Patent Grant
9211542
The present invention relates to sample support plates having a high density of sample recipient sites, and to methods of manufacturing and using such sample support plates. Such sample support plates may be employed in a variety of applications, including but not limited to MALDI mass spectrometry, emulsion PCR, singularization of cells for various analytical applications, microcrystallization of proteins etc.
For the analysis of analytes containing large molecules, in particular, biomolecules, mass spectrometry using matrix-assisted laser desorption and ionization (MALDI) has become a widely used standard method. In such methods, the analyte is dispersed in a crystalline organic matrix deposited on a sample support or on the boundary surface of such a matrix. The analyte is desorbed from the sample support and ionized by action of a desorption laser beam.
Various methods are known for applying the analyte and matrix to a sample support. In the simplest form, droplets of a solution containing the matrix and the analyte are pipetted onto a metal sample support plate. The solution wets the support plate and thus forms a sample spot whose size depends on droplet size, on the hydrophilic properties of the metal and on the properties of the solution. After the solution has dried, the sample spot consists of small matrix crystals in which analyte molecules are embedded. This however often results in an irregular distribution of analyte molecules over a relatively large spot size. As a consequence, ion yield and mass resolution fluctuate over the area of the sample spot, and the desorption laser beam must therefore normally be rastered over the sample spot in order to find “sweet spots” which provide a sufficiently high ion yield and mass resolution.
In U.S. Pat. No. 6,287,872 it has been suggested to coat the surface of the sample support plate with a highly hydrophobic coating, and to provide tiny hydrophilic sample recipient sites called “anchor areas” for the sample droplets on this hydrophobic surface. As the sample droplets are applied to the support plate, they are drawn to the hydrophilic anchor areas. Upon evaporation of the droplets, the matrix and analyte are concentrated to the anchor areas. Thereby sensitivity can be greatly improved, and the search for “sweet spots” becomes easier.
Sample support plates of this kind are commercially available under the name AnchorChip™ from Bruker Daltonik GmbH, Bremen, Germany. Typically, the anchor areas have lateral dimension in the range between 200 μm and 800 μm, with distances between anchor areas in the range of several millimeters. Typically the distance between adjacent anchor areas is more than one order of magnitude larger than the lateral dimensions of the anchor areas themselves, resulting in a relatively low density of sample recipient sites. Samples are normally applied to the anchor areas sites by pipetting droplets of the sample to each anchor area site individually. If a sample droplet is accidentally deposited midway between two anchor areas, it will generally not find its way to the next anchor area and will be wasted.
Conceptually similar sample support plates have also been disclosed in the following articles:
U.S. Pat. No. 7,619,215 discloses a MALDI sample support plate made of stainless steel, on which sampling areas with typical lateral dimensions of 1 to 5 mm are marked. The plate is coated with a hydrophobic organosilane coating. Tiny sample spots are provided in a central portion of each sampling area in which no coating is present, exposing the hydrophilic stainless steel surface of the sample support plate. The sample spots have lateral dimensions in the range of 100 μm to 1 mm. These sample spots are separated by the hydrophobic coating over distances that are typically at least an order of magnitude larger than the sample spots themselves. Also with such known sample support plates, if a sample droplet hits the area midway between two sample spots, the droplet will most likely be wasted. Therefore, such sample support plates still require exact pipetting of sample droplets to the correct spots on the sample support plate.
M. L. McLauchlin, Y. Dongqing, P. Aella, A. A. Garcia, S. T. Picraux and M. A. Hayes, “Evaporative properties and pinning strength of laser-ablated, hydrophilic sites on lotus-leaf-like, nanostructured surfaces”, Langmuir 23 (2007), 4871-4877 discloses MALDI sample support plates having a nanostructured superhydrophobic surface on which hydrophilic sample recipient sites have been prepared by laser ablation. The sites have diameters between 250 μm and 2 mm. No particular arrangements of such sites are disclosed.
A. Amantonico, J. Y. Oh, J. Sobek, M. Heinemann and R. Zenobi, “Mass spectrometric method for analyzing metabolites in yeast with single cell sensitivity”, Angew Chem Int Ed 47 (2008), pp. 5382-5385 shows that the amounts of metabolites corresponding to single yeast cells can be detected by MALDI-MS technique while the sample spot has a size corresponding to the focus size of the desorption laser beam in the MALDI-MS instrument. Picoliter amounts of a sample solution are pipetted onto a support plate precoated with a thin, homogeneous layer of matrix. This document does not disclose any means for concentrating the sample at predefined sample recipient sites.
A. Amantonico, P. L. Urban and R. Zenobi, “Facile analysis of metabolites by capillary electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry using target plates with polysilazane nanocoating and grooves”, Analyst 134 (2009), pp. 1536-1540 discloses MALDI sample plates coated with an omniphobic polysilazane coating. These sample plates are optimized for the deposition of analytes separated by capillary electrophoresis (CE). To this end, an array of parallel grooves acting as recipients for a separation effluent received from a CE apparatus is provided. No means for concentrating the effluent at predefined, point-like sample recipient sites are disclosed.
G. Marko-Varga, S. Ekstrom, G. Heildin, J. Nilsson and T. Laurell, “Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: I. Microstructure development and manufacturing.”, Electrophoresis 22 (2001), pp. 3978-3983 discloses MALDI sample support plates comprising arrays of nanovials fabricated in polymer substrates. The nanovials have diameters of 300, 400 or 500 μm. To improve chemical resistance, the sample plates may be covered with a homogeneous nitrocellulose/matrix layer or with a gold film. S. Ekstrom, J. Nilsson, G. Helldin, T. Laurell, and G. Marko-Varga, “Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: II. Biological applications.”, Electrophoresis 22 (2001), 3984-3992 discloses applications of such sample support plates. While these prior-art documents disclose MALDI sample support plates that have an increased density of sample recipient sites as compared to more traditional sample supports, it is still necessary to exactly dispense individual sample droplets into the individual sample recipient sites.
As will be apparent from the prior art, it is technically challenging to handle small volumes of liquid samples and suspensions of cells prior to analysis by MALDI-MS. An ideal sample support should simplify the sample preparation steps and provide seamless interference-free MS readout.
Similar challenges also exist in other analytical (chemical and biological) disciplines, whenever small volumes of liquid samples, including cell suspensions, are to be distributed to well-defined sample recipient sites.
In a first aspect, it is an object of the present invention to provide a sample support plate that enables a more facile application of small amounts of liquid samples to a plurality of sample recipient sites.
The invention provides a sample support plate comprising a substrate with a substantially flat surface, a plurality of spatially separated sample recipient sites being arranged on said surface. The recipient sites are mutually separated by areas that have a different degree of wettability than said recipient sites. They are arranged in a plurality of rows, each row consisting of a plurality of recipient sites whose centers are regularly spaced along a first direction with a predetermined periodicity, the rows being regularly spaced along a second direction perpendicular to said first direction with a predetermined centerline distance. Each recipient site has a minimum lateral dimension and a maximum lateral dimension, the maximum lateral dimension being less than or equal to 200 μm, preferably between 2 μm and 100 μm. The periodicity along the first direction and the centerline distance along the second direction are chosen such that each recipient site has a next neighbor within an edge distance that is less than or equal to three times said minimum lateral dimension, preferably less than or equal to twice said minimum lateral dimension.
In this manner, a bulk liquid sample may be applied to the surface of the sample support plate such that the bulk liquid will be drawn to nearby sample recipient sites with high probability and will automatically be split into droplets accumulating at these sample recipient sites. Thereby the need of exact pipetting of the liquid sample to individual sample spots is obviated. In other words, the present invention provides a sample support plate having a micro-array of densely spaced sample recipient sites, enabling facile distribution of liquid samples (including cell suspensions) among the sample recipient sites. The sample recipient sites have dimensions in a similar range as a typical dimension of the focus of a desorption laser beam. Using such micro-arrays, there is no need to address each sample recipient site individually during sample preparation.
One possible application is MALDI mass spectrometry, and the sample support plate may be adapted for such applications. In order to be compatible with MALDI procedures, at least a surface-near region of the substrate, preferably the bulk substrate or the surface of the substrate, is preferably substantially electrically conducting in order to enable charge transport away from the sample recipient sites. The term “substantially electrically conducting” is to be understood as including all situations where the substrate is sufficiently conducting to enable charge equilibration on the timescale of a few microseconds, including situations where only the areas between the sample recipient sites are electrically conducting, while the substrate is non-conducting at the sample recipient sites themselves. The substrate is preferably a metal plate, in particular, a stainless steel plate, a metallized plastic or glass plate, in particular, a plastic or glass plate coated with a noble metal, e.g., with gold, or a plastic or glass plate coated with an electrically conducting metal oxide, e.g., indium tin oxide.
The minimum and maximum lateral dimensions of each sample recipient site may be identical (as in the case of a circular sample recipient site) or different (as in the case of most other shapes). These dimensions are to be understood as being defined to be the length of a straight line drawn to the geometric center of each sample recipient site (i.e., as diametrical dimensions). The sample recipient sites of a single sample support plate may all have identical size and shape or may have different sizes and shapes, as long as the sites all are in a similar size range. The shape may be chosen according to need and may, e.g., be circular, elliptic, quadratic, triangular, rectangular, polygonal etc.
Efficient distribution of bulk liquid samples among sample recipient sites may be improved if adjacent rows are arranged so as to be shifted with respect to each other along the first direction. In some preferred embodiments, the rows are shifted by approximately half of said periodicity. This results in a (possibly distorted) “checkerboard”-type of arrangement of the sample recipient sites. When a bulk liquid sample is applied by moving some sort of application device relative to the sample support plate along the first or second direction, such a pattern increases the probability that the sample will be homogeneously distributed among adjacent sample recipient sites.
Often the sample support plate will have a substantially rectangular shape with two parallel longitudinal edges and two parallel transverse edges. Independently of the shape of the sample support plate, the sample recipient sites may be arranged in at least one array of substantially rectangular shape with two parallel longitudinal array edges and two parallel transverse array edges. The rows of sample recipient sites may then generally have an arbitrary orientation relative to the edges of the sample support plate and relative to the array edges.
In some embodiments, it may be preferred if the longitudinal edges of the sample support plate are parallel to the first direction and if the transverse edges are parallel to the second direction. If the recipient sites are arranged in a rectangular array, it may be preferred if the longitudinal array edges are parallel to the first direction and if the transverse array edges are parallel to the second direction. Either of these measures enables efficient usage of the sample recipient sites and simplifies distribution of a bulk liquid containing the sample onto the sample support if the liquid is applied by moving an application device along one of the edges of the sample support plate or of the array, respectively.
In other embodiments, the rows may have an angled orientation relative to the longitudinal edges of the sample support plate. The sample recipient sites may then form at least one array. The orientation of the rows is then preferably chosen such that, if a straight line is drawn parallel to the longitudinal edges and at an arbitrary position along the transverse edges of the sample support plate within the array, there are a plurality of sample recipient sites, preferably at least three or four sample recipient sites, which are cut by said straight line. This enables efficient and homogeneous spreading if a bulk liquid sample is applied by moving some sort of application device relative to the sample support plate along the longitudinal edge of the sample support plate, without the possibility that the application device would not move over any sample recipient site at all.
The centerline distance between adjacent rows and the periodicity within the rows preferably generally have a ratio between 0.3 and 3.0. In particular, if adjacent rows are shifted with respect to each other along the first direction by approximately half of the periodicity, it is preferred if this ratio is between 0.3 and 1.0. In particular, a ratio of 0.5 then corresponds to a “true”, undistorted checkerboard-type arrangement. A ratio of √{square root over (3)}/2≈0.87 corresponds to a trigonal arrangement wherein adjacent sample recipient sites form equilateral triangles. These values are particularly preferred. In preferred embodiments, the distance from any point within the array of sample recipient sites to the edge of the closest sample recipient site is less than 1.5 times the minimum lateral dimension of each sample recipient site, preferably less than this lateral dimension itself. In terms of absolute numbers, this distance is preferably less than 300 μm, in particularly preferred embodiments, less than 100 μm, so as to ensure that droplets will find their way to the closest sample recipient site with high probability. The edge distance from each sample recipient site to the closest adjacent sample recipient site (the next neighbor) is, in absolute numbers, consequently preferably less than 600 μm, in particularly preferred embodiments, less than 200 μm.
As already stated above, the areas between sample recipient sites generally have a different wettability than the sample recipient sites themselves. The wettability of these areas for a selected solvent is preferably lower than the wettability of the sample recipient sites. In particular, for a polar solvent, this situation is normally referred to by saying that the areas between the recipient sites are more hydrophobic than the recipient sites. For a nonpolar solvent, this situation is normally referred to by saying that the areas between the recipient sites are more lyophobic than the recipient sites. If the areas between sample recipient sites have a lower wettability than the sample recipient sites themselves for both polar and nonpolar solvents, the areas between sample recipient sites are said to be omniphobic. This situation is particularly preferred. Hydrophobic and/or lyophobic or omniphobic surfaces interrupted by less hydrophobic, lyophobic or omniphobic sample recipient sites, respectively, can be obtained by a wide range of methods, as detailed further below. In particular, in some embodiments, the sample support plate may comprise a hydrophobic and/or lyophobic or preferably omniphobic coating on the substrate, and the recipient sites interrupt said coating. This can be achieved, in particular, by first applying the coating uniformly to the substrate surface and subsequently removing the coating selectively at the sample recipient sites, e.g., by laser ablation or any other suitable method, as detailed further below.
In another aspect, the present invention also encompasses a MALDI mass spectrometer comprising a sample support plate as described above. A laser is arranged to direct a laser beam to the sample support plate so as to illuminate a beam spot on the sample support plate. It is then preferred that the beam spot has an area on said sample support plate that is at least 50% of the area of any one of said recipient sites. In even more preferred embodiments, the beam spot has an area on said sample support plate that exceeds the area of any one of said recipient sites. In this manner, it is possible to illuminate a complete sample recipient site at the same time, and rastering of the beam becomes unnecessary.
Nowadays, beam spots having a diameter as small as 10 μm have become possible; therefore, the maximum dimension of each sample recipient site may be as small as 10 μm or even smaller, e.g. 5 μm or even 2 μm. A practical lower limit might be approached if the sample recipient sites become smaller than the diffraction limit for the laser wavelength employed, e.g., smaller than 500 nm.
In addition, any MALDI mass spectrometer will generally comprise an ion extractor to extract ions generated by said laser beam in said beam spot, and a mass analyzer to analyze the mass-over-charge ratio of said ions. Both for ion extractors and for mass analyzers, a large number of designs are known in the art, and the invention is not limited to any particular design. In particular, the mass analyzer may be of the time-of-flight (TOF) type, of the (Fourier transform) ion cyclotron resonance (ICR or FT-ICR) type, or of other types, such as the ion trap type.
In yet another aspect, the present invention provides a method of manufacturing a sample support plate, the method comprising:
If the sample support plate is intended to be used for MALDI mass spectrometry, the substrate is preferably substantially electrically conducting at least in a surface-near region.
The invention further encompasses a method of sample preparation comprising:
The present invention further provides a method of sample preparation. In this method, a bulk liquid containing the sample is distributed onto a sample support plate as described above, causing the bulk liquid to separate into discrete droplets located at the sample recipient sites. The sample may be applied by continuously moving an application device over the surface of the sample support plate, the application device acting to continuously distribute the bulk liquid over the surface. This method may be employed in the context of MALDI mass spectrometry, but is by no means limited to such procedures and is equally applicable in the context of other applications.
The bulk liquid may be selected from the group consisting of solutions, suspensions and emulsions of organic molecules in a carrier liquid, and suspensions of cells in a carrier liquid.
The present invention further relates to a method of preparing a plurality of samples on a sample support plate, each sample comprising a first and a second reagent. The method comprises:
The application devices for the first and second reagents may be identical or different. The first and second reagents themselves may be identical or different. In some applications, at least one of the reagents may be a cell suspension, or cells may additionally be distributed to the sample recipient sites before distribution of the first reagent, between distribution of the first and the second reagent, or after distribution of the second reagent.
This method is particularly useful for high-throughput combinatorial screening. Again, this method may be employed in a variety of different applications and is not limited to sample preparation for MALDI-MS.
The above methods of sample preparation may be used, in particular, to apply cell suspensions to the sample support plate. The methods will then result in only a limited number of cells being deposited onto each sample recipient site. The exact number of cells at each site will generally fluctuate statistically. The mean number of cells deposited at each sample recipient site in this manner may be relatively low, e.g., it may be 1-100, preferably 1-10, most preferably 1-3. In one possible group of applications, the cells at each sample recipient site may be subjected to one or more analytical procedures, including mass spectrometry, fluorescence spectroscopy and other spectroscopic techniques. In this manner, similar results as with flow cytometry may be obtained. Another possible application is the distribution of cells to the sample recipient sites for the production of certain substances, e.g. the distribution of hybridoma cells for producing monoclonal antibodies.
The above methods of sample preparation may also be used in the context of amplification reactions for nucleic acids, in particular, for the distribution of fragments, oligos and/or primers to the sample recipient sites for carrying out amplification reactions such as PCR, LCR etc., in particular, emulsion PCR.
As will have become apparent from the above examples, the sample support plates and procedures described above may be used in a variety of different applications that require a defined aliquotation or defined parallel aliquotation either by volume or cell count, including but not limited to the following applications:
The volumes that can be applied to each sample recipient site may range from the picoliter range to the microliter range. The samples may have a widely varying polarity, depending on the specific design of the sample support plate.
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
For the purposes of the present invention, a “hydrophobic” surface is to be understood to be a surface which is not easily wettable by a polar liquid, in particular, by an aqueous liquid, specifically, water, repelling such a liquid. Generally, the contact angle of the liquid on a hydrophobic surface is more than 90 degrees. Conversely, a “hydrophilic” surface is to be understood to be surface with a comparatively small contact angle for such a liquid, certainly less than 90 degrees. A first area is “more hydrophobic” than a second area if the contact angle of such a liquid in the first area is larger than the contact angle of the same sample liquid in the second area. Similar definitions may also be used for the terms “lyophilic” and “lyophobic” for non-polar liquids. A surface is said to be “omniphobic” if it is both hydrophobic and lyophobic, i.e, if it is not easily wettable for a wide range of polar and non-polar liquids.
By the way of example, clean stainless steel surfaces exposed to normal air humidity are normally slightly hydrophilic for normal aqueous sample solutions due to hydroxy groups being created under the influence of the air. The same is true for most other metals, including precious metals. By the way of example, a plastic plate coated with a gold coating will generally be hydrophilic. Examples for omniphobic materials include polytetrafluorethylene (PTFE) and polysilazanes.
In
For carrying out MALDI mass spectrometry using such a sample support plate, the sample recipient sites are loaded with a MALDI matrix and with the actual sample, e.g., as described further below with reference to
The sample recipient sites 101 are preferably smaller than the beam spot 105. In other words, it is preferred that the beam spot illuminates a complete sample recipient site at the same time. In this way, an optimum sensitivity can be achieved. The beam spot size can vary considerably, depending on the laser and laser optics employed in the particular mass spectrometer. Typical beam spot diameters range from 10 to 200 μm. Consequently, the maximum lateral dimension of the sample recipient sites is preferably in the same range or below.
A third embodiment of a sample support plate according to the present invention is schematically illustrated in
Whereas the sample recipient sites of the above examples are of square and circular shape, respectively, other shapes are possible, including elliptic, rectangular, triangular, and regular polygonal and irregular polygonal shapes.
a) illustrates a first example of how a sample may be applied to a sample support plate according to the present invention. A bulk aqueous liquid, in the present example a cell suspension comprising cells 151 that are to be investigated, is spread onto a sample support plate 100 with the aid of a spreading device in the form of a glass slide 154. To this end, the glass slide is moved over the sample support plate 100 along the x direction (arrow 155). During the spreading of the liquid, liquid droplets 153 accumulate at the hydrophilic sample recipient sites 101, while the liquid is repelled from the hydrophobic polysilazane coating between the sample recipient sites.
Two different kinds of arrangements of sample recipient sites are illustrated in
If such a spreading method is employed with the sample support plates of
For cell analysis, the following protocol for loading the cells and the MALDI matrix to the sample support plate may be employed: First, the cell suspension is spread onto the sample support plate, while the plate is preferably kept at constant temperature with the aid of a Peltier element. The cells may then optionally be quenched by application of a quenching liquid, e.g., ethanol. Only then the matrix may be applied by the usual methods, e.g. by immersion into a matrix solution (possibly under the action of ultrasound), by spraying, by electrospraying or casting. Alternatively, it is also possible to first load the sample recipient sites with the matrix and then apply the cell suspension. A preferred matrix material is 9-aminoacridine (9AA).
The sample support plate of the present invention may be manufactured as follows: First, a substrate having a flat surface is provided. For MALDI applications, it is generally necessary that the surface of the substrate defines a substantially constant electric potential, since the MALDI process requires a substantially homogeneous electric field for acceleration and a dissipation of charges accumulating at the surface. Therefore, MALDI sample plates often have a substrate made of a metal, of a metallized plastic or glass plate or of a plastic or glass plate coated with gold. However, for other applications, the substrate may be non-conducting.
The surface of the substrate is then coated with a thin hydrophobic and/or lyophobic, preferably omniphobic, coating, which may or may not be a monolayer of functional molecules. Such coatings are well known in the art. The coating may preferably comprise a polysilazane. Polysilazane-based coating materials are available commercially, e.g., the coating CAG 37 available from Clariant Advanced Materials GmbH, Sulzbach, Germany.
In the simplest case, such coatings may be applied by spraying. Examples of other polysilazane-based coatings are described, e.g., in US 2005/0169803 and references contained therein. However, many other types of coatings may also be used, including silicones, alkylchlorosilanes, tin-organic compounds, alkane thioles or fluoroalkane thiols, etc. Many different methods of coating are known. For example, for a possible coating process with a thiol-based coating, explicit reference is made to the coating process described in U.S. Pat. No. 6,287,872. Preferably the coating is very thin, preferably less than 10 micrometers. Ideally, the coating preserves a certain amount of electric conductivity of the coated surface.
The sample recipient sites are then preferably created by ablating the coating at the desired locations, e.g., by application of a laser beam. Other possibilities include spark erosion, reactive ion etching and other ablation methods or photolithographic methods. Alternatively, it is also possible to cover the desired locations on the substrate surface with a hydrophilic or lyophilic lacquer before application of the coating or to attach hydrophilic or lyophilic spots to the coating, e.g., by application of amphiphilic substances which bond to the coating and create a surface having different wetting properties at these locations. In yet another embodiment, the coating may be destroyed at the desired locations by applying disintegrating chemicals. In some of these methods, the relevant substances may be applied to the surface or the coating in the same manner as in an inkjet printer.
In other embodiments, the surface of the substrate may be micro- and/or nanostructured to obtain a superhydrophobic surface, as described, e.g., in M. Groenendijk, “Fabrication of super hydrophobic surfaces by fs laser pulses”, Macro Material Processing, May 2008, pp. 44-47. The sample recipient sites may then be obtained, e.g., by destroying the superhydrophobic surface structure at these sites. Many other fabrication methods are possible.
The recipient sites of the sample support plate of
The functional design of the MALDI plate according to this invention offers fast unsupervised distribution of MALDI matrix, cell suspensions and liquid samples among multiple sites on MALDI plates. It also provides seamless deposition of effluents from microfluidic devices on MALDI plates enabling sensitive and high-throughput mass spectrometric analysis.
Unlike in most prior-art sample-focusing supports for MALDI-MS, the diameter of each sample site may be made smaller or equal to that of the MALDI laser beam; therefore, there is no need for rastering the sample deposit. This speeds up analysis while preserving high sensitivity of the measurement. It also eliminates issues due to inhomogeneous matrix crystallization: the whole sample is scanned at the same time.
The proposed design of the arrays enables seamless distribution of biological cells among the recipient sites. This can be done in a short period of time, manually or with a simple mechanical aid.
Deposition of liquids on the high-density mass spectrometry micro-arrays does not require alignment. Misalignment is compensated by the geometry of the recipient site pattern. For example, the micro-array can be used for deposition of effluents from nanoflow LC columns. This is important, e.g., for applications in proteomics where liquid chromatography (LC) separations are often coupled with MALDI spotters; using nanoflow LC would limit expenditure of costly chemicals.
Use of micro-arrays for analysis of liquid samples by MALDI-MS increases homogeneity of the sample deposit, minimizes consumption of the sample and of the toxic MALDI matrix.
Possible applications of the high-density micro-arrays described here include:
a) Collection of effluents from microscale capillaries or microfluidic devices: The present invention enables facile transfer of liquid samples, for example, when delivered with microscale capillaries onto the surface, prior to mass spectrometric analysis. This is important in a range of bioassays incorporating microfluidics for sample preparation and treatment.
b) High-throughput combinatorial screening: The invention enables performing biochemical reactions in nano- and pico-liter volume in a high-throughput manner prior to mass spectrometric analysis, potentially lowering the costs of drug discovery process. It can allow for the screening of drug interactions with target proteins that are only available in small quantities, thus speeding up the lead compound selection. Various types of information on the reaction progress can be obtained from such analysis; this stays in contrast with the affinity/activity screening methods incorporating optical detection. Moreover, the screening can be conducted also with live cells that can readily be patterned in the high-density micro-array, as outlined below.
c) Analysis of cancer and microbial cells: Mass spectrometry, including MALDI-MS enables analysis of numerous chemical species at the same time. However, technical obstacles exist that hinder direct application of mass spectrometry in high-throughput analysis of individual (and even small numbers of) cells. The dual function of the high-density mass spectrometry micro-array facilitates unsupervised handling of cells and subsequent mass spectrometric analysis.
d) Sensitive and reproducible analysis of solutions delivered in nanoliter and microliter volumes: Conventional MALDI-MS analysis of liquid samples is generally not reproducible enough to perform routine quantitative analyses of biological samples. High-density mass spectrometry micro-array can address this problem to some extent.
Whereas preferred embodiments have been described mainly in the context of MALDI-MS applications, the invention is by no means limited to MALDI-MS, and sample support plates according to the present invention may also be employed in various other applications.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10163610 | May 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/058273 | 5/20/2011 | WO | 00 | 2/1/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2011/144743 | 11/24/2011 | WO | A |
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|---|---|---|---|
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| 19923761 | Feb 2001 | DE |
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| Number | Date | Country | |
|---|---|---|---|
| 20130146758 A1 | Jun 2013 | US |
