Mass spectrometry measures the mass-to-charge ratio (m/z) of ions formed from analyte molecules. As shown in FIG. 1, mass spectrometers have three principal components: an ion source, a mass analyzer and an ion detector. The function of the ionization source is to convert analyte molecules into gas phase ions. Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized. The ions are accelerated under vacuum in an electric filed and separated by mass analyzers according to their m/z ratios. Exemplary mass analyzers include triple-quadrupole, time-of-flight (TOF), ion trap, quadrupole-TOF, and Fourier transform ion cyclotron resonance (FTICR) analyzers. As individual ions reach the detector, they are counted. During the last several years, technological developments in mass spectrometers have greatly improved the mass accuracy, resolution and sensitivity. However, there still remain great opportunities and challenges in mass spectrometry.
Proteomics and metabolomics as are generating new knowledge-bases for hypothesis-driven biochemical and bioimaging studies. Mass spectrometry has been the enabling technology for much of this research. Yet, the sensitivity and dynamic range have not been sufficient for all analyses. Thus far, Fourier transform ion cyclotron resonance mass spectrometry has achieved some of the best results—a detection limit of 10 zmole (i.e., 6000 molecules) for tryptic peptides of bovine albumin and a dynamic range of at least 6 orders of magnitude. This sensitivity suffices for the detection of the most abundant proteins in a single mammalian cell. However, due to the huge dynamic range (106-109) of proteins in the cell, it still remains a challenge to detect many of the less abundant proteins, even those that are present in large amounts. Revolutionary innovations can help to achieve proteomics and metabolomics of single cells. Mass spectrometry analysis that uses a very small sample size and has excellent ionization efficiency and extremely high resolution, accuracy, and dynamic range would be useful for performing such analyses.
One dominant soft-ionization method that can ionize large biomolecules such as peptides and proteins without significant fragmentation is electrospray ionization (ESI). In ESI, a volatile liquid containing an analyte moves through a very thin, charged capillary. The liquid is dispersed into a mist of small charged droplets by applying a high electric potential between the capillary and a counter electrode. As the liquid evaporates, highly charged analyte molecules explode out from the droplets. The smaller the droplets, the larger the explosion. In general, for conventional electrospray, the smallest capillaries have an inner diameter of approximately 1 μm and generate droplet diameters in the range of 1-2 μm with a flow rate of 20-40 nanoliters per minute (nL/min).
At sufficiently low flow rate and concentration, there is on average less than one analyte molecule per droplet. Ionization efficiencies can approach 100% as the analyte is dispersed in very small, easily desolvated, charged droplets. This limit is reached only if the concentration is low enough. The abundance of sample ions created by ESI can reach a plateau at certain concentrations and does not increase beyond the plateau even with increased sample concentration. Thus there is a limited dynamic range, which can be a serious drawback. In a complex mixture this problem can be especially severe. This ion suppression effect, which seems to occur at flow rates in the range of 50 nL/min and higher and is effectively absent at flow rates below approximately 20 nL/min, can render less abundant ions undetectable. Further reduction in the droplet size can alleviate this problem, increasing both the dynamic range and sensitivity of mass spectrometry.
In order to achieve lower flow rates, in the range of picoliters per minute (pL/min) or less, much smaller capillaries with sub-micron inner-diameters are needed. But there are difficulties associated with fabrication of such small capillaries, and there can be problems with increased back pressures from such extremely small channels. In addition, very small capillaries can lead to low throughput because of the need for careful and tedious alignment in the mass spectrometer.
There have been efforts to fabricate ESI emitters using polymeric materials, such as parylene (Licklider, L.; Wang, X. Q.; Desai, A.; Tai, Y. C.; Lee, T. D. Anal. Chem. 2000, 72, 367-375; Yang, Y. N.; Kameoka, J.; Wachs, T.; Henion, J. D.; Craighead, H. G. Anal. Chem. 2004, 76, 2568-2574), poly(dimethylsiloxane) (PDMS) (Kim, J. S.; Knapp, D. R. J. Am. Soc. Mass Spectrom. 2001, 12, 463-469), poly(methyl methacrylate) (PMMA) (Schilling, M.; Nigge, W.; Rudzinski, A.; Neyer, A.; Hergenroder, R. Lab Chip 2004, 4, 220-224), and negative photoresist SU-8 (Le Gac, S.; Arscott, S.; Rolando, C. Electrophoresis 2003, 24, 3640-3647; Nordstrom, M.; Marie, R.; Calleja, M.; Boisen, A. J. Micromech. Microeng. 2004, 14, 1614-1617). However, inherent properties of these hydrophobic polymers, such as strong binding affinity to proteins and incompatibility with organic solvents, have limited their usefulness for electrospray applications.
There have also been efforts to fabricate ESI emitters using silicon-based materials, such as silicon nitride (Desai, A.; Tai, Y.; Davis, M. T.; Lee, T. D. International Conference on Solid State Sensors and Actuators (Transducers '97), Piscataway, N.J., 1997, 927-930) and silicon/silica (Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063; Griss, P.; Melin, J.; Sjodahl, J.; Roeraade, J.; Stemme, G. J. Micromech. Microeng. 2002, 12, 682-687). In-plane silicon nitride emitter fabrication has failed due to intrinsic clogging problems arising from the etching of phosphosilicate glass between silicon nitride layers. Out-of-plane fabrication of silicon/silica has been limited critically because of the additional assembly steps needed to attach emitters to the end of a microfluidic channel. More recent efforts to make nanoelectrospray emitters from nanofluidic capillary slot and micromachined ultrasonic ejector arrays have faced similar challenges (Arscott, S.; Troadec, D. Appl. Phys. Lett. 2005, 87, 134101). None of these emitters has achieved desired flow rates of pL/min or less. In addition, no monolithic multinozzle emitter for nanoelectrospray mass spectrometry has ever been fabricated.
To increase the sensitivity of ESI further and to interface the emitters with lab-on-a-chip, breakthroughs in the design and fabrication of ultra-narrow emitters are clearly needed.