Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-03-01
2004-10-26
Berman, Jack (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C436S180000, C422S105000, C422S063000, C435S030000, C073S864000, C073S864810
Reexamination Certificate
active
06809315
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to methods and devices for preparing and optionally analyzing a cellular sample surface. More particularly, the invention relates to the use of nozzleless acoustic ejection to deposit droplets from a reservoir containing an analysis-enhancing fluid to designated sites on a cellular sample surface. The invention is particularly useful in enhancing the compositional analysis of the sample at the designated sites and in the mass spectrometric imaging of tissue surfaces.
BACKGROUND
Cellular assays are carried out to provide critical information for the understanding of complex cell functions. One commonly employed technique used in cellular assays involves the immobilization of sample cells on a substrate surface and the controlled exposure of the cells to one or more fluids. Particularly when the sample is small, such assays may require the precise and accurate handling of small volumes of fluid.
A number of techniques have been developed in order to meet the need for precise and accurate handling of small volumes of fluids, but most suffer from one drawback or another. 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 wall thickness to channel diameter ratio exacerbates fluid waste. Also, the pool has a minimum required volume driven 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.
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. Mass spectrometry has been employed for samples that have been prepared as an array of features on a substrate surface. Surface-based mass spectrometry has been used, for example, 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 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 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 an element or small molecule in biological specimens, including tissue slices and 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 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 controlled or uniform coating of a mass-spectrometry matrix material to the tissue surface, either as a series of features or as a continuous coating. 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. Most current small-volume dispensing techniques are not reliable for matrix material application as needed, due to limitations in volume or in accuracy of placement.
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. Ser. Nos. 09/669,996 and 09/964,212 for “Acoustic Ejection of Fluids from a Plurality of Reservoirs”, inventors Ellson, Foote and Mutz, filed Sep. 25, 2000 and Sep. 25, 2001, respectively, and assigned to Picoliter Inc. (Mountain View, Calif.). 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.
While nozzleless fluid ejection has generally been appreciated for ink printing applications, acoustic dep
Caprioli Richard Michael
Ellson Richard N.
Mutz Mitchell W.
Berman Jack
Picoliter Inc.
Reed & Eberle LLP
Wu Louis L.
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