Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-05-28
2004-03-16
Berman, Jack (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C436S180000, C422S105000, C422S063000, C435S030000, C073S864000, C073S864810
Reexamination Certificate
active
06707038
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to methods and devices for selectively depositing fluids on a nonuniform sample surface according to variations in a surface characteristic. More particularly, the invention relates to the use of nozzleless acoustic ejection to deposit droplets of analysis-enhancing fluid on sample surface sites selected according to the surface characteristic at the sites. The invention is especially useful in enhancing the compositional analysis of the biological samples and in the mass spectrometric imaging of tissue surfaces.
BACKGROUND
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. As the requirements for surface analytical techniques have become more exacting, [advances in mass spectrometry have made it possible to obtain in-depth information regarding a wide variety of sample surface types. In the semiconductor industry, for example, secondary ion mass spectrometry has been used to determine the composition of a microscopic region of a wafer surface. In addition, in the biotechnology arena, surface-based mass spectrometry has been 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 often 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 nonbiomolecular 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 enhance 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 has been 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 surfaces selectively interact with analytes and immobilize 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. U.S. Pat. No. 5,808,300 to Caprioli, for example, describes a method for imaging biological samples with MALDI mass spectrometry. 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 specific analysis of 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 the 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 profiling and imaging as described above is the application of a mass spectrometry matrix material to the tissue surface at each site of laser ablation. As described in 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. 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. In particular, U.S. patent application Ser. No. 10/087372, entitled “Method and System Using Acoustic Ejection for Preparing and Analyzing a Cellular Sample Surface,” filed Mar. 1, 2002, by inventors Ellson, Mutz, and Caprioli, describes the use of nozzleless acoustic ejection 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 some instances, different analysis-enhancing fluids may be applied to an analyte to optimize experimental parameters.
As with many types of samples, cellular samples are not typically uniform in composition, and the distribution of materials on the surface of such samples may vary. In addition, the sample surfaces may exhibit inhomogeneous morphologies. Since certain analysis-enhancing fluids are appropriate for use with certain analytes, there is a need to selectively deposit the analysis-enhancing fluid according to the surface characteristics of the cellular sample at that site. Acoustic ejection provides a means for carrying out such fluid deposition with unparalleled accuracy, precision, and efficiency.
SUMMARY OF THE INVENTION
Accordingly, one embodiment of the invention relates to a method for selectively depositing an analysis-enhancing fluid on a sample surface. The method involves providing a sample having a surface that exhibits variations in a specific characteristic, which corresponds to its desirability for receiving an analysis-enhancing fluid. Once a site on the sample surface has been selected according to the desired surface characteristic, focused radiation, typically acoustic radiation, is applied in a manner effective to eject a droplet of the analysis-enhancing fluid from a reservoir. As a result, the droplet is deposited on the sample surface at the selected site. In some instances, a plurality of sites is selected, and a droplet of the analysis-enhancing fluid is deposited onto each selected site. The sites may form a single contiguous region on the sample surface or a plurality of noncontiguous
Caprioli Richard Michael
Ellson Richard N.
Mutz Mitchell W.
Berman Jack
Picoliter Inc.
Reed & Eberle LLP
Wu Louis L.
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