Mass spectrometry is a well-established analytical technique in which sample molecules are ionized and the resulting ions are sorted by mass-to-charge ratio. Advances in mass spectrometry have made it possible to obtain detailed information regarding a wide variety of sample surface types. In the semiconductor industry, for example, secondary ion mass spectrometry is used to determine the composition of microscopic regions of wafer surfaces. As another example, in the biotechnology arena, surface-based mass spectrometry is used to analyze single nucleotide polymorphisms in microarray formats. See, e.g., U.S. Pat. No. 6,322,970 to Little et al.
Matrix-Assisted Laser Desorption Ionization (MALDI) is an ionization technique commonly used for mass spectrometric analysis of large and/or labile biomolecules, such as nucleotidic and peptidic oligomers, polymers, and dendrimers, as well as for analysis of non-biomolecular compounds, such as fullerenes. MALDI is considered a “soft” ionizing technique in which both positive and negative ions are produced. The technique involves depositing a small volume of sample fluid containing an analyte on a substrate comprised of a photon-absorbing matrix material selected to enhanced desorption performance. See Karas et al. (1988), “Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons,” Anal. Chem. 60:2299-2301. The matrix material is usually a crystalline organic acid that absorbs electromagnetic radiation near the wavelength of the laser. When co-crystallized with analyte, the matrix material assists in the ionization and desorption of analyte moieties. The sample fluid typically contains a solvent and the analyte. Once the solvent has been evaporated from the substrate, the analyte remains on the substrate at the location where the sample fluid is deposited. Photons from a laser strike the substrate at the location of the analyte and, as a result, ions and neutral molecules are desorbed from the substrate. MALDI techniques are particularly useful in providing a means for efficiently analyzing a large number of samples. In addition, MALDI is especially useful in the analysis of minute amounts of sample that are provided over a small area of a substrate surface.
Surface Enhanced Laser Desorption Ionization (SELDI) is another example of a surface-based ionization technique that allows for high-throughput mass spectrometry. SELDI uses affinity-capture reagents, such as antibodies, to collect samples from a complex mixture, which allows in situ purification of the analyte followed by conventional MALDI analysis. Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a “protein chip” to interact with analytes, e.g., proteins, in solution. Such a surface selectively interacts with analytes and immobilizes them thereon. Thus, analytes can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing different reactive moieties at different sites on a substrate surface, throughput may be increased.
Recently, mass spectrometry techniques involving laser desorption have been adapted for cellular analysis. Cellular assays such as mass spectrometry are carried out to provide critical information for the understanding of complex cell functions. U.S. Pat. No. 5,808,300 to Caprioli, for example, describes a method for imaging biological samples with mass spectrometry using surface-based ionization. This method allows users to measure the distribution of a specific element or small molecule within biological specimens such as tissue slices or individual cells. In particular, the method can be used for the analysis of specific peptides in whole cells, e.g., by obtaining signals for peptides and proteins directly from tissues and blots of tissues. In addition, the method has been used to desorb relatively large proteins from tissues and blots of tissues in the molecular weight range beyond about 80 kilodaltons. From such samples, hundreds of peptide and protein peaks can be recorded in the mass spectrum produced from a single laser-ablated site on the sample. When a laser ablates the surface of a sample at multiple sites and the mass spectrum from each site is saved separately, a data array is produced, which contains the relative intensity of any given mass at each site. An image of the sample surface can then be constructed for any given molecular weight, effectively representing a compositional map of the sample surface.
One important issue to successful MALDI and MALDI-like profiling and imaging as described above is the controlled application of a mass-spectrometry matrix material to the tissue surface, either as a series of features or as a continuous coating so as to provide mass spectrometry matrix material at each site of laser ablation. For example, as described in U.S. Pat. No. 5,808,300 to Caprioli, the mass spectrometry matrix material may be applied as a continuous and uniform coating of less than about 50 micrometers in thickness. In order to apply the mass spectrometry matrix material in a controlled manner, carefully metered amounts of sample fluids should be accurately and precisely placed on a sample surface. The ability to closely compare relative abundances of a given protein between two tissues is dependent on the application of matrix in exactly the same way to both tissues.
Most current small-volume dispensing techniques, however, are not suitable for precise and reproducible matrix material application, due to limitations in volume or in accuracy of placement. For example, capillaries having a small interior channel (e.g., Eppendorf-type capillaries) are often used to transfer fluids from a pool of fluid. Their tips are submerged in the pool in order to draw fluid therefrom. In order to provide sufficient mechanical strength for handling, however, such capillaries must have a large wall thickness as compared to the interior channel diameter. Thus, the physical dimensions of such capillaries limit their fluid-handling capability. In addition, since any wetting of the exterior capillary surface results in fluid waste, the high ratio of wall thickness to channel diameter exacerbates fluid waste. Also, the pool has a minimum required volume determined not by the fluid introduced into the capillary but, rather, by the need to immerse the large exterior dimension of the capillary. As a result, the fluid volume required for capillary submersion may be more than an order of magnitude larger than the fluid volume transferred into the capillary.
A number of patents have described the use of acoustic energy in printing. For example, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquid drop emitter that utilizes acoustic principles in ejecting droplets from a body of liquid ink onto a moving document to form characters or bar codes thereon. As described in a number of U.S. patent applications, acoustic ejection provides for highly accurate deposition of minute volumes of fluids on a surface, wherein droplet volume—and thus “spot” size on the substrate surface—can be carefully controlled, and droplets can be precisely directed to particular sites on a substrate surface. See, e.g., U.S. Patent Application Publication No. 2002037579 to Ellson et al. In other words, nozzleless fluid delivery provides high fluid-delivery efficiency through accurate and precise droplet placement. Nozzleless fluid ejection also provides a high level of control over ejected droplet size.
Acoustic ejection is a technique that is well suited for depositing minute volumes of fluids on a surface because the technique allows for control over droplet volume and thus “spot” size on the surface, as well as control over the trajectory of ejected droplets and the precise location of the deposition sites on the surface. See, e.g., U.S. Patent Application Publication No. 20020037579 to Ellson et al. While nozzleless fluid ejection has generally been appreciated for ink printing applications, acoustic deposition is a generally unknown technique in the field of cellular analysis. Recently, focused acoustic energy has been used to manipulate cells and engage in cell sorting. See U.S. Patent Application Publication Nos. 20020064808, 20020064809, 20020090720, and 20020094582 to Mutz et al. In addition, as cellular assays often involve the immobilization of sample cells on a substrate surface and the controlled exposure of the cells to one or more fluids, there exist opportunities to improve cellular assay and analysis techniques through the use of acoustic ejection, particularly when such assays require the precise and accurate handling of small volumes of fluid. For example, U.S. Patent Application Publication No. 20020171037 to Ellson et al. describes the use of acoustic ejection for preparing and analyzing a cellular sample surface. Nozzleless acoustic ejection is used to deposit mass spectrometry matrix material at designated sites on a sample surface to form either a uniform matrix material layer or an array of individual sites. In addition, U.S. Patent Application Publication No. 20020195538 to Ellson et al. describes the use of acoustic ejection to selectively deposit analysis-enhancing fluid according to the surface characteristics of the cellular samples.
As alluded to above, conventional analysis-enhancing fluids for use in mass spectrometry are typically comprised of a mass spectrometry matrix material dissolved in a volatile carrier fluid. Once deposited on a sample surface, the carrier fluid is evaporated, thereby allowing the matrix material to precipitate and crystallize with the sample. It has recently been discovered, however, that such conventional analysis-enhancing fluids are not optimal for use in mass spectrometry when dispensed as low-volume droplets under ordinary dispensing conditions, because such fluids do not allow the matrix material to properly crystallize with the sample.
Accordingly, there is a need for methods and systems that overcome the disadvantages and limitations associated with previously known technologies.