Chemistry: analytical and immunological testing – Optical result – Spectrum analysis
Reexamination Certificate
1998-10-09
2003-08-12
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
Spectrum analysis
C239S690000, C250S288000, C204S601000, C204S604000, C436S086000, C436S087000, C436S088000, C436S089000, C436S090000, C436S091000, C436S093000, C436S094000, C436S173000, C436S174000, C436S177000, C436S178000, C436S180000
Reexamination Certificate
active
06605472
ABSTRACT:
FIELD OF THE INVENTION
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
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.
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 &mgr;ESMS. The flow rates used and potentials applied in &mgr;ESMS are compatible with CE, and this has led to development of CE-&mgr;ESMS instruments, capable of initial separations followed by mass spectral analysis. A drawback of this approach is the 15-40 min. seperation on times often required, which tends to underutilize the spectrometer.
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.
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.
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-&mgr;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-&mgr;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 &mgr;L/min.
The limited sample volume used in CZE (2% of capillary volume), results in concentration detection limits of approximately 1 &mgr;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 &mgr;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.
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., sever
Bings Nicolas
Harrison D. Jed
Li Jianjun
Ocvirk Gregor
Skinner Cameron
Bereskin & Parr
Soderquist Arlen
The Governors of the University of Alberta
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