Electricity: measuring and testing – Particle precession resonance – Spectrometer components
Reexamination Certificate
1998-06-19
2001-02-27
Oda, Christine (Department: 2862)
Electricity: measuring and testing
Particle precession resonance
Spectrometer components
C324S318000
Reexamination Certificate
active
06194900
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to miniaturized liquid phase sample processing and analysis. More particularly, the invention relates to a miniaturized planar sample preparation and analysis device with an integrated on-chip miniature nuclear magnetic resonance (“NMR”) radiofrequency coil. Both the sample preparation and analysis device and the NMR radiofrequency coil are manufactured using a variety of means suitable for microfabrication of substrate materials such as, but not limited to ablation, molding and embossing. The device is intended to be used with high-field, low-cost miniature magnets with the intention of achieving high throughput, fast time-to-result analysis of biological liquids in a truly integrated fashion.
BACKGROUND
Real time identification of analytes in a complex biological fluid is difficult, and requires careful thought as to (a) the preparation of the sample, (b) whether a separation step is required to simplify the signal, (c) whether a detection method can be employed which has no effect on the sample itself, (e.g. non-destructive). An ideal device would allow rapid detection of a wide range of simple or complex molecules in the liquid phase, at biological concentrations, and yield information about chemical structure and composition. A desirable feature of the detection method would be to enable the separation criteria to be relaxed such that sample preparation and detection could occur in series, without the need for complex separation technology. An on-line detector is particularly advantageous when sample size is limited, and additional analysis of the sample is required. Moreover, mass spectrometry (“MS”) and NMR are detection methods well suited to yielding high quality chemical information for multi-component samples, requiring no a priori knowledge of the constituents.
Though much has been discussed in the literature towards realizing integrated separation technology including sample preparation and separation devices, and associated fluidics so that low yield or precious samples may be prepared and analyzed, little has been realized to date. In sample analysis instrumentation, particularly in separation systems involving capillary electrophoresis or liquid chromatography, smaller dimensions of the sample handling conduits and separation compartments result in improved performance characteristics, while reducing cost of production and analysis. Miniaturization of the sample preparation or separation region, to result in small sample volume requirements, necessarily means a greater demand on the detection method both by virtue of sample volume and potentially, sensitivity.
There are many types of detection methods possible. Optical transmission methods such as refractive index, ultraviolet-visible (“UV-VIS”) and infrared (“IR”) are relatively inexpensive, but are unable to give complex chemical structure and composition information. Furthermore they are path-length limited and sensitivity of detection is limited. Infrared spectroscopy is relatively insensitive, particularly to contaminants, and yields only functional group or fingerprint identification. MS is a sensitive method giving mass information; however, MS has the drawback of requiring sample preparation for nonvolatile analytes, as well as being destructive to the sample.
One of the most powerful analytical methods for molecular structure information is NMR. NMR provides spectral information as a function of the electronic environment of the molecule and is nondestructive to the sample. In addition, reaction rates, coupling constants, bond-lengths, and two- and three-dimensional structure can be obtained with this technique. The strength of both NMR and MS is the ability to derive fundamental chemical structure information, which is high resolution in terms of either chemical shift or mass, yielding the possibility of simultaneous analysis of multiple species. The inherent insensitivity of the NMR method however, has limited its usefulness as a detection method for liquid phase analysis of very small samples, such as effluent from a liquid chromatography or capillary electrophoretic separation.
NMR combined with liquid chromatography or capillary electrophoresis was demonstrated as early as 1978 using stopped flow (Watanabe et al. (1978)
Proc. Jpn. Acad
. 54:194), and in 1979 with continuous flow (Bayer et al. (1979)
J. Chromatog
. 186:497-507), though limitations due to solvent as well as inherent sensitivity curtailed the use of the method. See Dorn et al. (1984)
Anal. Chem
. 56:747-758 for a review.
Recent experiments using NMR as a detector for nanoliter sample volumes, suggests that NMR could provide a greater detection sensitivity than in previous investigations. Wu et al. (1994a)
J. Am. Chem. Soc
. 116:7929-7930; Olson et al. (1995)
Science
270:1967-1970; Wu et al. (1994b)
Anal. Chem
. 66:3849-3857; and Wu et al. (1995)
Anal. Chem
. 67:3101-3107. Unfortunately, though observations were made on nanoliter volumes, the findings translate into millimolar levels of detection sensitivity.
A number of areas can be targeted to increase the sensitivity of NMR detection for liquid phase analysis. Resistive losses, operating temperature, sample ionic strength, filling factor, and coil geometry affect the sensitivity of the coil. Cooling the radiofrequency coil and using superconducting coil material have resulted in some gain in signal-to-noise through reduction in coil resistance and thermal properties. However, it is difficult to achieve the theoretical maximum, since detecting signal from a room temperature liquid sample using a cryogenically cooled radiofrequency probe has proven difficult.
The NMR signal-to-noise is directly proportional to the sample volume (V
s
) interrogated by the detection coil (filling factor), the magnetization per unit volume (M
o
), and the strength of the radiofrequency (“RF”) field (B
1
) per unit current, and inversely proportional to the square root of the coil resistance (R):
Signal∝(V
s
×M
o
×B
1
)/R
Signal-to-noise can be maximized by decreasing the coil radius, and matching the coil inner diameter as close to the size of the sample as possible. Inadequate filling factor will generally be an issue when standard radiofrequency NMR coils are used to detect signal from very small sample volumes, e.g., from a microcolumn or other miniaturized sample preparation technology. Reduction in the size of NMR radiofrequency coils to the diameter of the fused glass capillary used for these types of separations, has allowed detection of signal from nanoliter volumes from on-line capillary electrophoretic separations Wu et al. (1994a), supra; Olson et al. (1995), supra; Wu et al. (1994b), supra; Wu et al. (1995), supra.
A solenoid microcoil detection cell formed from a fused silica capillary wrapped with copper wire has been used for static measurements of sucrose, arginine and other simple compounds. Wu et al. (1994a), supra; Olson et al. (1995), supra. Coil diameter has been further reduced by the use of conventional micro-electronic techniques in which planar gold or aluminum R.F. coils having a diameter ranging from 10-200 &mgr;m were etched in silicon dioxide using standard photolithography. Peck (1995)
J. Magn. Reson
. 108(B) 114-124. The signal-to-noise ratio (SNR) of these planar micro-coils for analyzing solid samples, e.g., silicon rubber was increased by a factor of 10 over other coils. For significant advancement in hyphenating NMR with LC or CE methods, an approach allowing micromolar or even nanomolar limits of detection is required however.
Factors affecting the limit of detection can also be attributed to bulk susceptibility shifts, which become dominant when the sample volume is of the order of the size of the sample chamber and coil. This is in addition to the coil geometry, resistive losses, sample ionic strength, filling factor and operating temperature considerations previously mentioned. We have constructed susceptibility-matched microcoils using 50 &mgr;m copper wire with an inner diameter of
Freeman Dominique M.
Swedberg Sally A.
Agilent Technologie,s Inc.
Beck Michael J.
Fetzner Tiffany A.
Oda Christine
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