Anal. Chem. 1998, 70, 645-650
High-Resolution Microcoil NMR for Analysis of Mass-Limited, Nanoliter Samples Dean L. Olson, Michael E. Lacey, and Jonathan V. Sweedler*
Beckman Institute and Department of Chemistry, University of Illinois, Urbana, Illinois 61801
An improved nanoliter-volume NMR probe design places the microcoil and capillary at the magic angle (54.7°) with respect to the external magnetic field. Using an NMR probe which requires a total sample volume of just 200 nL, high-resolution 300-MHz 1H-NMR spectra (line width, 0.6 Hz) are presented of 10 mM r-bag cell peptide for an observe quantity of 45 ng (50 pmol in 5 nL). For the volume of sample inside the microcoil (the observe volume, Vobs), the 3σ limit of detection (LOD) is 9 ng (10 pmol, 2 mM) for data obtained in 15 h. To reduce the data acquisition time, a probe with a greater Vobs is developed. As an example of a rapid, mass-limited analysis, a concentration corresponding to 400 ng of menthol dissolved in Vobs ) 31 nL (82.6 mM) yields a spectrum in 9 min (LOD ) 6.9 ng, 44 pmol, 1.4 mM). To illustrate improvements in concentration sensitivity, a spectrum is acquired in 45 min for 400 ng of menthol dissolved in a total sample volume of 200 nL (12.8 mM). Compared to a commercial nanoprobe for the same mass of menthol, these two examples reduce data acquisition time by at least 95%. Both model compounds demonstrate substantially improved concentration LODs compared to those obtained in previous high-resolution, microcoil NMR work. These advances illustrate the utility of enhanced sensitivity provided by NMR microcoils applied to nanoliter volumes of mass-limited samples. The sensitivity of conventional NMR spectroscopy often hinders its applicability in trace analyses. Relatively poor limits of detection (LODs) compared to those of other methods can limit the lower range of sample quantity and concentration and impede rapid analysis. Microcoil NMR offers enhanced mass sensitivity and smaller sample requirements compared to those of the best available commercial methods for mass-limited NMR analyses. We demonstrate here improved microcoil NMR techniques, probe designs, and LODs for a biologically relevant peptide and a model compound. R-Bag cell peptide (R-BCP) is found in the sea snail Aplysia californica; this peptide and organism prove interesting in a wide array of neurological studies.1-3 Menthol is used as a model compound to evaluate overall probe performance and to (1) Scheller, R. H.; Kaldany, R. R.; Kreiner, T.; Mahon, A. C.; Nambu, J. R.; Schaefer, M.; Taussig, R. Science 1984, 225, 1300-8. (2) Conn, P. J.; Kaczmarek, L. K. Mol. Neurobiol. 1989, 3, 237-73. (3) Sigvardt, K. A.; Rothman, B. S.; Brown, R. O.; Mayeri, E. J. Neurosci. 1996, 6, 803-13. S0003-2700(97)00972-4 CCC: $15.00 Published on Web 02/01/1998
© 1998 American Chemical Society
illustrate decreases in concentration LODs and required total sample amount. The history and development of radiofrequency microcoils in magnetic resonance was recently reviewed extensively.4 Previous studies5-7 employed microcoils as detectors in capillary electrophoresis and considered the effect of flowing analyte on the NMR signal. Other work8 developed a high-resolution NMR microcoil and explored its relative sensitivity and application in static analyses. The first high-resolution NMR spectra were obtained with line widths of 0.6 Hz using a 300-MHz spectrometer and a microcoil observe volume, Vobs (sample volume inside the coil), of 5 nL. An additional report9 investigated key microcoil design parameters and concluded that concentration LODs could be significantly reduced by increasing the fraction of volume inside the microcoil occupied by sample, but a concomitant increase in line widths was also seen. Another investigation10 described an alternative means of fabricating microcoils using microcontact printing and electrodeposition. In contrast to the conventional definition of NMR sensitivity at constant concentration, mass sensitivity, Sm, is defined as the signal-to-noise ratio (S/N) for any constant amount of sample, such as S/N per micromole.8,11 Such a definition enables absolute performance comparisons of NMR probes. For instance, compared to that of a typical 10-mm probe used with a spinning tube, the Sm of an NMR microcoil is enhanced by a factor of 130.8 That study employed a microcoil Vobs of 5 nL and a sample concentrations of 500 mM. The 3σ LODs for arginine and sucrose were 11 (52 pmol) and 6 ng (19 pmol), respectively, for data acquired in 10 min. The present peptide investigation decreases the sample concentration to 10 mM, reduces the LOD compared to the earlier result, but increases the data acquisition time to 15 h. To decrease analysis time, increase concentration sensitivity, and maintain high spectral resolution, an improved microprobe is developed with a larger Vobs of 31 nL. In addition, the new probe requires a total (4) Webb, A. G. Prog. Nucl. Magn. Reson. Spectrosc. 1997, 31, 1-42. (5) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. J. Am. Chem. Soc. 1994, 116, 7929-30. (6) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-57. (7) Wu, N.; Webb, A.; Peck, T. L.; Sweedler, J. V. Anal. Chem. 1995, 67, 31017. (8) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-70. (9) Webb, A. G.; Grant, S. C. J. Magn. Reson. B 1996, 113, 83-7. (10) Rogers, J. A.; Jackman, R. J.; Whitesides, G. M.; Olson, D. L.; Sweedler, J. V. Appl. Phys. Lett. 1997, 70, 2464-6. (11) Derome, A. E. Modern NMR Techniques for Chemistry Research; Pergamon Press: New York, 1987; pp 51-4.
Analytical Chemistry, Vol. 70, No. 3, February 1, 1998 645
Figure 1. Diagram of NMR microprobe for trace analysis. The required total sample volume, Vtot, was 200 nL. A microsyringe attached to a separate capillary functions as both sample volumemeasuring device and solution transfer tube. The plugs are 8-mmlong segments of capillary with an outside diameter matched to that used for the probe; the plug ends are sealed with epoxy. A perfluorinated liquid fills the container and acts as a magnetic susceptibility matching medium.
sample volume, Vtot, of just 200 nL. Modeling of static line shapes for a cylinder of material placed in a magnetic field indicates that a magic angle configuration yields the best result.12 Due to the placement of this probe at the magic angle (54.7°) with respect to the external magnetic field, adjustment of field homogeneity (shimming) is easier and faster and produces a better line shape than with previous microcoils. EXPERIMENTAL SECTION Chemicals. R-BCP (1-7) (molecular weight 922.2 g/mol; purity 99.0%) was purchased from American Peptide (Sunnyvale, CA). D2O (99.9 atom % D) and CDCl3 (99.8 atom % D) were both from Cambridge Isotope Laboratories (Andover, MA). H2O was generated from a Milli-Q (Millipore, Bedford, MA) water purification system. Spectra are referenced to 0 ppm using 1% tetramethylsilane (TMS; Aldrich, Milwaukee, WI) for organic solutions or 10% H2O at 4.8 ppm for aqueous work. Fluorinert FC-43 (3M, St. Paul, MN) was used as a magnetic susceptibility matching medium to surround the microcoil and sample capillary region. FC-43 is a mixture of perfluorinated C12branched tributylamines with an average molecular mass of 670 g/mol and a formula of C12F27N. NMR Microprobe. Figure 1 shows a layout of probe components. The solenoidal microcoil is fabricated from 50-µmdiameter polyurethane-coated copper wire. The wire is manually wound directly onto a fused silica capillary (Polymicro Technologies, Inc., Phoenix, AZ) with the polyimide coating intact, which has been demonstrated not to affect 1H-NMR data from solutions.8 The ends of the coil are affixed to the capillary using cyanoacrylate adhesives (Krazy Glue, Borden, Inc., Columbus, OH; Ross Super Glue, Conros Corp., Taylor, MI). The coil consists of 17 turns and is 1 mm long. The coil-wrapped capillary rests on a pair of grooved, Plexiglas shoulders placed about 1 cm apart on a circuit board. For soldering the nonmagnetic electrical components to (12) Barbara, T. M. J. Magn. Reson. A 1994, 109, 265-9.
646 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
the board, a simple pattern is printed of tin-coated copper. The board (0.5 in. × 3 in.) is mounted on a cylindrical aluminum probe stand, which places the microcoil at the axial center of the magnetic field. The microcoil region is contained in a 20-mL lowdensity polyethylene bottle filled with FC-43. To eliminate possible damage from uneven heating of the microcoil, conductive silver epoxy (Epo-Tek 410E, Epoxy Technology, Inc., Billerica, MA) is used instead of solder for electronic connection. The microcoil mounted on the circuit board is heated at 60 °C for 12 h to cure the epoxy. The completed microcoil has a dc resistance of 0.3 Ω. A microcoil identical in design to that used for menthol experiments (below) was wrapped on an empty 338-µm-o.d. capillary. It was attached via 3-mm leads to a calibrated platform on an HP4195A network analyzer (HewlettPackard, Palo Alto, CA). At 300 MHz, the measured Q value was 26, and the inductance was 39 nH. This yields an inductance per unit coil volume of 39 nH/90 nL ) 0.43 H/L. Additional details on the microprobe appear elsewhere.8 Peptide Analysis and Conditions. The microprobe employed here consists of a 40-mm length of capillary (75-µm i.d., 357-µm o.d.) which yields Vtot ) 200 nL (5 nL/mm). These capillary dimensions are similar to those used in a previous study8 and result in a coil fill factor, fc (fraction of coil volume occupied by the sample) of 4.4%, and Vobs ) 5 nL, which yields an observe factor, fo (Vobs/Vtot) ) 2.5%. A small segment of 30-gauge TFE tubing (Cole-Parmer, Vernon Hills, IL) is attached to each end of the capillary. A 10-µL syringe (No. 87404, Model 1701RNFS; Hamilton Co., Reno, NV) is fitted with an identical piece of capillary, which functions as a delivery needle and a volumemeasuring device. Samples are loaded from the syringe via the segment of tubing into the microprobe capillary. Both ends of the NMR receiving capillary are then plugged with a short segment of identically sized capillary. These plugs have epoxy tapped into them to seal their ends; they hold the solution in place and prevent evaporation (see Figure 1). For the peptide experiment, the sample can be prepared by two different means. First, an appropriate volume of liquid can be delivered directly to the lyophilized powder in its original ampule to attain the desired concentration (here, 10 mM). Then, 200 nL of this solution can be used for NMR analysis as described in this work. Alternatively, the sample can be prepared from an appropriate mass of dried peptide (such as a lyophilized HPLC fraction)13 and diluted to the desired volume. In this procedure, a syringe is fitted with a capillary, and a 200-nL volume of solvent is drawn up just to fill the 40-mm-long capillary section. While being observed with a stereomicroscope, this volume is pushed into the sample vial to form a nanodroplet, which is manipulated to dissolve the sample material in the vial. The resultant solution is then withdrawn back into the syringe capillary and injected into an identical section of capillary which is wrapped with a microcoil for NMR analysis. The choice of preparation procedure depends on the nature and availability of the original sample. In the present investigation, a stock solution of peptide is prepared. Menthol Analysis and Conditions. The menthol analysis employs a 197-µm-i.d., 338-µm-o.d. capillary which results in a Vobs of 31 nL and an fc of 34%, which is 7.7-fold larger than the previous (13) Sweedler, J. V.; Olson, D. L.; Peck, T. L.; Webb, A. G. Presented at the HPLC Symposium, San Francisco, CA, June 1996.
fc in the peptide study. Since Vtot remains unchanged at 200 nL, fo ) 15.5%, a 6.2-fold increase compared to the value found in the earlier peptide work. Solutions are made of menthol at 12.8 and 82.6 mM in CDCl3. These concentrations represent 400 ng of menthol dissolved in either 200 or 31 nL, respectively. Since handling and analyzing a volume of just 31 nL is not yet practical in the present system, a 200-nL aliquot of the corresponding concentration is injected with a syringe as before. However, some details of the procedure differ from that used for peptide because the capillary inside diameter is now 197 µm (31 nL/mm). To maintain Vtot ) 200 nL, a small segment of epoxy-sealed capillary is inserted into each end of the NMR sample capillary to occupy excess volume and, thereby, maintain the total sample required at 200 nL. The inserted segments are made from polyimide-coated fused silica capillary of 146-µm-o.d. and slide easily inside the sample capillary with a larger inside diameter. Each insert extends into the sample capillary to within about 3 mm of the NMR microcoil. After the sample and inserts are in place, the NMR capillary is sealed with plugs as before. The inserts do not affect the quality of the NMR data. NMR Spectroscopy. All NMR spectra and data are obtained and analyzed on a General Electric 300-MHz (7.05 T) spectrometer with an 89-mm-wide bore. A custom probe stand (made in-house) is employed in all microcoil work. The present magnet is not vibrationally isolated, and neither the console, software, nor magnet is modified in any way to accommodate the microcoil probe. Experiments are run at ∼18.5 °C within the magnet bore. Gentle purging of the NMR probe body with N2 minimizes H2O diffusion into the sample. Addition to organic stock solutions of a few prerinsed and dried (110 °C, 24 h) molecular sieve beads (Type 13x, 4-8 mesh; Grace Davison, Baltimore, MD) also minimizes H2O contamination. The FC-43 spectrum is obtained from a sample in a 5-mm spinning tube using a 10-mm high-resolution broadband solution probe. For the initial procedure used to adjust the position of the microcoil within the magnet, the capillary contains the sample in either 10% H2O or 1% TMS. These conditions yield sufficiently large, single-scan free-induction decays (FIDs) for shimming. Blanks of solvents only, including the capillary plugs and inserts, showed no contamination peaks. For each coil, a 90° pulse width is employed to maximize signal amplitude. Line width is measured as the signal width at half-height without the use of line broadening (smoothing) for a single scan of 1% TMS or 10% H2O. The perfluorinated fluid used as a microcoil surrounding medium has a volume magnetic susceptibility which nearly matches that of copper. This nonconducting fluid reduces field inhomogeneities in the coil region, narrows the spectral line width, improves its shape, and thus increases the S/N. The small size of the NMR microcoil results in an enhanced mass sensitivity due to an increased inductance per unit volume from the radiofrequency excitation pulse.14 Unlike conventional NMR (especially for small molecules), sample spinning is not employed with the microprobe, yet line widths of 0.6 Hz are obtained with both coil sizes used here. (14) Peck, T. L.; Magin, R. L.; Lauterbur, P. C. J. Magn. Reson. B 1995, 108, 114-24.
Background Suppression and Subtraction. A spatially selective, composite 90° radio frequency pulse15 is used to suppress the signals originating from protons in the FC-43 fluid, which is outside the microcoil. This pulse sequence is designed to limit the effective sample volume to the region within the microcoil. Where R represents the 90° tip angle, and (x and (y represent the direction and axis of the pulse, the sequence is as follows: Rx-Ry-R-x-R-y-R-x-R-y-Rx-Ry. RESULTS AND DISCUSSION Improved Procedure for Microprobe Positioning and Shimming. To obtain the optimum line width, line shape, and sensitivity from a new microcoil probe, it is necessary to adjust the magnetic field in the sample region through a procedure called shimming. Initial shimming of a new, commercial NMR probe often requires several hours, although often a probe can be replaced in the magnet bore and optimized more quickly by loading its previous shim settings. We have developed a greatly improved shimming procedure that is customized for microcoils. Prior to insertion of the probe into the magnet, the shim stack is adjusted so that the Z1 shim center coincides with the center of the external magnetic field. This is the normal operating configuration for the NMR system. A solenoidal microcoil usually has its axis perpendicular to the magnetic field and should be located at the vertical and axial center of the Z1 shim field. The positioning method derives from the fact that a variation in the Z1 shim setting changes the magnetic field strength at regions in space that are not at the geometric center of the Z1 shim. Consequently, the signal frequency in the spectrum depends on the value of Z1 if the coil position is located anywhere other than the shim center. The microcoil positioning strategy aims to minimize the change in peak location when Z1 is changed (10% of its range. The Z1 shim on the spectrometer ranges between (2048, a value proportional to a direct current through a coil in the magnet bore which results in a corrective magnetic field. To aid probe positioning, a macro program initially sets all 22 shims to zero, except the coarse Z1 ) +200; then the macro acquires and stores a spectrum. The macro then sets coarse Z1 ) -200, acquires another spectrum, adds it to the first, and then displays the summed spectrum on the console screen. The macro continuously repeats to allow interactive vertical adjustment using the two probe set screws. When the two signals for Z1 ) (200 overlap, the microcoil is positioned at the shim center (see Figure 2). Once the microcoil is at the Z1 shim center, deviation from a symmetrical, Lorentzian line shape may still be observed. Further fine adjustment of the horizontal angle of the probe body and microcoil provides a good line shape to complete the positioning procedure. After this point, coarse shimming yields a signal line width of
