glass rod pierced with
approximately 4000 capillaries of 10 .mu.m diameter at 105.degree.
C. The multicapillary rod is rinsed with toluene, tetrahydrofuran
and methanol, and dried with a nitrogen stream.
Example 20
[0089] Polybutadiene Stationary Phase
[0090] The 10% solution of vinyltrichlorosilane in isooctane is
pumped at 20 .mu.L/min for six hours through a 1 mm outer
diameter.times.25 mm long multicapillary glass rod pierced with
approximately 4,400 capillaries of 10 .mu.m diameter at 90.degree.
C. The multicapillary rod is rinsed with isooctane,
tetrahydrofuran, methanol, toluene and isooctane. A solution of 100
mg polybutadiene (MW 3,400) and 0.5 mg dicumyl peroxide in 100 mL
of isooctane is pumped at 10 .mu.L/min for six hours through the
multicapillary rod at 90.degree. C. The multicapillary rod is
rinsed with toluene, tetrahydrofuran and methanol, and dried with a
nitrogen stream.
Example 21
[0091] Biotin Stationary Phase
[0092] Twenty five 1 mm outer diameter.times.2.5 mm long
multicapillary glass rods pierced with approximately 4400
capillaries of 10 .mu.m diameter are placed in a flask containing
50 mL of a 5% solution of 3-aminopropyltrimethoxysilane in toluene
and equipped with a reflux condenser. The mixture is slowly
refluxed for five hours while the condenser is maintained at a
temperature of 70.degree. C. The liquid phase is separated and the
multicapillary rods are repeatedly washed with toluene, methanol
and dimethylformamide. 15 mL of a saturated dimethylformamide
solution of biotin, a solution of 0.3 g of 1-hydroxybenzotriazole
hydrate in 10 mL of dimethylformamide, and a solution of 0.25 g of
N, N-dicyclohehylcarbodiimide in 10 mL of dimethylformamide are
added, and the mixture is slowly agitated for five hours at room
temperature. The liquid phase is separated and the multicapillary
rods are repeatedly washed with dimethylformamide, methanol, water,
methanol and methylene chloride, and dried at room temperature.
Example 22
[0093] Heparin Stationary Phase
[0094] Twenty 2.3 mm outer diameter.times.2.5 mm long
multicapillary glass rods pierced with approximately 1400
capillaries of 40 .mu.m diameter are placed in a flask containing
50 mL of a 5% solution of 3-aminopropyltrimethoxysilane in toluene
and equipped with a reflux condenser. The mixture is slowly
refluxed for five hours while the condenser is maintained at a
temperature of 70.degree. C. The liquid phase is separated and the
multicapillary rods are repeatedly washed with toluene, methanol,
water and 0.05 M 2-morpholinoethane sulfonic acid buffer, pH 5.6.
The 10 mL solution of 0.2 g heparin (from bovine kidney), 1 g
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide, and 0.5 g
N-hydroxysuccinimide in 0.05 M 2-morpholinoethane sulfonic acid
buffer, pH 5.6, is added. The mixture is slowly agitated for three
hours at room temperature. The liquid phase is separated, and the
multicapillary rods are repeatedly washed with water, phosphate
buffer, pH 8, and 20% sodium chloride, and then washed with water
and stored at 4.degree. C.
Example 23
[0095] Glycoprotein Stationary Phase
[0096] Twenty 2.3 mm outer diameter.times.3 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 20 mL of a 5% solution of
(3-glycidoxypropyl)trimethoxysilane in toluene and equipped with a
reflux condenser. The mixture is slowly refluxed for five hours
while the condenser is maintained at a temperature of 70.degree. C.
The liquid phase is separated and the multicapillary rods are
repeatedly washed with toluene, methanol and water. The 4 mL
solution of 100 mg alpha1 acid (from bovine plasma) in a 1:1
mixture of 0.4 M sodium chloride and 0.2 M borate buffer, pH 8.5,
is added. The mixture is slowly agitated at room temperature for 48
hours. The liquid phase is separated. The multicapillary rods are
repeatedly washed with a 1:1 mixture of 0.4 M sodium chloride and
0.2 M borate buffer, pH 8.5, then washed with water and stored at
4.degree. C.
Example 24
[0097] Trypsin Stationary Phase
[0098] Twenty 2 mm outer diameter.times.5 mm long multicapillary
glass rods pierced with approximately 4600 capillaries of 25 .mu.m
diameter are placed in a flask containing 20 mL of a 5% solution of
(3-glycidoxypropyl)trimethoxysilane in toluene and equipped with a
reflux condenser. The mixture is slowly refluxed for five hours
while the condenser is maintained at a temperature of 70.degree. C.
The liquid phase is separated and the multicapillary rods are
repeatedly washed with toluene, methanol and water. A 7 mL solution
of 100 mg trypsin (from bovine pancreas) in 0.2 M phosphate buffer,
pH 7.0, is added to the flask. The mixture is slowly agitated at
room temperature for 15 hours, and the liquid phase is separated.
The multicapillary rods are repeatedly washed with 0.2 M phosphate
buffer, pH 7.0, water, 0.2 M Tris buffer, pH 7.5, for two hours,
and then washed with water and stored at 4.degree. C.
Example 25
[0099] Avidin, Lectin, and Protein A Stationary Phases
[0100] In accordance with the conditions described in Example 24,
avidin, lectin and protein A stationary phases are prepared using
avidin (from egg white), lectin (from Agaricus bisporus), and
protein A (from Staphylococcus aureus), correspondingly.
Example 26
[0101] Antibody Stationary Phase
[0102] Twenty 2.3 mm outer diameter.times.3 mm long multicapillary
glass rods pierced with approximately 1400 capillaries of 40 .mu.m
diameter are placed in a flask containing 50 mL of a 5% solution of
10-(carbomethoxy)decyldimethylmethoxysilane in toluene and equipped
with a reflux condenser. The mixture is slowly refluxed for three
hours while the condenser is maintained at a temperature of
70.degree. C. The liquid phase is separated, and the multicapillary
rods are repeatedly washed with toluene, methanol and methylene
chloride. A 35 mL solution of 1 mL trimethyliodosilane in methylene
chloride is added. The mixture is slowly agitated for 72 hours at
room temperature.
[0103] The liquid phase is separated and the multicapillary rods
are repeatedly washed with methylene chloride, methanol, 90%
methanol and water. 20 mL of 0.02 M
ethyl(dimethylaminopropyl)-carbodiimide in 0.1 M
2-morpholinoethanesulfonic acid buffer, pH 4.5, is added, and the
mixture is slowly agitated for one hour at room temperature. The
liquid phase is separated, and 5 mL of the 5 .mu.g/mL solution of
ricin antibody in 0.1 M phosphate buffered saline, pH 7.2, is
added. The mixture is slowly agitated for two hours at room
temperature. The liquid phase is separated. The multicapillary rods
are repeatedly washed with phosphate buffered saline, pH 7.2, then
washed with water and stored at 4.degree. C.
Example 27
[0104] Polypropylene Pipette Tip Format Multicapillary Sample
Preparation Device
[0105] A 1 mm to 5 mm outer diameter multicapillary element 10,
prepared as described in Example 6, is tightly pushed to the lower
end of a polypropylene 10 .mu.L to 5,000 .mu.L volume micropipette
tip. The lower portion of the pipette tip is placed for ten minutes
in an oven thermostated at a temperature at which polypropylene
begins to soften. Polypropylene pipette tip format multicapillary
devices for sample preparation 12, 20 are shown in FIGS. 4A and
4B.
Example 28
[0106] Polyethylene Pipette Format Multicapillary Sample
Preparation Device
[0107] A 1 mm to 5 mm outer diameter multicapillary element 10,
prepared as described in Example 6, is tightly placed in the lower
end of a disposable polyethylene transfer pipette. The lower
portion of the pipette is placed for ten minutes in an oven
thermostated at a temperature at which polyethylene begins to
soften. A polyethylene pipette format multicapillary device for
sample preparation 12, 20 is shown in FIG. 4C.
Example 29
[0108] Syringe Format Multicapillary Sample Preparation Device
#1
[0109] A 2 mm outer diameter multicapillary element 10, prepared as
described in Example 5, is attached to a heat shrinkable tubing. A
removable syringe needle is attached to the second end of the heat
shrinkable tubing. The heat shrinkable tubing zone is heated at a
temperature recommended for the shrinkable tubing for ten minutes A
representative syringe format multicapillary device for sample
preparation 12, 22 is shown in FIG. 5A.
Example 30
[0110] Syringe Format Multicapillary Sample Preparation Devices #2
and #3
[0111] A 2.3 mm outer diameter multicapillary element 10, prepared
as described in Example 5, is tightly placed in a removable
thermoplastic syringe hub. The hub is heated for ten minutes in an
oven thermostated at a temperature at which thermoplastic begins to
soften. Syringe format multicapillary devices for sample
preparation 12, 22 are shown in FIGS. 5B and 5C.
Example 31
[0112] Separation of Three Component Mixture
[0113] A three component mixture (uracil, fluorene, and
phenanthrene) is separated on the multicapillary device for sample
preparation prepared as described in Example 5 and installed on a
model LC-600 Shimadzu liquid chromatographic instrument using
standard HPLC fittings. The chromatographic conditions and
chromatogram are reproduced in FIG. 6. The chromatogram shows a
uracil peak at about 1.8 minutes, a fluorene peak at about 2.1
minutes, and a phenanthrene peak at about 2.4 minutes. The example
illustrates a liquid chromatographic application using the
multicapillary device for sample preparation of the present
invention, which enables a typical organic mixture to be analyzed
in less than three minutes.
Example 32
[0114] Sample Enrichment for HPLC Analysis
[0115] Referring to FIG. 7A, a 2.3 mm outer diameter.times.100 mm
length multicapillary C-18 device for sample preparation containing
approximately 1,400 capillaries of 40 .mu.m diameter prepared as
described in Example 5 is used for sample enrichment prior to HPLC
analysis. At present, short HPLC columns known as solid phase
extraction ("SPE") cartridges are commonly used for sample
preparation. Compared to SPE cartridges, multicapillary devices for
sample preparation 12 are much faster, simpler and reusable.
Performance of the present multicapillary device for sample
preparation 12 (FIG. 7A) as compared to a standard SPE cartridge
(FIG. 7B) is shown in Table 1. TABLE-US-00001 TABLE 1
Multicapillary Device vs. Conventional SPE Cartridge.
Characteristics SPE Cartridge Multicapillary Device 1 Collection
time 20-40 minutes 1-5 minutes 2 Amount 1-5 ml samples 100-200
.mu.l samples 3 Processing the Extract volume No concentration
extract 5-20 ml or evaporation of Requires the extract required
evaporation for analysis 4 Reconditioning Cannot be Reconditioned
by reconditioned washing with methanol and water for 2 min. 5
Reusability Cannot be Can be reused at reused least 50-100 times
depending on the sample to be extracted 6 Silica particles Fine
silica No silica or other in the sample particles in the particles
in the samples samples 7 Auto samplers Cannot be Easily adaptable
adaptability used in auto to auto samplers samplers 8 Field use
Difficult Requires only syringes to transport Costly equipment/
accessories
Example 33
[0116] Hydrolysis of Bovine Serum Albumin
[0117] A 10 .mu.L volume of 0.25 mg/mL bovine serum albumin
solution in a 40:60 mixture of acetonitrile and 0.2 mM ammonium
bicarbonate, pH 7.5, is aspirated in the multicapillary sample
preparation device, prepared as described in Example 24, and
thermostated at 37.degree. C. After five minutes, the digested
sample is dispensed in a vial and stored at 4.degree. C.
Example 34
[0118] Desalting of Peptide Samples
[0119] The bovine serum albumin digest, prepared as described in
Example 34, is dried with a nitrogen stream and dissolved in 10
.mu.L of 0.1% trifluoroacetic acid (TFA) in water. 1 .mu.L of this
sample is aspirated and dispensed from the multicapillary sample
preparation device, as described in Examples 5 and 27. Three 10
.mu.L portions of 0.1% trifluoroacetic acid (TFA) in 5%
acetonitrile/water are pumped in and out of the multicapillary
sample preparation device. The sample is eluted from the
multicapillary sample preparation device with a 5 .mu.L portion of
0.1% TFA in 70% acetonitrile/water and analyzed by MALDI-MS. The
MALDI-MS spectra of the sample before and after desalting are shown
in FIGS. 8A and 8B.
Example 35
[0120] Fractionating of Peptides
[0121] A 3 .mu.L volume of the 100 pmole/.mu.L peptide mixture
obtained by the enzymatic hydrolysis of bovine serum albumin, as
described in Example 34, is introduced into a 10 cm long C-18
multicapillary sample preparation device. The sample is eluted at
100 .mu.L/min with 100 .mu.L of 0.1% TFA in water followed by 30
.mu.L of 0.1% TFA in 40% acetonitrile/water. Ten 3 .mu.L 40%
acetonitrile/water fractions are collected and analyzed by
MALDI-MS. The mass-spectra of fractions 3 and 6 are shown in FIGS.
9A and 9B. This example illustrates the fractionating ability of
the multicapillary sample preparation device of the present
invention, prior to MALDI-MS analysis of a complex peptide
mixture.
Example 36
[0122] Gel Electrophoresis of Proteins
[0123] FIGS. 10A and 10B depict gels produced to evaluate the
sample capacity, recovery, and reproducibility of a multicapillary
device for sample preparation according to the present invention,
as compared to commercially available porous silica-based devices.
Lanes 1 and 10 are protein standards inserted for purposes of
comparison. Lanes 2 to 5 represent the conventional devices, while
Lanes 3 to 10 denote the multicapillary sample preparation device
of the instant invention.
[0124] The test sample used for electrophoresis is a mixture of the
following four representative proteins: Phosphorylase B (MW
97,400), Bovine Serum Albumin (MW 66,200), Carbonic Anhydrase (MW
31,000), and Lysozyme (MW 14,400). These proteins were selected due
to their varying size and generally known properties. Moreover,
these proteins are commonly used as standards. All results were
reproduced and retested several times to ensure accuracy. Testing
was conducted at ChromBA, Inc. (State College, Pa.), The Materials
Research Institute (University Park, Pa.), The Milton Hershey
Medical Center (Hershey, Pa.), Huck Institute (University Park,
Pa.), APD LifeSciences Inc. (State College, Pa.), and MassTech Inc.
(Columbia, Md.). In total, over 40 gels were used, including both
C.sub.18 and C.sub.4, the two most popular phases on the market. In
addition, more than 500 sample preparation devices were tested.
[0125] In both figures, the conventional silica-based devices (FIG.
10A: Lanes 2-5; FIG. 10B: Lanes 1-2) show faint and varying bands.
In contrast, the device of the present invention (FIG. 10A: Lanes
6-9; FIG. 10B: Lanes 3-10) shows bold and dark bands, indicating
vastly superior binding capacity, and recovery. Moreover, the bands
of the present device are clearly more identical from lane to lane
demonstrating superior reproducibility. As shown in Table 2,
further quantification reveals that the sample preparation device
of the present invention, on average, binds and releases nearly
twice the protein bound and released by conventional devices, and
demonstrates half the margin of error (i.e., variance in amount of
sample bound). TABLE-US-00002 TABLE 2 Sample Capacity, Recovery
Performance, and Reproducibility of Multicapillary Device vs.
Conventional Devices. MEAN VALUE % OF AVERAGE OF BAND INCREASED
MARGIN INTENSITY CAPACITY OF ERROR Convent. Multicap. Multicap.
Convent. Multicap. PROTEIN Device Device Device Device Device FIG.
Phosphorylase B 45.7 56.2 23% 5.8% 3.4% 10A Bovine Serum 85.2 162.1
91% Albumin Carbonic 86.8 131.4 51% Anhydrase Lysozyme 84.2 151.6
79% FIG. Phosphorylase B -- -- -- -- -- 10B Bovine Serum 85.5 135.1
107% 6.3% 3.8% Albumin Carbonic 70.1 108.6 55% Anhydrase Lysozyme
77.0 157.6 104%
[0126] FIG. 10C shows a gel produced to evaluate the speed of use
(time performance) of a multicapillary device for sample
preparation according to the present invention relative to
commercially available silica-based devices. As recommended by the
manufacturers, a sample must be passed through the tip of the
silica-based devices approximately ten times to achieve suitable
results. This is largely due to the fact that most of the sample
travels through large voids in the filtration material and is
subsequently not adsorbed and cleaned. Specifically, as illustrated
by Lanes 1-4 of FIG. 10C, the conventional devices show faint bands
even after ten passes through the tip.
[0127] In contrast, Lanes 5-10 show nearly identical spots for
samples passed through the multicapillary device of the present
invention only once. Notably, the bands are much darker and broader
than those of the silica-based devices, denoting the presence of
vastly greater amounts of protein.
[0128] In FIGS. 10A-10C, the darkness and size of the bands on the
gels developed by electrophoresis indicate the amount of protein
bound by and recovered from the sample preparation devices; namely,
pipette tips with integral silica-based plugs versus multicapillary
elements. The superior performance of the multicapillary device 12
of the present invention is due, in part, to the uniform,
consistent and virtually identical pathways for sample passage
through the device, along with excellent tip-to-tip duplication, as
shown in FIG. 2. FIG. 3 and FIG. 11, in contrast, depict
cross-sections of commercially available (market leader) porous
silica and silica fiber based sample preparation devices. The
silica based devices reveal irregular particle sizes and fiber
diameter, large voids (dark areas), and vastly different sample
pathways. Moreover, the conventional devices demonstrate poor
tip-to-tip duplication (i.e., tremendous variance).
Example 37
[0129] Isolation of Nucleic Acids
[0130] The following experiments, featuring both syringe 22 and
pipette tip 20 format multicapillary devices 12, demonstrate the
advantage of using a multicapillary device for sample handling and
extraction of DNA from various biological samples, such as tissue,
blood, bacterial cells, urine, etc.
[0131] A multicapillary device for sample preparation 12 (1 to 2.5
mm outer diameter, 0.25 to 3 cm long, approximately 4000
capillaries of 10 to 40 .mu.m diameter, inner volume 2 to 90 .mu.L)
is loaded twice with 10 to 1,000 .mu.L lysed biological sample.
After discarding the loading solution, the multicapillary device 12
is rinsed with a washing buffer to dispose of proteins and other
non-DNA type materials. Adsorbed DNA is then eluted with 10 to 300
.mu.L elution buffer. The eluted DNA is analyzed using a variety of
methods including Yo-Pro fluorescence on a Packard FluoroCount
instrument at 530 nm (for yield determination), Agarose gel
electrophoresis (for quality determination), and pulsed field gel
electrophoresis (for DNA size and quality determination).
[0132] FIGS. 13A and 13B, respectively, are bar graphs comparing 16
rat liver samples' purification results in terms of DNA yield and
time required for sample preparation using 25 .mu.m.times.1
cm.times.200 .mu.L pipette tip format multicapillary devices 12, 20
according to the present invention and 16 commercially available
(market leader) spin columns. The data for DNA yield was obtained
using Yo-Pro fluorescence, and time was recorded with a stopwatch
for each sample. The experiments demonstrate that the
multicapillary devices 12 of the present invention are comparable
to conventional spin columns in terms of DNA yield, but require
significantly less time for processing. Notably, use of the present
multicapillary devices results in a seven-fold decrease in
"hands-on" labor time for DNA isolation sample preparation.
[0133] Agarose gel electrophoresis and pulsed field gel
electrophoresis analyses were performed to determine DNA quality
and size. FIG. 12B shows a 0.8% agarose gel run to analyze DNA
extracted from whole bovine blood using the pipette tip format
multicapillary devices (Lanes 1-7) and commercially available spin
columns (Lanes 8-9), demonstrating comparable DNA yields and
quality. Further, as shown in FIG. 12A, pulsed field gel
electrophoresis was used to size DNA strands of purified samples
using the multicapillary device 12 of the present invention and
commercially available spin columns. As compared to the leading
spin columns, samples extracted using the present multicapillary
device 12 demonstrate considerably larger DNA fragments, which
suggests a significant decrease in DNA breakage or "shearing"
during sample processing. Larger fragments of DNA reflect a better
quality of the purified sample, which is highly desirable for many
downstream applications such as PCR and sequencing.
[0134] As demonstrated in FIGS. 12 and 13, significant DNA shearing
is unavoidable in specimens processed using the commercially
available spin columns, which are operated by means of a
centrifuge. In the multicapillary device 12 of the present
invention, however, DNA shearing is largely avoided through the use
of an insoluble, surface-mediated mechanism of separation, which
ensures that capillary channels (lumens) remain open and
unobstructed throughout sample processing. Use of a gentle
pipetting procedure for sample processing, in lieu of the commonly
employed centrifuge, further contributes to the quality and size of
DNA processed by means of the multicapillary devices 12 disclosed
herein.
[0135] Table 3 shows the adsorbance reading of five multicapillary
devices 12 prepared according to the method described above. The
average DNA recovery yield is 50-60%, which demonstrates that the
multicapillary device for sample preparation 12 is efficacious for
sample handling, purification and isolation of nucleic acids.
TABLE-US-00003 TABLE 3 DNA Isolation on Multicapillary Device.
Fluorescence Reading Sample (Units) Blank elution buffer 21 Elution
buffer spiked with 16 .mu.g/mL DNA 214 Eluted DNA from MC 211
Eluted DNA from MC 211 Eluted DNA from MC 217 Eluted DNA from MC
238 Eluted DNA from MC 213
Example 38
[0136] Sample Handling of Cells
[0137] To demonstrate the use of a multicapillary device 12 for
sample handling of cells, the following cells are grown and
evaluated: mouse myeloma Sp2/0-Ag14, approximately 25 .mu.m size;
mouse macrophage J774A1, approximately 15 .mu.m size; and human
prostrate tumor THP-1 approximately 20 .mu.m size. A syringe format
multicapillary device for sample preparation 12, 22 (2.3 mm outer
diameter, 150 mm long, approximately 1400 capillaries of 40 .mu.m
diameter, inner volume 262 .mu.L) is attached to a syringe and
rinsed with 1 mL of balanced salt solution creating an environment
conductive to cell viability. At room temperature, a 1.5-3.0 mL
volume of cell suspension is passed through the multicapillary
device at a flow rate of 1 mL/min, followed by a 0.5 mL volume of
balanced salt solution. The eluted cell suspension is concentrated
by centrifugation and cells are counted using a hemacytometer. The
change in cell viability after passage through the multicapillary
device is presented in Table 4. TABLE-US-00004 TABLE 4 Cell
Viability Upon Passage Through Multicapillary Device. Viable %
Viable Suspension Concentration Cells Viable Cells Cells Cell
Volume of Cells Initial Recovered Recovered Sp2/0-Ag14 3.0 mL 1
.times. 10.sup.6 cells/mL 2.73 .times. 10.sup.6 2.09 .times.
10.sup.6 76.5 J774A1 2.0 mL 3.5 .times. 10.sup.6 cells/mL 6.30
.times. 10.sup.6 3.57 .times. 10.sup.6 56.6 THP-1 2.0 mL 3.5
.times. 10.sup.6 cells/mL 6.65 .times. 10.sup.6 5.06 .times.
10.sup.6 76.1
[0138] The data reveal that the vast majority of each cell type
passed through the device emerges unharmed. Therefore, the
multicapillary device for sample preparation is highly efficacious
for sample handling of cells.
[0139] Manufacturers of existing pipette tips state in their
technical literature that 40-60% sample loss is average for a
purified product. In comparison, the increased capacity and
recovery afforded by the present invention substantially reduces
and/or eliminates the critical problem of sample loss in the
pipette tip. Moreover, equipment wear is reduced, since the present
invention eschews repeat sample passage, as required by
conventional silica-based devices.
[0140] As demonstrated by the foregoing Examples 1-38, the
multicapillary device 12 of the present invention enables a user to
achieve superior sample preparation results in significantly less
time. Indeed, the device's 12 ability to return quality results
quickly and reproducibly permits a user to increase the throughput
of available sample preparation stations at least several times,
depending upon the sample and system employed.
[0141] In short, the multicapillary sample preparation device 12 of
the present invention advantageously increases sample throughput
and decreases variance in a highly reproducible fashion. The
multicapillary device 12 may be used in an array of applications
without departing from the scope of the invention. These include,
but are not limited to, sample handling of small molecules,
polymers, viruses and cells; the isolation, purification,
concentration, desalting and fractionation of biological samples
and nucleic acids, including DNA and RNA; solid phase extraction;
head space analysis; gas chromatography; liquid chromatography
(e.g., HPLC); supercritical chromatography; electrochromatography;
and capillary electrophoresis.
[0142] Representative examples of the above-mentioned applications
include: sample preparation of biological samples such as proteins,
peptides, and polynucleotides, fractionation of peptide mixtures
prior to mass-spectrometric analysis, desalting of samples prior to
instrumental analysis, desalting of peptide solutions, desalting of
protein solutions, sample concentration prior to instrumental
analysis, and peptide concentration prior to mass-spectrometric
analysis.
[0143] While the invention has been particularly shown and
described with reference to the examples and preferred embodiments
thereof, it will be understood by those skilled in the art that
various alterations in form and detail may be made therein without
departing from the spirit and scope of the invention.
* * * * *