U.S. patent application number 10/464657 was filed with the patent office on 2003-11-13 for microfluidic devices connected to capillaries with minimal dead volume.
Invention is credited to Bings, Nicolas, Harrison, D. Jed, Li, Jianjun, Ocvirk, Gregor, Skinner, Cameron, Tang, Thompson, Thibault, Pierre, Wang, Can.
Application Number | 20030211631 10/464657 |
Document ID | / |
Family ID | 22614404 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211631 |
Kind Code |
A1 |
Skinner, Cameron ; et
al. |
November 13, 2003 |
Microfluidic devices connected to capillaries with minimal dead
volume
Abstract
A method is provided for joining a microchip device to a
capillary tube. The microchip device has a capillary channel
opening onto an edge surface of the device. A short hole is drilled
into the edge surface, aligned with the capillary channel. The
drilling is done with a flat bottom, preferably by a two-step
drilling process. Then, the end of the capillary can be inserted
into the hole so that its end is substantially flush with the flat
bottom of the hole, thereby eliminating dead volume. Testing has
shown that this connection provides very little band broadening of
samples transported through the capillary channel into the
capillary tube. The tip of the capillary tube can be tapered, so
that it is suitable for use as an electrospray source for a mass
spectrometer.
Inventors: |
Skinner, Cameron; (Edmonton,
CA) ; Tang, Thompson; (Edmonton, CA) ;
Harrison, D. Jed; (Edmonton, CA) ; Bings,
Nicolas; (Bloomington, IN) ; Wang, Can;
(Edmonton, CA) ; Ocvirk, Gregor; (Edmonton,
CA) ; Li, Jianjun; (Hull, CA) ; Thibault,
Pierre; (Aylmer, CA) |
Correspondence
Address: |
BERESKIN AND PARR
SCOTIA PLAZA
40 KING STREET WEST-SUITE 4000 BOX 401
TORONTO
ON
M5H 3Y2
CA
|
Family ID: |
22614404 |
Appl. No.: |
10/464657 |
Filed: |
June 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10464657 |
Jun 19, 2003 |
|
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09169146 |
Oct 9, 1998 |
|
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6605472 |
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Current U.S.
Class: |
436/174 ;
436/180 |
Current CPC
Class: |
B01L 2300/0838 20130101;
B01L 2200/12 20130101; G01N 30/6095 20130101; Y10T 436/2575
20150115; G01N 30/7266 20130101; Y10T 436/143333 20150115; G01N
30/6034 20130101; B01L 2400/0421 20130101; Y10T 436/25375 20150115;
B01L 2200/027 20130101; Y10T 436/14 20150115; Y10T 436/24 20150115;
Y10T 436/142222 20150115; G01N 27/44791 20130101; Y10T 436/25
20150115; Y10T 436/255 20150115; B01L 3/502707 20130101; B01L
3/502715 20130101; B01L 2300/0816 20130101 |
Class at
Publication: |
436/174 ;
436/180 |
International
Class: |
G01N 001/00 |
Claims
1. An article of manufacture prepared by a process comprising: (a)
drilling a hole into a microchip having at least one capillary
channel opening onto an edge surface thereon, such that a hole
extends from the edge surface into the microchip and is axially
aligned with the channel, with a conically tipped drill bit to
establish a hole with a conical bottom; (b) removing the conical
end face by drilling with a flat tipped drill bit having a diameter
equal to the diameter of the first mentioned drill bit and larger
than the diameter of the capillary channel, to provide the hole
with a flat-bottom and a larger cross-section than the
cross-section of the capillary channel; and (c) mounting a
capillary tube in the hole, such that the capillary tube abuts the
flat bottom, and is bonded to the microchip.
2. The article of manufacture of claim 1, further including a side
channel that can be used for at least one of: introducing at least
one of a buffer and a solvent immediately upstream of the capillary
tube; and applying a voltage.
3. The article of manufacture of claim 1, wherein the capillary has
a square end surface located substantially adjacent to the flat
bottom of the hole.
4. The article of manufacture of claim 1, wherein the capillary
tube is secured by means of an adhesive substance between the
capillary tube and the hole.
5. The article of manufacture of claim 4, wherein the adhesive
substance does not extend to an area between the end surface of the
capillary tube and the flat bottom of the hole.
6. The article of manufacture of claim 1, wherein the hole has a
length which is in the range of 2 to 5 times the diameter of the
hole.
7. The article of manufacture of claim 6, wherein the hole has a
diameter of 200 microns.
8. The article of manufacture of claim 1, wherein the microchip
comprises two glass sheets bonded together, with a network of
channels etched into one glass sheet including a main channel to
which the capillary tube is connected with the other channels
connected to the main channel.
9. The article of manufacture of claim 8, wherein the other glass
sheet includes a plurality of openings, aligned with free ends of
said other channels, providing reservoir connections.
10. The article of manufacture of claim 1, in combination with a
mass spectrometer.
11. The article of manufacture of claim 10, which includes a power
supply connected to the free end of at least one or said other
channels, for providing a potential, for both electrophoresis and
for generating an electrospray from the capillary tube.
12. The article of manufacture of claim 10, which includes a power
supply connected to the free end of at least one or said other
channels, for providing a potential for electrophoresis, and a
second power supply connected to the free end of the capillary
tube, for providing a potential for electrospray ionization.
13. The article of manufacture of claim 1, which includes a
connection T at the free end of the capillary tube and an inlet to
the connection T for a sheath buffer fluid.
14. A method of joining a capillary tube to a microchip including
at least one capillary channel that opens onto an edge surface of
the microchip, the method comprising: (a) filling the end of the
channel adjacent the edge surface of the microchip with a
substantially solid substance having a melting point of at least
49.degree. C., to prevent substantial penetration of glass chips
into the capillary channel; (b) drilling a hole into the edge
surface of the microchip, the hole being aligned with the capillary
channel; (c) removing the substantially solid substance from the
capillary channel; and (d) inserting an end of a capillary tube
into the hole and bonding the capillary tube to the microchip.
15. The method of claim 14, wherein removing said substantially
solid substance comprises the step of melting the substantially
solid substance and removing the melted substance from the end of
the capillary channel using a vacuum.
16. The method of claim 14, wherein said substance is soluble by a
solvent and (c) includes rinsing said capillary channel with said
solvent to wash away the residues of said substance.
17. The method of claim 14, further including dyeing the substance
to aid in locating the capillary channel for alignment with the
hole.
18. A method of joining a capillary tube to a microchip including
at least one capillary channel that opens onto an edge surface of
the microchip the method comprising: (a) providing a stream of
fluid through the capillary channel such that a continuous flow of
fluid out from the free end of the channel is produced; (b)
drilling a hole into the edge surface of the microchip, the hole
being aligned with the capillary channel such that the flow of
fluid flushes away drilling debris; and (c) inserting an end of a
capillary tube into the hole and bonding the capillary tube to the
microchip.
Description
[0001] This application is a continuation application of
application Ser. No. 09/169,146 filed on Oct. 9, 1998.
FIELD OF THE INVENTION
[0002] This invention relates to microfluidic devices, and more
particularly, relates to an apparatus for and a method of coupling
a microfluidic device to an electrospray or other interface of a
mass spectrometer.
BACKGROUND OF THE INVENTION
[0003] Glass microfluidic devices have shown their vast potential
in the field of analytical chemistry in the last decade and enable
a certain amount of separation and analysis to be carried out.
However, to augment the analytical capabilities of microfluidic
chip systems, it is necessary to couple these devices to other
instruments, and in particular it is desirable to be able to couple
them to mass spectrometers. Other uses for this type of connection
include, but are not limited to, coupling of the microfluidic
device to conventional Capillary Electrophoresis (CE) detectors,
sample introduction to a device, automation of a device and
interconnections between devices. In large part, these microfluidic
devices find their greatest utility in high performance
separations. Therefore, connections to the devices must have
minimal dead volumes so that the efficiency of the system is not
compromised.
[0004] The coupling of separation methods with mass spectrometry
provides a powerful tool for rapid identification of target
analytes present at picogram levels in biological matrices, and
structural characterization of complex biomolecules ranging from
small pharmaceuticals to complex antibodies. Furthermore, mass
spectrometry using electrospray ionization (ESMS) has emerged as a
sensitive technique in a number of applications including the
sequencing of peptides comprising common or modified amino acids,
and the analysis of short DNA oligomers. Further modification of
ESMS has improved sensitivity substantially through the use of
ionization techniques operating at sub-microliter flow rates,
giving .mu.ESMS. The flow rates used and potentials applied in
.mu.ESMS are compatible with CE, and this has led to development of
CE-.mu.ESMS instruments, capable of initial separations followed by
mass spectral analysis. A drawback of this approach is the 15-40
min. Separation times often required, which tends to underutilize
the spectrometer.
[0005] Microchip technology has recently been applied to CE,
generating an extremely powerful separation and sample pretreatment
tool (chip-CE) with analysis time of a few seconds. Separations
have been combined on-chip with sample dilution, derivatization,
enzyme digestion, and a set of independent manifolds for separation
have been integrated on to a single chip to give a form of
multiplexed analysis. Thus, sample pretreatment can be automated
within an integrated device, a feature which could offer
significant advantages in sample preparation for mass spectrometry,
particularly if the chip could be designed as an ion source within
an ESMS system.
[0006] Mass spectrometry using electrospray ionization has emerged
as a sensitive technique, providing peptide analysis in the low
nanogram range for digested protein using sequence tags and data
base searching (Mann, M., Wilm, M., Anal. Chem., 66, 4390-4399
(1994)). Sequence information can be obtained from tandem mass
spectrometric analysis where a given multiply-charged precursor ion
is selected by the first mass analyzer and the fragment ions
resulting from collisional activation with a neutral target gas
(e.g. Argon) are transmitted into the second mass analyzer. The
product ion spectra are characterized by easily identifiable series
of fragment ions, and can be interpreted in the absence of protein
or DNA sequence. Even in situations where only partial sequence is
obtained, the sequence tag plus the peptide molecular weight can be
used to locate the peptide in a given protein or data base. This
combined approach was recently presented for the characterization
of proteins from silver-stained polyacrylamide gels (Shevchenko,
A., Wilm, M., Vorm, O., Mann, M., Anal. Chem., 68, 850-858 (1996)).
Such advances have been facilitated by the introduction of
micro-electrospray ionization operating in the low nL/min flow rate
regime (Wilm. M. Mann, M., Int. J. Mass Spectrom. Ion Proc., 136,
167-180 (1994)). Although this mode of sample introduction does not
require any prior analyte separation (e.g. Liquid chromatography or
CE), the sensitivity of the micro-electrospray technique can be
adversely affected by the presence of salts used in proteolytic
digestion or by the simultaneous ionization of a large number of
different peptides isolated from digestion or by the simultaneous
ionization of a large number of different peptides isolated from
these digests. In addition, the mass spectra of unseparated digests
are further complicated by the appearance of multiply-protonated
molecules (M+nH)n+ for each peptide, which significantly compromise
interpretation if more than one peptide is initially present. The
combination of a high resolution separation technique to
micro-electrospray sources thus confers a unique advantage in
situations where both sensitivity and selectivity are desired.
[0007] The production of stable ionization conditions from
micro-electrospray sources requires critical adjustment of low
liquid flow rate (10-300 nL/min), column diameter, and field
strength at the micro-electrospray tip. Consequently, the coupling
of separation techniques to micro-electrospray is best achieved
using CE, which typically operates in a flow rate regime of less
than 300 nL/min. Recent reports have demonstrated the applicability
of the capillary electrophoresis-micro-electrospray mass
spectrometry (CE-.mu.ESMS) approach for peptides and protein
digests (Wahl, J. H., Gale, D. C., Smith, R. D., J. Chromatogr,
659, 217-222 (1994); Kriger, M. S., Cook, K. D., Ramsey, R. S.,
Anal. Chem.; 67, 385-389 (1995); Kelly, J. F., Ramaley, L. R.,
Thibault, P., Anal. Chem. 69, 51-60 (1997)). As a result of the
high separation efficiencies obtainable with CE, analyses conducted
using CE-.mu.ESMS typically yield 20-100 femtomoles mass detection
limits in full-mass scan acquisition mode and 100-200 femtomoles
for tandem mass spectrometric analyses. This is a 10-fold
enhancement of sensitivity compared to more conventional (i.e.
non-micro) CE-ESMS interface, using a coaxial sheath design
operating at flow rates of 2-10 .mu.L/min.
[0008] The limited sample volume used in CZE (2% of capillary
volume), results in concentration detection limits of approximately
1 .mu.M, even at 20 femtomole mass detection limits. Improvement in
sample loadings can be achieved using isotachophoretic
preconcentration (Foret, F., Szoko, E., Karger, B. L., J.
Chromatogr, 608, 3 (1992); Foret, F., Sustacek, V., Bocek, P., J.
Microcol. Sep., 2, 127 (1990); Mazereeuw, M., Tjaden, U. R.,
Reinhoud, N. J., J. Chromatogr. Sc., 33, 686 (1995)). This approach
was successfully applied to the analysis of paralytic shellfish
poisoning toxins present at low nM concentration levels in
contaminated shellfish tissues, and enabled the injection of up to
1 .mu.L on a single capillary arrangement (Locke, S. J., Thibault,
P., Anal. Chem., 66 6436 (1994)). On-line trace enrichment can also
be obtained by loading large volumes of sample using microcolumns
containing adsorptive media, followed by elution or
electromigration onto a CE column. A review of different
chromatographic preconcentrators has been presented recently
(Tomlinson, A. J., Guzman, N. A., Naylor, S., J. Cap. Elect., 6,
2247 (1995)). These methods provide satisfactory means to overcome
many detection limit problems.
[0009] Capillary Electrophoresis is a well established method and
provides a number of separation formats thus giving flexibility for
the analysis of different biomolecules. It is well suited as a
sample introduction device to a mass spectrometer (Banks, J. F.,
Recent Advances in Capillary Electrophoresis/Electrospray/Mass
Spectrometry. Electrophoresis, 18, 1997; and Cai, J. and Henion, J.
Capillary Electrophoresis--Mass Spectrometry. J. Of Chromatography
A., 703, 1995) At the most recent High Performance Capillary
Electrophoresis Conference in Orlando Fla., several research groups
reported on their efforts to directly interface microfluidic
devices to mass spectrometers (Ramsey, R. S. And Ramsey, J. M. New
Developments in Microchip ESI Mass Spectrometry; Figeys, D. And
Aebersold, R. Microfabricated Devices Coupled to an Ion Trap Mass
Spectrometer for the Identification of Proteins; and Liu, H.,
Foret, F., Zhang, B., Felten, C., Jedrzejewski, P. And Karger, B.
L. Development of Microfluidic Devices for High Throughput ESI/MS).
These groups have demonstrated that it is possible to obtain an
electrospray directly from the microfluidic device, but they did
not demonstrate high efficiency separations. The inventors'
experience with electrospray directly from the edge of a device, as
in these other proposals, has shown that the droplet formed on the
face of the device is sufficiently large that high efficiency
separations are not possible due to the large mixing volume.
[0010] The effect of dead volumes is to distort the peak shape and
increase band broadening. The maximum separation efficiency that
can be observed with a microfluidic CE system joined to a capillary
is limited by four principal sources of band broadening, namely
longitudinal diffusion and effects of both injection and detection
volume as well as any additional dead volumes.
[0011] One proposal has demonstrated reasonable separations with a
device that included a pneumatic nebulizer (Foret, F., Liu, H.,
Zhang, B. and Karger, B. L., Single and Multiple Channel
Microdevices for Microanalysis by ESI/MS. HPCE, Orlando, Fla., Feb.
1-5, 1998).
[0012] This still relies on forming an electrospray plume from the
edge of the device, but combines this with a pneumatic or gaseous
flow to improve nebulization of the emerging droplet, thereby
reducing the droplet size and assisting in volatilization. A built
in sprayer on the end of the chip is apparently simple and
advantageous. However, it is believed that this can never give the
same performance as a tapered capillary tip. Such a capillary tip
provides a smaller droplet size, thus less dead volume, and less
band broadening. The length of the capillary can be changed to meet
changing resolution needs since separation continues in the
capillary. These combined devices would be able to exploit
commercial micro electrospray interfaces, with independent control
over the electrospray operating parameters.
[0013] The literature has reported several methods used to join
capillaries to microfluidic devices but to date they have
shortcomings. Figeys et al. have constructed a butt joint to the
edge of the chip with the use of a piece of Teflon tubing glued to
the edge of the device as a guide sleeve and mooring point for the
capillary. The capillary was used as an electro-osmotic pump for
the introduction of protein digests to a MS device (Figeys, D.,
Ning, Y. And Aebersold, R., Anal. Chem., 69, 1997, p. 3153-3160).
This article gives information regarding the dead volume of the
connection. It also acknowledged the presence of contamination that
may have been due to the epoxy used to glue the capillary in
position. This method also requires that the capillary and the
channel be aligned to within a few microns and held in position by
the glue, a difficult task at best. This type of connection has the
additional shortcoming that it is not possible to directly examine
the joint for the presence of debris, glue or dead volume. These
problems render this joining technique impractical for most
applications.
[0014] With silicon it is possible to form a connection with
minimal dead volume. This was demonstrated by van der Moolen et al
(van der Moolen, J. N., Poppe, H. And Smit, H. C., Anal Chem., 69,
1997, P. 4220-4225). The SEM images of the interface presented in
their article showed a tight connection with no apparent dead
volume. The device was intended for correlation CE and no
investigations were made for presence of band broadening introduced
from the joint. Furthermore, the silicon device was used as an
injector and performed the separation on the capillary.
Unfortunately it is not possible to chemically etch deep structures
into glass while retaining flat surfaces suitable for joining to a
capillary so that the silicon procedure used by Moolen is
inappropriate for glass. The problem with silicon devices is their
inability to sustain the high electric fields that glass devices
exploit for rapid separations. Consequently, a method to make low
dead volume connections to glass devices is still needed.
SUMMARY OF THE INVENTION
[0015] Instrumental modifications are required to improve the
analytical performance of the CE-.mu.ESMS interface in terms of
ruggedness and speed of analysis. The present invention is based on
the development of a compact and versatile, micromachined chip
device to perform CE or other sample manipulation and then
introduce the sample to a .mu.ESMS system, giving a
chip-CE-.mu.ESMS hybrid system. The chips are thus an integral
component of the electrospray ion source for the mass spectrometer,
providing both sample treatment and ion source functions. The
intent of the present invention is to develop a chip-ES interface
which is easily manufactured, so that it can be made commercially
at lower cost than current methods, and can increase utilization
and sample throughput of rather powerful, but expensive instruments
such as ESMS systems. This ES interface will be reusable, but
readily replaced when required by the user.
[0016] Interfacing chips to .mu.ESMS would greatly expand the
potential of both CE and ESMS for biotechnological applications
requiring faster analysis time, enhanced sensitivity and
selectivity. On-chip separations will provide for sample clean-up
and separation of components to prevent interference in the mass
spectrum, with a substantial reduction in analysis time (less than
5 and typically under 2 minutes). Minute sample and reagent
consumption with less solvent and salt introduction at the
interface should also lead to increased performance and
efficiency.
[0017] To address this need, the inventors have developed a method
of connecting fused silica capillaries to microfluidic devices and
an associated article of manufacture. Silica capillaries were
chosen because electrophoretic separations begun on the device can
continue on the capillary and silica is transparent over a wide
wavelength range. The initial invention was to develop an interface
to MS that exploits the common micro electrospray. The results of
MS experiments are presented.
[0018] The present invention provides in one aspect a method of
joining a capillary tube to a microchip including at least one
capillary channel that opens onto an edge surface of the microchip,
the method comprising:
[0019] (a) filling the end of the channel adjacent the edge surface
of the microchip with a substantially solid substance having a
melting point of at least 49.degree. C., to prevent substantial
penetration of glass chips into the capillary channel;
[0020] (b) drilling a hole into the edge surface of the microchip,
the hole being aligned with the capillary channel;
[0021] (c) removing the substantially solid substance from the
capillary channel; and
[0022] (d) inserting an end of a capillary tube into the hole and
bonding the capillary tube to the microchip.
[0023] In another aspect, the present invention provides a method
of joining a capillary tube to a microchip including at least one
capillary channel that opens onto an edge surface of the microchip,
the method comprising:
[0024] (a) providing a stream of fluid through the capillary
channel such that a continuous flow of a fluid out from the free
end of the channel is produced;
[0025] (b) drilling a hole into the edge surface of the microchip,
the hole being aligned with the capillary channel such that the
flow of fluid flushes away drilling debris; and
[0026] (c) inserting an end of a capillary tube into the hole and
bonding the capillary tube to the microchip.
[0027] Another aspect of the present invention provides an article
of manufacture prepared by a process comprising:
[0028] (a) drilling a hole into a microchip having at least one
capillary channel opening onto an edge surface thereon, such that a
hole extends from the edge surface into the microchip and is
axially aligned with the channel, with a conically tipped drill bit
to establish a hole with a conical bottom;
[0029] (b) removing the conical end face by drilling with a flat
tipped drill bit having a diameter equal to the diameter of the
first mentioned drill bit and larger than the diameter of the
capillary channel, to provide the hole with a flat-bottom and a
larger cross-section than the cross-section of the capillary
channel; and
[0030] (c) mounting a capillary tube in the hole, such that the
capillary tube abuts the flat bottom, and is bonded to the
microchip.
[0031] The capillary is advantageously bonded in position by an
adhesive substance, which does not extend to an area between the
end surface of the capillary tube and the flat bottom of the
hole.
[0032] In yet another aspect of the present invention, this device
is provided in combination with a mass spectrometer.
[0033] It is to be appreciated that this invention is not limited
to application with mass spectrometers. More generally, the method
is applicable to any aspect of microfluidic technology in which it
is desired to connect a capillary tube to a microchip containing
capillary channels. Such a device can be used to carry out a wide
variety of different analytical and other techniques. The capillary
tube itself need not necessarily have a free end, but could
conceivably be connected at both ends to microfluidic chips, so as
to provide an inter-connection between them. The capillary tube
enables a channel of any desired length to be provided, and can
enable a variety of different processing to be carried out, e.g. by
irradiation of the tube and/or detection of substances travelling
through the tube.
[0034] A chip-ESMS interface also offers other advantages.
Microfabrication allows for multiple sample treatment manifolds on
a single chip, so that multiplexing of sample introduction into a
single MS is feasible, thereby increasing throughput. Also, regular
supply of mass calibration standards is possible via the chip,
giving improved mass accuracy. Finally, a variety of more complex
sample treatments, such as on-chip digestion of proteins or DNA can
further automate sample preparation and introduction in the
mESMS.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] For a better understanding of the present invention and to
show more clearly how it may be carried into affect, reference will
now be made, by way of example, to the accompanying drawings which
show a preferred embodiment of the present invention and in
which:
[0036] FIG. 1 is a schematic perspective view of a microfluidic
device and a capillary tube in accordance the present invention,
with FIG. 1a showing a detail of the microfluidic device;
[0037] FIG. 2 shows a perspective view of a drilling press
including a bracket in accordance with the present invention;
[0038] FIGS. 3a, 3b and 3c show sequentially steps in drilling a
hole in the edge of the microfluidic device;
[0039] FIG. 4 is an image of a capillary inserted into a hole with
a conical bottom, showing the dead volume;
[0040] FIG. 5 shows electropherograms obtained using the
combination device of FIG. 4;
[0041] FIG. 6 is an image of a capillary inserted into a hole with
a flat bottom;
[0042] FIG. 7 shows electropherograms obtained using the
combination device of FIG. 6;
[0043] FIG. 8a shows a schematic view of a microfluidic device-ESMS
coupled directly with a micro-electrospray emitter;
[0044] FIG. 8b shows a schematic view of a microfluidic device-ESMS
interface using a coaxial sheath flow;
[0045] FIG. 9a shows an ion electropherogram from the microfluidic
device-ESMS interface shown in FIG. 8a with extracted mass spectra
for peaks highlighted in FIGS. 9b-9e;
[0046] FIG. 10a shows an ion electropherogram from the microfluidic
device-ESMS interface in FIG. 8b with extracted mass spectra for
peaks highlighted in FIGS. 10b-10j; and
[0047] FIG. 11 shows schematically an apparatus for an alternative
method of drilling a hole in the edge of a microfluidic device.
DETAILED DESCRIPTION OF THE INVENTION
[0048] For the preferred embodiment of this invention, glass chips
designed for interfacing directly to mass spectrometer were used,
and these can have either a coaxial sheath flow arrangement or a
sheathless interface. The chip can have a side channel at the end
of the chip, forming a Y, that allows make up fluid flow and
electrical control to assist the electrospray function. The glass
chips were prepared using the techniques and procedures previously
discussed in the literature. The glass chips or microfluidic
devices were designed to have the separation channel exit the edge
of the chip to form an electrospray and as such are well suited to
the present experiments. FIG. 1 illustrates the overall layout of
the device and the experimental setup.
[0049] FIG. 1 shows a combination device 10 comprising a
microfluidic chip 12 and a capillary 14 connected thereto, in
accordance with the present invention and as detailed below.
Detectors 16 are indicated schematically, for detecting radiation
from the chip 12 and the capillary 14. A counter electrode 18 is
provided for test purposes, but its use would be replaced by a mass
spectrometer. As indicated, in this preferred embodiment, the
capillary 14 has a length, indicated at 20, of 5.5 cm. The end of
the capillary, as indicated at 22, is spaced at 5 mm from the
counter electrode 18.
[0050] The exact configuration of the microchip 12 is not critical
to the present invention, and indeed any configuration of channels
can be provided. Nonetheless, an exemplary channel configuration is
detailed below.
[0051] The glass microchip 12 is formed from two layers. The
channels are etched in one layer, and typically would have an
approximately trapezoidal shape, or a cylindrical, oval or
rectangular shape. A second glass sheet is then bonded to the top
of the first sheet with the etched channels. The top sheet is
usually formed with small holes, with a diameter of 1-2 mm,
centered over the ends of the channels 26-36, so as to provide
connections to reservoirs. Reservoirs can either be formed by
bonding directly onto the top glass sheet or by otherwise forming a
connection to the small holes.
[0052] A main microcapillary channel 22 extends from a junction
with the capillary 14 indicated at 24. A buffer channel 26 is
connected to the other end of the main micro channel 22. A sample
line is provided at 28, and on the opposite side of the channel 22
there is a sample waste 30. A further channel 32 is provided, but
this was not used for experiments detailed below. A floating
reservoir line 36 is connected to the main channel 22, adjacent the
junction 24.
[0053] Junctions between the channels 28, 30 and 32 and the main
channel 22 are shown in detail in FIG. 1a. As shown, each of the
channels 28, 30 and 32 forms a T-connection with the main channel
22, and they are all offset relative to one another. Between the
channels 28 and 30, this creates a short section, indicated at 34
of the main channel 22. The configuration of the sample supply line
and reservoir 28 and the sample waste 30, enables a portion of the
sample to be flowed through to 30, and, once the supply of sample
is cut off, there will be left a plug or fixed length of the sample
in the section 34.
[0054] Reference will now be made to FIGS. 2 and 3, to describe the
detailed manner in which the capillary tube 14 is joined to the
microfluidic device or chip 12.
[0055] The glass chip 12 was first prepared by filling one end of
the main or separation channel 22 with dyed Crystal Bond 509
(Aremco Products--Crystal Bond is a trade mark of Aremco Products)
to aid visualization and prevent plugging the channel 22. One to
two drops of black ink from a Staedtler Lumocolor permanent pen
were mixed with approximately 1-2 ml of melted Crystal Bond to make
the dyed Crystal Bond. Only the length of the separation channel 22
adjacent to the junction 24, where the device 12 was to be cut and
drilled, was filled, since the material was quite viscous. The
device 12 was placed on a hot plate (80.degree. C.) and by applying
vacuum on the side or reservoir channel 36, the desired segment of
the main channel 22 was filled.
[0056] Crystal Bond was selected to fill the channel 22, over other
possible options such as water and paraffin wax. It has a number of
desirable attributes, namely: it is readily available; accepts dyes
readily; it has a low melting point; forms free flowing chips
reducing binding on the drill; and is largely insoluble in water
yet readily soluble in acetone.
[0057] Crystal Bond does have a disadvantage that prolonged
exposure to water causes it to soften and expand. For this reason,
the Crystal Bond is only allowed on the outside of the capillary
and not to the area at the end of it, i.e. the dead volume 50 (FIG.
4). Other bonding materials can be used, but Crystal Bond does have
the advantage that it allows a joint to be disassembled by gentle
heating.
[0058] With the channel 22 filled with Crystal Bond, the device 12
was cut at the appropriate position perpendicular to the separation
channel 22 with a diamond glass saw. The newly cut edge surface 38
was sanded smooth and flat with 220 grit and then 600 grit silicon
carbide abrasive paper. This step facilitated locating the end of
the separation channel and reduced the risk of the drill catching
on the surface.
[0059] With reference to FIG. 2, the device was then clamped
vertically to a bracket 100 mounted on a horizontal Z axis
translation stage 102 (Newport, Irvine, Calif.) where the device is
movable in a horizontal XY plane and the drill is movable
vertically along the Z axis, with the main separation channel 22
held vertically and the end surface 38 on top.
[0060] As shown in FIG. 2, the translation stage 102 is mounted on
the base of a drill press indicated generally at 104. The drill
press 104 is a conventional, high quality drill press. The bracket
100 is secured, as by screws to the top of the translation stage
102. The bracket 100 includes a main body 106 and a clamping plate
108, and a number of screws 110 are provided, to provide a simple
clamping action. The chip 12 can then be clamped between the main
body 106 and the clamping plate 108, with its edge surface 38 at
the top, as indicated. Then, in known manner, the drill bit,
indicated at 114, and mounted in the chuck of the drill press 104
can be brought down vertically to engage the surface 38.
[0061] In accordance with the present invention, a flat bottomed
hole was cut, forming the junction 24. This was carried out using
200 .mu.m tungsten carbide drills and flat tipped drills which were
purchased from Tycom (Mississauga, Ontario) and had nominal
tolerances of +0-8 .mu.m. Alternatively flat tipped drills can be
prepared manually by grinding the tip of the drill flat with a fine
diamond wheel. A small jig was built to hold the drill bit and the
wheel was rotated manually to avoid breaking the delicate drill.
The manually flattened drill bits appear to produce better quality
holes than the commercially prepared bits.
[0062] When drilling the hole, it was found necessary to use a high
quality drill press 104 with no measurable runout. Such a press can
produce holes with less potential for cracking of the wall of the
hole and less drill bit breakage. The drill bit must follow the
channel 22 within the chip 12; it was found that finer drills (200
micron) were better able to follow channel 22, having a nominal
width of 45 microns, through the glass. It was also easier to
center the drill bit. Due to their small diameter and brittle
nature the glass powder must be removed from the drills.
[0063] The choice of capillaries compatible with the 200 micron
size of drill was limited to 185 .mu.m OD and 50 .mu.m ID.
Unfortunately this inside area of the capillary does not provide a
very good match to the cross sectional area of the separation
channel. The channel has a cross sectional area of approximately
450 .mu.m.sup.2 whereas the capillary has an area of 1960
.mu.m.sup.2 or about 4.4 times larger. Such large mismatch in areas
can lead to unexpected band broadening due to inhomogeneous
electric fields and distortions of the sample zone at the
interface. The observed separation efficiency was about 95% of the
predicted value for this volume mismatch, so the effect appears to
be minor when the flat-bottomed connection is used.
[0064] Capillaries with a wider selection of ID are available in
the 140 to 150 micron OD range but it was not possible to evaluate
these capillaries. The 150 micron nominal OD drill bits that were
tested in fact produced holes smaller than the available
capillaries because of the relatively large negative tolerances on
the drills. Larger drill bits (370 micron) produced holes suitable
for (365 micron) capillaries which are available with a wide range
of ID. These capillaries appear to have a larger dead volume
because of the larger diameter and hence larger area that is not
sealed with Crystal Bond. The larger capillaries were not evaluated
because interfacing the device to the mass spectrometer, the
impetus for this research, is more convenient with the smaller
diameter capillaries.
[0065] The Z axis translation stage 100 was used to center the
channel on the end of the drill bit 114. A 20.times. jewellers
loupe and side illumination were used, while the drill tip 114 was
about 0.2-0.5 mm above the surface of the chip, to facilitate
alignment. Once the drill 114 was accurately centered it was turned
on (4000 RPM) and lowered until the tip touched the edge surface 38
(FIG. 2), where it began removing glass. At this stage the drill
114 was raised and the face examined to ensure that the drill was
on the center of the channel 22; if not, the chip 12 was removed,
resanded and a new hole started.
[0066] If the hole was centered, a drop of water was placed on the
surface of the device to help lubricate and cool the drill. The
drill 114 was then lowered into the glass and allowed to bore
approximately 1-2 drill diameters into the glass before it was
raised. This process was repeated until a hole 40 of suitable depth
(600-800 .mu.m) was obtained (FIG. 3a). The face of the chip 12 was
then cleaned with a paper towel to remove the glass powder produced
during drilling. As detailed below both conventional conically
tipped holes and flat bottomed holes were drilled for comparison
purposes. For the flat bottomed holes, the conventional conically
tipped drill bit 114 was then replaced with the flat tipped drill.
A new drop of water was placed on the device, the flat tipped drill
was introduced and the bottom of the hole was flattened in one step
(FIG. 3b), as indicated at 42. FIG. 3c illustrates that the bottom
of the hole "fishtails" or widens out, if the flat tipped drill is
forced to drill beyond the end of the hole left by the pointed or
conical drill, as indicated at 66. The flat face of the drill bit
is not capable of removing the glass so the lower wall of the hole
is enlarged which results in a poor connection and possibly
increased dead volume.
[0067] The glass debris was removed from the hole by using one of
the two following techniques. If there were no air bubbles in the
hole then the device or chip 12 was inverted in a beaker of water
and the glass particles were allowed to settle out of the hole.
This required a few hours. Alternatively a capillary (at least 25
.mu.m smaller OD than the hole diameter) was used to flush the hole
with filtered water (0.45 .mu.m, Millipore). The chip was then
placed on a hot plate and the Crystal Bond was melted and removed
via the hole 40 at the end of the channel 22 with the aid of
vacuum. The device 12 was removed, allowed to cool and the residues
of the Crystal Bond were washed clean with reagent grade acetone
(Caledon Laboratories Ltd., Georgetown, Ontario).
[0068] The end of the capillary 14 for connection at the junction
24 was prepared by sanding the end flat and square with 600 and
1200 grit silicon carbide paper, glass particles were flushed out
with water. The capillary was glued into the device 12 by first
placing the device 12 on a 10.times.10 cm scrap of glass to
facilitate handling the hot assembly and then inserting the
capillary tube 14 into the hole. The whole assembly was placed on a
hot plate and allowed to heat to the Crystal Bond melting point
(80.degree. C.). A small amount of the Crystal Bond was applied
onto the surface 38 face of the joint and allowed to wick into the
hole 40 until it nearly reached the end of the capillary 14. The
rate of flow was controlled by adjusting the temperature. The
assembly was removed and cooled with forced air to freeze the
Crystal Bond.
[0069] Reference will now be made to FIGS. 4 and 6, which show the
hole with a conical end, as drilled by a conventional drill and a
flat-bottomed hole respectively. These holes in FIGS. 4 and 6 are
indicated at 40a and 40b to distinguish them from one another and
are also indicated as such in FIGS. 3a and 3b.
[0070] Referring first to FIG. 4, the hole 40a has a conical end
surface indicated at 41. The capillary 14 is shown inserted into
the hole 40a. The end plane of the capillary is indicated at 46,
i.e. the capillary 14 has a square end surface 46. The Crystal Bond
securing the capillary 14 in position is indicated at 48. As shown,
the conical end face 41 prevents the capillary 14 reaching the end
of the hole. Consequently, there is a relatively large dead volume
50, having a frusto-conical shape between the end plane 46 of the
capillary 14 and the capillary channel 22. This dead volume 50, as
shown, is relatively large compared to the dimensions of the
capillary channels, and is a significant multiple of the length
along any capillary.
[0071] Referring to FIG. 6, this shows a joint formed in accordance
with the present invention. Here the hole 40b has a plane end face
indicated at 43. Again, the capillary 14 is shown, secured in
position with the Crystal Bond 48. It can now be seen that the end
plane 46 with capillary 14 is very close to the bottom of the hole
43, so as to leave a relatively small dead volume 52. As explained
below, this reduces band broadening due to dead volume effects.
[0072] The chip and capillary assembly was flushed with water, 0.1
M NaOH and then with the running buffer for 30 min. for the
complete assembly or apparatus. Partially decomposed (due to age)
0.1 .mu.M FITC labelled arginine diluted with buffer was used as a
sample. Labelled arginine was prepared by mixing 7.63 mM arginine
with 1.52 mM FITC (Sigma) before allowing the mixture to stand
overnight at room temperature and stored at 40.degree. C. 50 mM
morpholine in deionized distilled water was used as a buffer with
an unadjusted pH 9.5. Morpholine was chosen for this project
because this buffer is suitable for MS detection of peptides.
[0073] A sample was injected (from sample line 30 in FIG. 1) by
applying -1.5 kV at the sample waste 28 with 26, 30 and 36 grounded
and the counter electrode 18 was left floating. With sample present
at 34, separation was performed by applying -7 kV to the counter
electrode 18, grounding the buffer reservoir 26, floating reservoir
36 and applying pushback voltages of approximately -550 V at 28 and
30, depending on liquid levels. During separation an electrospray
formed at the tip of the capillary 14. A flat piece of copper was
connected as a counter electrode with an electrospray gap 27 of 5
mm, as shown.
[0074] The channel 32, shown on the inset, is for a small volume
injector, and was not used in this study and was left to float
electrically.
[0075] Two devices, one where the hole was left with a conical
bottom 40a from the pointed drill bit (FIG. 4) and another one with
a flat bottomed hole 40b (FIG. 6) were used for the experiments and
the experimental parameters are set out in Table 1. The difference
in resolution caused by the different geometry of the two joints
was evaluated by comparing the resulting electropherograms. Two
separate 2 mW argon ion laser beams (448 nm) were focused to 40
.mu.m diameter spots on the capillary 14 and the device,
respectively. Fluorescence signals were collected during
separation, one on the separation channel 22, 4.4 cm from the
injector 34, 1 mm before the function 24 and the second on the
capillary 4.9 cm after the junction, 4 mm from the tip of the 5.3
cm long capillary.
[0076] As detailed below in relation to FIGS. 8a and 8b, for
electrospray operation an electrical connection needs to be made
such that droplets emerging from the emitter are charged. This can
be achieved by using the potential applied to the separation
reservoir, 30 for example, to initiate both the separation and the
ionization of analytes. As also detailed below, an electrospray
emitter can be prepared by tapering one end of the fused silica
capillary to a smaller diameter. Alternatively, electrospray
contact can be made using a gold-coating capillary emitter, butted
to the chip which also offers independent control of the
electrospray voltage.
[0077] The effect of dead volumes is to distort the peak shape and
increase band broadening for non-absorbing ions. The maximum
separation efficiency within a microfluidic CE system is limited by
longitudinal diffusion, the effects of both injection and detection
volume as well as any additional dead volumes. A measure of the
efficiency of a separation system is the ratio of the measured
plate numbers to the maximum plate numbers predicted by theory. The
number of plates predicted by theory can be expressed as: 1 N = L 2
2 ( 1 )
[0078] where L is the length of the capillary and .sigma..sup.2,
the variance of the peak, is given by:
.sigma..sup.2=2D.sub.it+L.sub.inj.sup.2/12+L.sub.det.sup.2/12+.sigma..sub.-
dv .sup.2 (2)
[0079] where D.sub.i is the diffusion coefficient of the analyte
and t is analysis time, L.sub.inj is the length of the injected
sample and L.sub.det is the detector spot size. The
.sigma..sub.dv.sup.2 term represents the effect of the joint's dead
volume, and is of an unknown form that depends on the geometric
shape of the dead volume and the electric field. Using equations
(1) and (2) we calculated the expected number of plates using a
separation column (injector to detector distance) length of L=4.4
cm on the chip and 9.4 cm on the attached capillary. A D.sub.i
value of 3.9.times.106 cm.sup.2/s was used for all of the
components in the labelled arginine sample. The detector length was
40 .mu.m and the injector length 34 was 400 .mu.m.
[0080] The plate numbers were calculated from the well resolved
peaks in FIGS. 5 and 7, the percentage ratio of the measured to
calculated peak efficiencies are presented in Table 2. From FIG. 5
it is clear that the tapered bottom hole left by the pointed or
conical drill bit (FIG. 4) introduces significant band broadening.
For example, the plate numbers for Peak 2 went from 40,000 to
15,500 on going from the device to the capillary. As a result, this
system only delivers about 12-17% of its theoretical efficiency.
This should be contrasted with the data that was collected from the
flat bottomed hole (FIG. 6) as shown in FIG. 7. In this case the
plate numbers increased significantly from the device to the
capillary and the observed efficiencies were in the range of 54-95%
of the maximum. For example, the number of theoretical plates went
from 47,000 to 112,000 and from 71,000 to 117,000 for peaks 1 and 3
respectively. Theory predicts that the increased length of
separation on going from the chip to the capillary should also give
increased total number of plates. The results presented in FIG. 7
agree with this and demonstrate that there is minimal dead volume
at the joint between device and capillary.
[0081] Given this data, it is clear that drilling into the device
represents a useful method for connecting microfluidic devices to
common CE capillaries. The requirements for minimized dead volume
in the flow path can be easily met by using tipped drills to create
flat bottomed holes.
[0082] This technique for connecting capillaries could be used for
attaching standard capillaries for a wide range of applications.
For example simple injectors could be attached to capillaries to
give standard CE instrumentation the high efficiencies and rapid
sample introduction currently enjoyed only by complete microfluidic
devices. Much of the current CE detector technology is based on UV
absorption. With a capillary attached to the device it is possible
to exploit UV absorption as a means of detection. Currently this is
a difficult task for microfluidic devices unless constructed from
fused silica and employing multi-reflection absorption cells.
[0083] The coupling of sample separation with mass spectrometry by
means of electrospray ionization provides a powerful tool for rapid
identification of analytes present in picogram levels in biological
matrices, and structural characterization of complex biomolecules.
Furthermore, ESMS has emerged as a sensitive technique in a number
of applications including the sequencing of peptides comprising
common or modified amino acids, and the analysis of short DNA
oligomers. Microfluidic devices, which could easily be connected to
commercial electrospray nebulizers by common CE columns applying
the here described coupling technique, would greatly expand the
potential of both CE and ESMS for biotechnological applications
requiring faster analysis time, enhanced sensitivity and
selectivity. The resulting on-chip separation and a wide variety of
sample treatments, e.g. the on-chip digestion of proteins or DNA,
would provide for sample clean-up and separation of components to
prevent interference in the mass spectrum, with a substantial
reduction in analysis time. Minute sample and reagent consumption,
with less solvent and salt introduction at the MS interface should
also lead to increased performance and efficiency.
[0084] For electrospray modes, the diameter of the tip is
critically important to the performance of the device. Hence, the
ability to change capillaries, and tip diameters, is particularly
useful, since the type of tip of the capillary is quite fragile and
susceptible to plugging and breakage.
[0085] Referring now to FIG. 8a, there is shown, schematically, a
microfluidic device 60 to which is attached a capillary tube 62, as
described above. The microfluidic device 60 is provided with three
wells, indicated at A, B and C, connected by short channels to a
main channel 64. A side channel 66 is connected to an additional
buffer D.
[0086] Here, the capillary tube 62 had a length of 1-5 cm, and a
180 micron outside diameter (50 micron inside diameter). It was
prepared by tapering one end of a fused silica capillary tube to a
diameter of approximately 50 microns o.d. or less.
[0087] For electrospray operation, an electrical connection needs
to be made such that droplets emerging from the emitter are
charged. This can be achieved by using the potential applied to the
separation reservoir to initiate both the separation and the
ionization of analytes (FIG. 8a).
[0088] A tapered tip can be provided by manually suspending a metal
weight (15 g) from one end of the capillary and melting the fused
silica with the flame from a microwelding torch (see K. P. Bateman,
R. L. White, and P. Thibault, Rapid Commun. Mass Spectrom., 11,
307-315 (1997)) to taper the free end of the capillary tube to an
inner diameter of 15 .mu.m (50 .mu.m o.d.). The other end of the
capillary 62 is then inserted in the chip 60 as described
earlier.
[0089] In use, a solution to be tested was supplied at well A in
FIG. 7a. Typically, well B contains the separation buffer, and well
C is the sample injection waste reservoir. A voltage applied
between wells A and C creates a sample volume at the intersection
of the channels. This plug is then driven towards the electrospray
tip with a voltage applied between well B and the mass
spectrometer. During the latter step a voltage can occasionally be
applied to well D to assist the electrospray step. Typically
voltages are in the range of 200 to 15,000 V on the chips, although
30,000 V has been demonstrated. Linear flow velocities of 0.01 to
15 mm/s can be achieved with these potentials. In order to improve
the stability of the electrospray, an additional buffer solution
was pumped from well D to increase the flow rate. The buffer
solution was applied from the side channel (well D) with a flow
rate of 50 nL/min. Also, a positive potential of the order of 5 kV
was applied to well B to effect for both separation and analyte
ionization. The configuration of the chip design required that the
flow from the side channel 66 be set to 150 nL/min or lower.
Increase of flow rate above 150 nL/min resulted in improved signal
stability, though the sensitivity was reduced as a result of peak
broadening and counter flow during the electrophoresis separation.
Alternatively, electrospray contact can be made using a gold-coated
emitter butted to the chip which also offers independent control of
the electrospray voltage (see K. P. Bateman, R. L. White, and P.
Thibault, Rapid Commun. Mass Spectrom., 11, 307-315 (1997)).
[0090] Reference will now be made to FIG. 8b which shows an
alternative arrangement. The microchip is indicated at 70 and the
capillary tube at 72. As for FIG. 8a, wells on the microchip are
indicated A, B and C and are connected to a main channel 74. A side
channel 76 is connected to a well D and an additional well E is
provided. Here, the capillary tube, a fused silica transfer line
72, was generally longer and had a length of the order of 10-15 cm,
with a 180 micron outside diameter and 50 micron inside diameter.
It is connected to a T connector 78 including an inlet 80 for a
sheath liquid. The sheath liquid flows in the inter-space between
the CE column and the electrospray tip or needle at the tip of the
connector 78 (see reference J. F. Kelly, S. J. Locke, L. Ramaley,
P. Thibault, J. Cromatogr. A, 720, 409-427 (1996)). This approach
provides an independent means of modifying the composition of the
electrospray buffer for enhanced sensitivity, while simultaneously
maintaining continuity of the voltage gradient across the CE
capillary.
[0091] An ESI power supply 82 is connected between ground and the T
connector 78. A CE power supply 84 is connected between ground and
the well A as indicated from which to provide a potential for
capillary electrophoresis.
[0092] As indicated schematically at 90, the electrospray could be
directed to the inlet of a quadrupole mass spectrometer having a
nitrogen curtain gas at the inlet, in known manner. The
spectrometer could be, for example, a PE/Sciex API 300 (supplied by
the Sciex Division of MDS Inc., of Concord, Ontario, Canada).
[0093] An example of separation conducted using the chip-ESMS
interface shown in FIG. 8a is presented in FIG. 9. The total ion
electropherogram for m/z 500-800 (FIG. 9a) corresponds to the
separation of a mixture of 9 peptides (injection of 64-180 fmol
each). The main separation channel on the chip was 4 cm in length
and neither the chip channels nor the electrospray emitter were
coated. The peak width for individual components ranges from 12-20
sec. It is noteworthy that the separation efficiencies could be
improved by increasing the length of the separation channel and
reducing the tip of the electrospray emitter to smaller inner
diameter. Extracted mass spectra taken for the peaks shown in FIG.
9(b)-9(e) are also presented on the right panels.
[0094] The versatility of the chip-ESMS interface was also
demonstrated for longer transfer line whereby this arrangement
enables coupling to other types of mass spectrometric interfaces.
An example of the chip-ESMS device using a 15 cm fused silica
capillary coupled to a coaxial sheath flow interface is presented
in FIG. 10 for the separation of the peptide mixture shown in FIG.
9. The coupling of the chip device via the co-axial sheath flow
solvent delivery provides an independent means of optimizing the
electrospray voltage or adding organic solvent to facilitate
desolvation of the analyte. In this case both the chip of the
capillary were coated with an amine reagent referred to as BCQ (see
K. P. Bateman, R. L. White, and P. Thibault, Rapid Commun. Mass
Spectrom., 11, 307-315 (1997)) which not only prevents analyte
absorption on the inner walls of the chip and capillary but enables
the use of acidic separation buffers. FIG. 10a shows the total ion
electropherogram for the full scan analysis (m/z 400-900) of a
mixture of 9 peptides each at 10 .mu.g/mL. The ion electropherogram
for the multiply-charged ions of each peptide are shown in FIGS.
10(b)-10(i). The mass spectrum for each peptide is dominated by
singly and/or doubly protonated molecules of the corresponding
peptides (peptides are designated in FIGS. 10(b)-10(j) using the
single letter amino acid).
[0095] Reference is now made to FIG. 11, which shows an alternative
apparatus and method for drilling a hole in a microchip device.
Here, the microchip device is indicated at 92, and is shown held in
a clamp 94. Separate tubes 96 are connected to a pump (not shown)
and are sealed by O rings 98 to the microchip device 92. A drill
bit 99 is brought up against an end of a channel 93 within the
microchip 92, as indicated.
[0096] Here, a steady stream of water or other fluid is supplied
through the tubes 96 from the pump, connected to the various wells
or inlets to the channel 93. This produces a continuous flow of
fluid out from the free end of the channel 93, as indicated in FIG.
11. Then, as the drill bit 98 is brought up against the channel, to
drill a hole, the flow of fluid flushes away chippings, ground
glass particles and the like.
[0097] It was found that this technique prevented plugging of the
exit channel, but did not always prevent the trapping of some
particles within the channels on the chip, particularly the
channels on the chip with less than a 100 micron diameter and 20
micron depth. In general, it is expected that this technique would
be better suited to larger size channels, where there is less
likelihood of particles becoming trapped and a larger flow rate of
flushing fluid can be maintained.
[0098] As will be apparent to those skilled in the art, various
modifications and adaptations of the structure described above are
possible without departing from the present invention, the scope of
which is defined in the appended claims.
1TABLE 1 Experimental parameters Parameter Value Laser Power 2 mW
per beam (nominal) Ar ion at 488 nm Microscope 5X Magnification
Photo Multiplier Tube Filter 530 nm 10 nm bandpass Separation
Voltage 7 kV Injection 1.5 kV for 1 s Buffer 50 mM Morpholine
Sample FITC Labeled dust and debris
[0099]
2TABLE 2 Average plate numbers and relative efficiencies of 6
selected peaks detected on the capillary (FIG. 4 and FIG. 6).
Comparison of pointed and flat bottomed hole junctions between the
device and the capillary. Pointed hole junction Flat bottomed hole
junction Average Relative Average Relative Peak plate number
efficiency plate number efficiency number (n = 4) (%) (n = 4) (%) 1
18000 17 112000 84 2 15500 15 121000 95 3 15000 16 117000 94 4
12000 12 118000 69 5 9000 12 53000 54 6 9000 14 66000 76
* * * * *