Anal. Chem. 2003, 75, 5030-5036
A Micromachined Double-Tuned NMR Microprobe J. H. Walton,*,‡ J. S. de Ropp,‡ M. V. Shutov,† A. G. Goloshevsky,§ M. J. McCarthy,§ R. L. Smith,† and S. D. Collins†
MicroInstruments and Systems Laboratory (MISL), University of Maine, Orono, Maine, NMR Facility, University of California-Davis, Davis, California, and Department of Food Science and Technology, University of California-Davis, Davis, California
A double-tuned NMR microprobe with dual RF microcoil assemblies was fabricated and tested. The coils for the proton and X (low frequency) nuclei were a variant of a loop gap resonator and Helmholtz pair, respectively. 1-D 31P spectra of phosphoric acid and 13C-labeled spectra of methanol were recorded from 1.4-µL samples. Signal was easily observable in one scan. A COSY spectrum of 13C-labeled acetic acid was acquired in ∼1 h, demonstrating sufficient sensitivity to perform 2-D 13C direct detect experiments. Thus, the ability to obtain direct detection spectra of low-γ nuclei is demonstrated. NMR spectroscopy, over the course of 50 years, has evolved from a simple laboratory curiosity used by physicists to observe the nuclear properties of water into a powerful analytical tool used to probe the atomic infrastructure and molecular dynamics of molecules from small organics to proteins, polymers, and biomolecular assemblies.1-6 The principal driving force for NMR is that it is the only analytical tool providing direct solution-based determination of molecular structure. This feature alone makes NMR an indispensable tool for structural determination of complex biomolecules, drug discovery, process monitoring, and even in vivo spectroscopy. Unfortunately, despite its diverse utility, NMR ranks as one of the least sensitive tools in the analytical arsenal. Advances in superconducting magnets, electronics, and probe design have greatly increased sensitivity, resolution, and ease of NMR spectroscopy. However, NMR still falls well behind its analytical counterparts in sensitivity and is usually reserved only for those samples that can be obtained in relatively large quantities. Other techniques, such mass spectroscopy, IR spectroscopy, fluorescence spectroscopy, etc. exhibit considerably higher mass sen†
University of Maine. NMR Facility, University of California-Davis. § Department of Food Science and Technology, University of California-Davis. (1) Sanders, J. K. M.; Hunter, B. K. Modern NMR Spectroscopy: A Guide for Chemists, 2nd ed.; Oxford University Press: Oxford, New York, 1993. (2) Wu ¨ thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York, 1986. (3) Evans, J. N. S. Biomolecular NMR Spectroscopy; Oxford University Press: Oxford, New York, 1995. (4) Roberts, G. C. K. NMR of Macromolecules: A Practical Approach; IRL Press at Oxford University Press: Oxford, New York, 1993. (5) Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G., III; Skelton, N. J. Protein NMR Spectroscopy: Principles And Practice; Academic Press: San Diego, 1996. (6) La Mar, G. N.; de Ropp, J. S. In Biological Magnetic Resonance; Berliner, L. J., Reuben, J., Eds.; Plenum Press: New York, 1993; Vol. 12.
sitivity and, as a result, require considerably less sample mass to analyze. This is a particularly troublesome limitation for NMR when rare or limited samples, such as critical biomolecules, are analyzed. Often, the lack of sufficient sample completely precludes their NMR investigation, despite the need. Clearly, the ability to perform NMR spectrometry on small sample volumes would constitute a significant advancement in analytical research. Recently, NMR spectroscopists have borrowed from telecommunication and electronic circuit fabrication to miniaturize the RF coils and their corresponding sample volumes. Microprobe sample volumes are generally on the order of 1 µL or less and significantly smaller than traditional commercial NMR probes. The coils generally shrink with the sample, because that is necessary for high sensitivity on small volumes. The first microcoils reported were planar RF coils7,8 micromachined on GaAs substrates. The motivation for using III-V substrates was to provide on-site integration of a high-frequency receiver preamplifier, which has subsequently been realized.9 Since then, many other surface microcoils have followed suit, each with its own distinctive contribution.10 However, while surface microcoils represent a significant advancement in NMR analysis, these microcoils generally exhibit poor shimming, field homogeneity, and magnetic susceptibility matching, which generally results in less than optimal sensitivity, resolution, and line width. Olsen et al.11 took a somewhat different approach with an impressive solenoid-based microcoil design that was capable of nanoliter detection. The microcoil was fabricated by wrapping a 50-µm diameter Cu wire around a 357-µm-diameter glass capillary tube. The primary impetus for this design was the inherently higher sensitivity of the solenoid configuration and the flowthrough construction of the capillary tube that allowed the NMR microprobe to be conveniently interfaced with other analytical instruments for simultaneous separation and detection,that is, capillary electrophoresis (CE),12-14 capillary isotachophoresis
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(7) Peck, T. L.; Magin, R. L.; Kruse, J.; Feng, M. IEEE Trans. Biomed. Eng. 1994, 41, 706-709. (8) Stocker, J. E.; Peck, T. L.; Webb, A. G.; Feng, M.; Magin, R. L. IEEE Trans. Biomed. Eng. 1997, 44, 1122-1127. (9) Boero, G.; Frounchi, J.; Furrer, B.; Besse, P. A.; Popovic, R. S. Rev. Sci. Instrum. 2001, 72, 2764-2768. (10) Massin, C.; Boero, C.; Vincent, F.; Abenhaim, J.; Besse, P. A.; Popovic, R. S. Sens. Actuators, A 2002, 97-98, 280-288. (11) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (12) Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Proceedings of 16th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Baltimore, MD, Nov. 3-6, 1994; 986. 10.1021/ac034073n CCC: $25.00
© 2003 American Chemical Society Published on Web 08/30/2003
(cITP),15,16 or high-pressure liquid chromatography (HPLC).17 The solenoid RF microcoil has successfully demonstrated 1-D and 2-D 1H NMR results,15,18 including COSY17,19 and HMQC19,20 spectra. Although initial reports12 for solenoid microcoils lacked resolution, bathing them in a fluorinated susceptibility matching fluid reduced 1H line widths to 0.6 Hz11,18 in favorable cases. To date, solenoid microcoils have enjoyed considerable success in high-resolution nanoliter 1H spectroscopy and promise to provide significant advances for the future. Unfortunately, hand assembly of microcoils and bathing them in susceptibility matching fluids is not particularly conducive to microsystem integration. Microfabrication brings an impressive cache of advantages to NMR spectroscopy that are not readily accessible through macro techniques, such as hand assembly of coils, probes, or mechanical machining. These advantages include submicrometer precision, micrometer-sized structures, high reproducibility, and easy integration of several microcomponents into a microsystem. Using microfabrication, an integrated NMR microsystem is easily envisioned, complete with multiple RF coils/components for shimming, gradients, tuning, decoupling, lock, and simultaneous multinuclear detection. Additionally, by appending the NMR microprobe to other microsystems, the “laboratory on a chip” or µ-TAS (micro total analysis system) becomes a tangible reality. Such a microsystem would, of necessity, contain a diversity of controlled microfluidic separation and detection components, including microanalysis instruments, such as NMR. Such dramatic advances in analytical science certainly demand further attention. If completely integrated NMR microsystems are to become a practical reality, microfabrication will inevitably provide the tools necessary to achieve that reality. Recently, microfabricated solenoids21,22 have surfaced with varying degrees of success, but none yet appear poised for practical microsystem utilization. With one exception, previous NMR microcoil reports have focused on 1H detection, delegating NMR spectra for low-γ nuclei to indirect detection16,17,19,20 methods. Advances in structural determination via NMR have placed renewed emphasis on 13C, 15N, and 31P NMR investigations. Low-γ nuclei direct detection avoids problems associated with water suppression. For 13C studies, carbonyls have no directly bonded protons, making direct detection advantageous.23 Thus, there is a need to perform experiments on low-γ nuclei. (13) Olson, D. L.; Lacey, M. E.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 1999, 71, 3070-3076. (14) Trumbull, J. D.; Glasgow, I. K.; Beebe, D. J.; Magin, R. L. IEEE Trans. Biomed. Eng. 2000, 47, 3-7. (15) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 2306-2313. (16) Kautz, R. A.; Lacey, M. E.; Wolters, A. M.; Foret, F.; Webb, A. G.; Karger, B. L.; Sweedler, J. V. J. Am. Chem. Soc. 2001, 123, 3159-3160. (17) Subramanian, R.; Kelley, W. P.; Floyd, P. D.; Tan, Z. J.; Webb, A. G.; Sweedler, J. V. nal. Chem. 1999, 71, 5335-5339. (18) Olson, D. L.; Lacey, M. E.; Sweedler, J. V. nal. Chem. 1998, 70, 645-650. (19) Zhang, X.; Sweedler, J. V.; Webb, A. G. J. Magn. Reson. 2001, 153, 254258. (20) Subramanian, R.; Sweedler, J. V.; Webb, A. G. J. Am. Chem. Soc. 1999, 121, 2333-2334. (21) Malba, V.; Evans, L. B.; Yan, K.; Cosman, M.; Maxwell, R. S. Experimental NMR Conference, Orlando, FL, March 11-16, 2001. (22) Rogers, J. A.; Jackman, R. J.; Whitesides, G. M.; Olson, D. L.; Sweedler, J. V. Appl. Phys. Lett. 1997, 70, 2464-2466. (23) Serber, Z.; Richter, C.; Moskau, D.; Bohlen, J. M.; Gerfin, T.; Marek, D.; Haberli, M.; Baselgia, L.; Laukien, F.; Stern, A. S.; Hoch, J. C.; Dotsch, V. J. Am. Chem. Soc. 2000, 122, 3554-3555.
In this paper, we present a naissance microsystem NMR probe for the detection of low-γ nuclei. The microsystem integrates a Helmholtz RF microcoil, a loop gap resonator, and a 1.4-µL micromachined spherical sample chamber with accompanying microchannel fluidic interconnects for static or flow-through measurements. The Helmholtz RF microcoil and loop gap resonator are the first reported use of these coil designs in an NMR microprobe and provide an inherent improvement in field uniformity over previously reported planar surface microcoils. The micromachined spherical sample chamber and associated microfluidics also offer ideal NMR geometries for field shimming. The viability of the microprobe system was demonstrated by using the Helmholtz microcoil for the direct detection of 13C (labeled) and 31P and using the 3/4 turn loop gap resonator microcoil for 1H decoupling of a 1.4-µL sample volume. A 2-D 13C COSY spectrum of fully labeled acetic acid was also obtained, demonstrating this probe has sufficient sensitivity to perform standard 2-D experiments on low-γ nuclei. EXPERIMENTAL SECTION Microprobe Fabrication. Materials. Analytical grade phosphoric acid (85%) was purchased from J. T. Baker and used without further purification. 13C-labeled methanol (99%) and acetic13C acid (99%) were purchased from Aldrich Chemical and also 2 used without further modifications. All processing chemicals were of either MOS or reagent grade. Water was 18 MΩ deionized. All microcoils were fabricated on 1-mm Pyrex borosilicate 7740 plate (Corning) and diced to the appropriate size. Copper microcoils were electroplated from a solution of copper sulfate electroplating solution: Techni Copper U (Catalogue no. 060559), at current densities of 25 mA/cm2, giving a plating rate of 0.7-0.8 µm/min. Copper plate (5 N) was used as the anode. Helmholtz Microcoil and Sample Chamber. Fabrication of the planar Helmholtz microcoil is illustrated diagrammatically in Figure 1. A 50 nm/1000 nm Cr/Au metal layer was e-beamevaporated and photolithographically patterned on a standard 4-in. Pyrex substrate (Figure 1A), using a KI/I2 etchant for the Au and a Ce2+/Ce3+-based etchant, Transcene CR-7, for the Cr. The Cr/ Au metal served as an etch mask during subsequent etching of the Pyrex substrate. The glass was then isotropically etched in 49% HF through a small circular mask opening to form a hemispherical pit, which eventually served as the spherical sample chamber (Figure 1B). Process steps A and B were repeated to etch microchannels into the substrate for fluidic I/O to the sample chamber. The microchannel cross section was an oval shape with major and minor axis dimensions of 150 µm (wide) by 110 µm (deep), respectively. The etch relief for the sample chamber and fluidic channels is shown in the bottom half of Figure 1J. Figure 2 shows a cross-sectional photograph of a typical hemispherical HF etch relief. The Cr/Au etch mask was then stripped in aqua regia, and the two etched substrates were then aligned and fusionbonded at 650 °C for 1 h to form the complete fluidic module (Figure 1C). During bonding, the furnace temperature was ramped from 100 °C to 650 °C at 1-5 °C/min and allowed to cool at similar ramp rates. During bonding, a pressure of ∼0.1 kg/ cm2 was applied to the substrates to facilitate bonding. The machined and bonded glass was then used as the substrate for subsequent Helmholtz microcoil fabrication (Figure 1D-I). A 15 nm/200 nm Ti/Cu seed layer was e-beam-evaporated Analytical Chemistry, Vol. 75, No. 19, October 1, 2003
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Figure 1. Fabrication process for the multiturn Helmholtz microcoil. Details given in text.
on both sides of the substrate, and a 10-µm thick photoresist layer, Hoechst Celanese AZ 4620, was spun at 2500 rpm and patterned (Figure 1D). The RF microcoils along with corresponding bonding pads were then formed by electroplating 8 µm of Cu into the photoresist relief (Figure 1E). The photoresist was then stripped in acetone or standard process photoresist strippers, and the Ti/ Cu seed layer was removed by etching in 1:1:20 acetic acid/ hydrogen peroxide/water (etch rate, 150 nm/min) to remove the Cu and a quick etch in 50:1 HF (etch rate, 50 nm/min) to remove the Ti layer (Figure 1F). To provide electrical connection between the interior and exterior of the microcoils, a dual layer Cu metallization was required to cross over the microcoils. A sacrificial photoresist layer, Hoechst Celanese AZ 4620, was spun and patterned to expose contact to the underlying copper, and a 5 nm/100 nm Ti/ Cu seed layer was again e-beam-evaporated onto the sacrificial photoresist (Figure 1G). Another layer of photoresist was patterned on the seed layer, and copper was electroplated under the same conditions as described above (Figure 1H). Both layers of photoresist were then stripped to form a suspended metal “bridge” 5032
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over the microcoil lines (Figure 1I). Figure 3 shows the electrical bridge of a microcoil suspended over the turn lines of a microcoil. A photograph of a completed Helmholtz microcoil, etched spherical sample chamber, and fluidic microchannels is shown in Figure 4. Although a number of different microcoil designs and sizes were fabricated, all data presented in this report were taken on the three-turn Helmholtz design shown in Figure 4. The copper line widths were 130 µm at a 160 µm pitch with an inner microcoil diameter of 3.5 mm and an outer diameter of 3.95 mm. The spacing between the two microcoils, i.e., substrate thickness, was 2 mm nominal. The microcoil was designed primarily (and thus, the coil dimensions set) for RF field homogeneity and, as such, only had a fill factor of 6%. The filling factor can be increased if sensitivity, rather than RF homogeneity, is the primary design goal. The coil dimensions can easily be shrunk to submillimeter with micromachining techniques. Proton Resonator. The microcoil used for proton decoupling is a variant of a loop gap resonator.24,25 The vertical sections of (24) Hardy, W. N.; Whitehead, L. A. Rev. Sci. Instrum. 1981, 52, 213-216.
Figure 2. Cross-sectional relief of a typical hemispherical etch pit formed in the glass substrates. Cross section is not exactly at the center of the pit and shows some eccentricity.
Figure 3. SEM of a suspended electrical “bridge” to cross over the turn lines of the microcoil. Shown here is a square, 200-µm surface coil configuration with 8-µm-thick electroplated Cu lines.
the loop gap resonator were 6.6 mm apart. As shown in Figure 5, this implementation of the resonator does not have a current path below the sample. The vertical legs were connected at the top remotely around the lateral edges to avoid contributions to the RF field from the connection and provide for microfluidic access. No attempt was made to optimize the resonator coil for 1H detection because its primary function was as a decoupling coil. This allowed the decoupling coil to be fabricated by standard chemical etching of copper-plated polyimide tape. The polyimide tape was then attached to glass substrates, which in turn were mounted around the Helmholtz microcoil assembly. Dual Coil Assembly. The entire assembly of RF microcoils, proton resonator and the multiturn planar Helmholtz pair, were mounted on a standard NMR probe housing with variable tuning/ (25) Froncisz, W.; Hyde, J. S. J. Magn. Reson. 1982, 47, 515-521.
Figure 4. Photograph of a Helmholtz microcoil used for NMR testing. The perspective view shows both legs of the Helmholtz microcoil, both in front of and behind the spherical sample chamber. Fluidic microchannels are seen on top and bottom to interface sample chamber with the external world.
matching capacitors. The resonator was mounted around the Helmholtz coil so that the 1H RF resonator plane was in the plane of the Helmholtz coil. Thus, the RF of the 1H and X channels were orthogonal to and decoupled from each other by virtue of geometry. Crosstalk between Helmholtz and resonator microcoils was further decoupled by virtue of their different frequencies, 500 MHz for the loop gap resonator proton decoupling and 125 MHz/ 202 MHz for 13C and 31P, respectively. A diagram of the loop gap resonator and dual coil assembly is shown in Figure 5. Electrical Characterization. The measured inductance of the Helmholtz pair was 41 nH and the decoupling coil was 16 nH. These small values were obtained by introducing different chip capacitors across the coils. The resonant frequency, f, was determined by loose inductive coupling to a small loop on the end of a piece of coax driven by a spectrum analyzer (HP 8752A). A plot of (2πf)-2 vs capacitance yielded a straight line whose slope is the inductance. The three-turn spiral planar coils each made up half of the Helmholtz pair. They were connected in parallel (Figure 6). This arrangement has the advantage of a lower total inductance due to parallel addition of inductance. Although not absolutely necessary for carbon detection at the relatively low frequency of 125 MHz, lower inductance becomes a critically important advantage at higher frequencies. The parallel microcoil arrangement has the immediate disadvantage of allowing asymmetric current distribution through the two halves and, hence, asymmetric RF field distributions if the impedances of the two microcoils are mismatched. Fortunately, the precision of microfabrication provides sufficient uniformity that this was not generally an issue. The variable capacitors, CM and CT, were 1-30 pf, Johanson Part no. 5641. The bump capacitor was an 8.2-pf ATC chip capacitor and was used to reach the carbon frequency, but it was not present for the phosphorus experiment. The Helmholtz pair had a resistance of ∼1 ohm so that Q was ∼30 at 125 MHz. This Q is comparable to that reported by Massin10 et al. for a planar Analytical Chemistry, Vol. 75, No. 19, October 1, 2003
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Figure 5. Diagram of dual microcoil assembly.
Figure 6. Helmholtz pair circuit connection diagram. The two coils of the pair were connected in parallel.
micromachined coil and calculated by Grant26 et al. for multiturn hand-wrapped coils. Diameters in both cases were ∼1 mm, a little smaller than the case here. Q was ∼300 for the half-turn loop resonator. Test and Measurement. NMR spectra were obtained on a Bruker Avance-DRX spectrometer operating at 500 MHz with a standard narrow bore (52 mm) magnet. The coils were mounted on a custom probe housing constructed to fit the narrow bore spectrometer. Rods attached to each capacitor permitted in situ tuning. Application of current to the standard shim set had little affect on the line widths. Spectra were taken without use of an NMR lock. The magnet drift rate is substantially less than 2 Hz/h and, thus, did not contribute appreciably to the line widths. Onedimensional spectra were acquired on the X nuclei of interest by applying a single pulse and recording the free induction decay (FID). Proton decoupling was applied in selected cases as described below. RESULTS AND DISCUSSION Figure 7 shows a 1-D single scan 31P spectrum of 1. 4 µL of 85% phosphoric acid with a S/N of 52/1. The 90° pulse (200 W into the probe) is 7.5 µs, comparable to the values found on commercial 5-mm probes. An exponential apodization resulting in 1 Hz of line broadening has been applied, resulting in a line width of 12 Hz at full width half max (fwhm). (26) Grant, S. C.; Murphy, L. A.; Magin, R. L.; Friedman, G. IEEE Trans. Magn. 2001, 37, 2989-2998.
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Figure 8 contains 13C spectra of 34 µmol of fully labeled methanol acquired with eight scans. The spectrum shown in 8A was acquired without, and 8B with, proton decoupling. Figure 8A exhibits the familiar quartet from J coupling (J ) 140 Hz) between proton and carbon, which is removed in 8B via application of a GARP decoupling pulse scheme with a 2.5-kHz RF field. It is clear from the collapse of the quartet in Figure 8B that decoupling is efficient. The 13C 90° pulse value was 11 µs and about what is expected on the basis of the ratio between 13C and 31P γ values. The line width was 15 Hz fwhm and 35 Hz at 12.5% of full height. Signal-to-noise was 12/1 forA and 49/1 for B. All signal-to-noise measurements included measurement over 1 ppm of noise using standard software supplied by Bruker. With improvements in shim, these numbers will increase, and direct detection of natural abundance 13C may be possible. The spectral line widths achieved here, 15 and 12 Hz, respectively, for 13C and 31P, while larger than is typical of 3- or 5-mm probes, are more than sufficient to resolve the spectra of small molecules. The required resolution for chemical identification using low-γ nuclei is less stringent than that required for proton spectroscopy because of the larger chemical shift dispersion of low-γ nuclei. The gradients generated by the shim coils were insufficient to counteract the local gradients in the microprobe. This situation has been reported previously.8,27,28 The line widths here and in other planar microcoil studies with proton samples are determined by local field inhomogeneity rather than T2. These gradients may be attributed to the bulk magnetic susceptibility difference between Pyrex (-11.0 × 10-6)29 and methanol (-6.66 × 10-6)29 or phosphoric acid (-9.5 × 10-6).30 Note that methanol has the larger line width, corresponding with (27) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. J. Am. Chem. Soc. 1994, 116, 7929-7930. (28) Wu, N. A.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-3857. (29) Doty, F. D.; Entzminger, G.; Yang, A. Concepts Magn. Reson. 1998, 10, 133-156. (30) Lide, D. R. CRC Handbook of Chemistry and Physics, 3rd electronic ed.; CRC Press: Boca Raton, FL, 2001.
Figure 7. 31P spectrum of phosphoric acid; single scan using a 7.5-µs 90° pulse. Exponential apodization of 1 Hz has been applied. The line width is 12 Hz, and the S/N is 52/1.
Figure 8. Eight-scan Hz.
13C
spectra of labeled methanol (A) without and (B) with decoupling. The 90° pulse was 11 µs. The line width was 15
the larger susceptibility difference between it and the surrounding Pyrex. When NMR lines are inhomogeneously broadened, the line widths scale with γ within the sample. This fact is used to make qualitative comparisons to other proton results. The line widths achieved here compare well with other planar microcoil studies.7,9,10 For instance, 12 Hz on 31P at 202 MHz scales to 18 Hz on proton at 300 MHz, which is close to the 25 Hz reported by Boero9 et al. and the 17 Hz reported by Massin10 et al. Recent work8 has demonstrated a line width of 2.5 Hz for protons using a single planar microcoil without susceptibility matching fluid that was reduced to 1.8 Hz with susceptibility matching fluid. However, this was achieved only at the expense
of moving the sample away from the coil by a distance of three coil diameters, reducing the resultant signal. Although Trumbull et al.14 have shown smaller line widths,
